Introduction to Biotechnology and Biostatistics 1774073668, 9781774073667

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INTRODUCTION TO BIOTECHNOLOGY AND BIOSTATISTICS

INTRODUCTION TO BIOTECHNOLOGY AND BIOSTATISTICS

Khushboo Chaudhary

www.delvepublishing.com

Introduction to Biotechnology and Biostatistics Khushboo Chaudhary Delve Publishing 2010 Winston Park Drive, 2nd Floor Oakville, ON L6H 5R7 Canada www.delvepublishing.com Tel: 001-289-291-7705 001-905-616-2116 Fax: 001-289-291-7601 Email: [email protected] e-book Edition 2020 ISBN: 978-1-77407-508-1 (e-book)

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ABOUT THE AUTHOR

Khushboo Chaudhary is presently a Ph.D scholar in Banasthali Vidyapith (Raj) and having one year of teaching experience. Recently, she is working on phytoremediation of Fluoride. She has published seven research papers in the peer review journals of international repute and three book chapters. She has also attended several national and international conferences/ symposia/ seminars in the various fields of biotechnology and agriculture sciences and presented her research findings. Her current research includes microbiology, and soil chemistry of the plants. She has got best poster award by ISSGPU CIRG Makhdoom. She has published several gene banks in NCBI.

TABLE OF CONTENTS

Glossary ........................................................................................................... xxiii List of Figures .................................................................................................... xxix List of Tables ....................................................................................................xxxiii List of Abbreviations ........................................................................................ xxxv Preface........................................................................ ....................................xxxix Acknowledgment.................................................................................................xli Chapter 1

Introduction of Biotechnology .................................................................. 1 1.1. Scope ................................................................................................. 1 1.2. Importance ........................................................................................ 2 1.3. Biotechnology Century ...................................................................... 3 1.4. History Of Biotechnology ................................................................ 10 1.5. Traditional Biotechnology ................................................................. 11 1.6. Modern Biotechnology ..................................................................... 29 1.7. Global Impact And Current Excitement Of Biotechnology ................ 33 1.8. Biotechnology Impact On Health Care ............................................ 33 1.9. Biotechnology Impact On Environment ............................................ 34 1.10. Biotechnology Impact On Agriculture ............................................ 35 1.11. Biotechnology In India ................................................................... 36 1.12. Biotechnology In World .................................................................. 38 1.13. Future Development Achievements Of Biotechnology .................... 38 1.14. Misuse Of Biotechnology ............................................................... 40 1.15. Biodiversity And Its Conservations .................................................. 41 1.16. Introduction To Biodiversity ............................................................ 41 1.17. Definition And Explanation ............................................................. 41 1.18. Alpha And Beta Biodiversity ........................................................... 41 1.19. Levels Of Biodiversity ..................................................................... 41 1.20. Rate Of Loss Of Biodiversity ........................................................... 42

1.21. Causes For The Loss Of Biodiversity ................................................ 42 1.22. Uses Of Biodiversity ....................................................................... 43 1.23. Extent Of Biodiversity In Plant ........................................................ 43 1.24. Exploration And Germplasm Collection .......................................... 43 1.25. Introduction And Exchange Of Pgr (Plant Genetic Resources) ......... 44 1.26. Red Data Book And Endangered Plant Species................................ 44 1.27. Plant Genetic Resources ................................................................. 44 1.28. Plant Quarantine Aspects ................................................................ 45 1.29. Sanitary And Phytosanitary Systems (SPS) ........................................ 45 1.30. In-Situ Conservation ....................................................................... 45 1.31. Ex-Situ Conservation ....................................................................... 46 1.32. Cryopreservation ............................................................................ 46 1.33. Gene Banks .................................................................................... 46 1.34. Cryobanks ...................................................................................... 46 1.35. IPGRI (International Plant Genetic Resources Institute) ................... 46 1.36. FAO (Food And Agriculture Organization Of The United Nations)... 47 1.37. NBPGR (National Bureau Of Plant Genetic Resources) ................... 47 1.38. CGIAR (Consultative Group For International Agricultural Research)47 1.39. Plant Breeders Rights ...................................................................... 48 1.40. Farmers Rights ................................................................................ 48 1.41. Protection Of Plant Varieties ........................................................... 48 1.42. Farmers Rights Acts ......................................................................... 49 Chapter 2

Genes And Genomes And Genetic Engineering....................................... 51 2.1. Nature Of DNA ................................................................................ 51 2.2. Composition Of DNA ....................................................................... 51 2.3. Nucleosides ..................................................................................... 52 2.4. Nucleotides ...................................................................................... 52 2.5. Polynucleotide ................................................................................. 53 2.6. Chargaff-Equivalence Rule ................................................................ 53 2.7. Physical Nature Of DNA................................................................... 54 2.8. Watson And Crick’s Model Of DNA ................................................. 54 2.9. Circular And Super Helical DNA ..................................................... 55 2.10. Organization Of DNA ................................................................... 56 2.11. Structure Of Ribonucleic Acid (RNA) ............................................. 57

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2.12. Gene Concept ................................................................................ 57 2.13. Units of A Gene ............................................................................. 58 2.14. Cistron ............................................................................................ 58 2.15. Recon ............................................................................................ 59 2.16. Muton............................................................................................. 60 2.17. Split Genes (Or Introns) ................................................................. 60 2.18. RNA Splicing ................................................................................. 60 2.19. Ribozymes ...................................................................................... 61 2.20. Overlapping Genes (Genes Within Genes) ..................................... 61 2.21. Gene Organization ........................................................................ 62 2.22. Gene Expression ............................................................................. 62 2.23. Gene Regulation ............................................................................. 63 2.24. Transcription ................................................................................... 63 2.25. Translation ...................................................................................... 63 2.26. Polymerase Chain Reaction (PCR) .................................................. 64 2.27. Polymerase Chain Reaction Application.......................................... 68 2.28. DNA Fingerprinting ........................................................................ 72 Chapter 3

Recombinant DNA Technology And Tools Of Genetic Engineering ........ 75 3.1. Chimeric DNA.................................................................................. 75 3.2. Restriction Enzymes For Cloning ...................................................... 75 3.3. Technique Of Restriction Mapping ................................................... 76 3.4. Restriction Cleavage And Gel Electrophoresis ................................... 77 3.5. Construction Of A Restriction Map .................................................. 77 3.6. Use Partial Digests, End Labeling Hybridization In Restriction Mapping ...................................................................... 78 3.7. Construction Of Chimeric DNA ....................................................... 79 3.8. Adding Poly Da At The 3’ Ends Of The Vector And Poly Dt At The 3’ Ends Of The DNA Clone ................................................. 79 3.9. Blunt End Ligation By T4 DNA Ligase ............................................... 80 3.10. DNA Cloning In Bacteria And Eukaryotes ....................................... 81 3.11. Cloning In Bacteria ......................................................................... 81 3.12. Cloning In Eukaryotes ..................................................................... 81 3.13. Molecular Probes............................................................................ 82 3.14. Preparation Of Probes..................................................................... 82 3.15. Genomic DNA Probes ................................................................... 82 ix

3.16 CDNA Probes .................................................................................. 83 3.17. Synthetic Oligonucleotides As Probes ............................................. 84 3.18. Rna Probes Or Riboprobes.............................................................. 84 3.19. Labeling Of Probes ......................................................................... 84 3.20. Radiolabeled Probes ....................................................................... 84 3.21. Amplification Of DNA Probe Signals ............................................. 87 3.22. Techniques Used In Molecular Probing .......................................... 88 3.23. Separation Of DNA Fragments Using Agarose Or Polyacrylamide Gel Electrophoresis ............................................... 88 3.24. Separation Of Large DNA Molecule Molecules Using (PFGE) ......... 88 3.25. Southern, Northern, And Western Blotting ...................................... 89 3.26. Dots And Slot Blots ......................................................................... 90 3.27. Applications Of Molecular Probes .................................................. 91 3.28. Construction And Screening Of Genomic And CDNA Libraries ...... 94 3.29 CDNA Library From Mrnas ............................................................. 95 3.30. Colony (Plaque) Hybridization For Screening Of Libraries ............. 96 3.31. Chromosome Walking And Characterization Of Chromosome Segments ............................................................................................ 97 3.32. Reverse Genetics And Chromosome Jumping (Or Hopping Libraries) ................................................................... 97 3.33. Isolation, Sequencing, And Synthesis Of Genes .............................. 99 3.34. Maxam And Gilbert’s Chemical Degradation Method .................. 105 3.35. Sanger’s Dideoxynucleotide Synthetic Method.............................. 105 3.36. Direct DNA Sequencing Using PCR (Also Called Ligation Mediated PCR LMPCR) ............................................................... 106 3.37. Synthesis Of Genes ...................................................................... 107 3.38. Gene Synthesis Machines ............................................................ 110 3.39. Synthesis Of Genes From MRNA .................................................. 111 3.40. Gel Permeation ............................................................................. 112 3.41. Chromatography ........................................................................... 112 3.42. Ion Exchanged Chromatography ................................................... 112 3.43. Electrophoresis ............................................................................. 113 3.44. Agarose Gel Electrophoresis.......................................................... 113 3.45. Pulsed Field Gel Electrophoresis (PFEG)........................................ 113 3.46. Two-Dimensional Electrophoresis ................................................ 115

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3.47. Spectrometery .............................................................................. 115 3.48. Matrix-Assisted Layer Desorption-Ionization (MALDI) ................... 116 3.49. Surface Enhanced Laser Desorption-Ionization (SELDI) ................. 117 3.50. Electrospray Ionization (ESI) ......................................................... 117 3.51. Fluorescence Spectroscopy ........................................................... 118 3.52. Polymerase Chain Reaction ......................................................... 119 3.53. DNA Amplification Fingerprinting (DAF) ...................................... 119 3.54. Application Of PCR ..................................................................... 120 Chapter 4

Recombinant DNA Technology And Tools Of Genetic Engineering ...... 123 4.1. Exonuclease.................................................................................... 123 4.2. Endonuclease ................................................................................. 123 4.3. Restriction Endonucleases .............................................................. 124 4.4. Nomenclature................................................................................. 124 4.5. Examples Of Some Enzymes ........................................................... 125 4.6. Nuclease ........................................................................................ 126 4.7. DNA Ligases .................................................................................. 126 4.8. Alkaline Phosphatase ..................................................................... 126 4.9. Reverse Transcriptase ..................................................................... 127 4.10. DNA Polymerase ......................................................................... 128 4.11. T4 Polynucleotide Kinase ............................................................. 128 4.12. Terminal Transferase ..................................................................... 128 4.13. Use Of Linkers And Adaptors ....................................................... 128

Chapter 5

Tools Of Genetic Engineering (Cloning Vectors) ................................... 131 5.1. Cloning And Expression Vectors ...................................................... 131 5.2. Cloning Vector For Recombinant DNA............................................ 131 5.3. Plasmids As Vectors ........................................................................ 132 5.4. Pbr322 And Pbr327 Vectors ............................................................ 133 5.5. Puc Vectors .................................................................................... 133 5.6. Yeast Plasmid Vectors ...................................................................... 134 5.7. Retriever Vectors ............................................................................ 135 5.8. Ti And Ri Plasmids As Vector For Higher Plants ............................... 136 5.9. Bacteriophages As Vectors .............................................................. 136 5.10. Lambda Phage Vectors .................................................................. 137 5.11. Cosmids As Vectors ....................................................................... 139 xi

5.12. Phagemids As Vectors ................................................................... 139 5.13. P1 Cloning Vectors Foe Cloning Large DNA Segments .................. 140 5.14. Plant And Animal Viruses as Vectors.............................................. 140 5.15. Transposones As Vectors ............................................................... 142 5.16. Artificial Chromosome (Yac And Mac) Vectors For Cloning Large DNA Segments ...................................................... 142 5.17. Promoters ..................................................................................... 143 5.18. Nopaline Synthase (Nos) Promoter From T-DNA ........................... 143 5.19. Expression Cassettes ..................................................................... 144 5.20. Virus Expression Vector For Mammalian Cells ............................... 145 5.21. Binary And Shuttle Vectors ............................................................ 145 5.22. Ac-Ds Elements ............................................................................ 146 5.23. P Element ..................................................................................... 146 5.24. Expression Vector ......................................................................... 147 Chapter 6

Techniques Of Genetic Engineering ...................................................... 149 6.1. Gene Cloning ................................................................................. 149 6.2. Cloning In Prokaryotes.................................................................... 150 6.3. Strategies Of Recombinant DNA Technology ................................. 150 6.4. Gene Library................................................................................... 150 6.5. Genomic Library............................................................................. 151 6.6 CDNA Library .................................................................................. 151 6.7. Isolation Of MRNA ......................................................................... 151 6.8. Preparation Of CDNA. .................................................................... 152 6.9. Oligo-Dg Primer ............................................................................ 152 6.10. Synthesis Of Second Strand Of CDNA .......................................... 153 6.11. Insertion Of DNA Into Vector ........................................................ 153 6.12. Use Of Restriction Enzyme Linkers .............................................. 154 6.13. Use Of Homopolymer Tails........................................................... 154 6.14. Transfer Of Recombinant DNA Into Bacterial Cell ........................ 154 6.15. Transformation ............................................................................. 154 6.16. Transfection ................................................................................. 155 6.17. Selection (Screening) Of Recombinants ....................................... 155 6.18. Direct Selection Of Recombinants ................................................ 155 6.19. Insertional Selection Inactivation Method ..................................... 156 6.20. Blue-White Selection Method ...................................................... 156

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6.21. Colony Hybridization (Nucleic Acid Hybridization) Technique .... 157 6.22. In Vitro Translation ........................................................................ 157 6.23. Immunological Tests ..................................................................... 158 6.24. Blotting Techniques ....................................................................... 158 6.25. Southern Blotting Techniques ....................................................... 158 6.26. Northern Blotting Technique ........................................................ 159 6.27. Western Blotting ........................................................................... 160 6.28. Recovery Of Cells ........................................................................ 160 6.29. Expression Of Cloned DNA .......................................................... 161 6.30. Shine-Dalgarno Sequence ............................................................ 161 6.31. Detection Of Nucleic Acids ......................................................... 161 6.32. Radioactive Labeling ................................................................... 161 6.33. Random Primed Radiolabeling Of Probes ..................................... 162 6.34. Non-Radioactive Labeling ............................................................ 163 6.35. Digoxigenin (Dig) Labeling System ............................................... 163 6.36. Biotin-Streptavidin Labeling System .............................................. 164 6.37. Gene Cloning In Eukaryotes ......................................................... 164 6.38. Transformation In Filamentous Fungi............................................. 165 6.39. Gene Transfer In Dicots By Using Agrobacterium Ti-DNA As Vector ........................................................................ 165 6.40. Gene Transfer In Monocots ........................................................... 166 6.41. Plant Cell Transformation By Ultrasonication ............................... 166 6.42. Liposome-Mediated Gene Transfer ............................................... 166 6.43. Animal Cells ................................................................................ 167 6.44. Electroporation ............................................................................. 168 6.45. Particle Bombardment Gun .......................................................... 168 6.46. Microinjection .............................................................................. 169 6.47. Direct Transformation ................................................................... 169 6.48. Gene DNA Sequencing ............................................................... 170 6.49. Maxam And Gilbert’s Chemical Degration Method ....................... 170 6.50. Cleavage Of Purine ....................................................................... 170 6.51. Cleavage Of Pyrimidine ................................................................ 171 6.52. Sanger Method (Dideoxynucleotide Chain Termination Method) . 171 6.53. Automatic DNA Sequencers ........................................................ 171 6.54. Site-Directed Mutagenesis ........................................................... 172 xiii

6.55. Methods Of Mutagenesis ............................................................. 172 Chapter 7

Genetic Engineering For Human Welfare .............................................. 175 7.1. Production Of Chemicals And Cloned Genes ................................. 175 7.2. Human Peptide Hormone Genes .................................................... 175 7.3. Insulin ............................................................................................ 175 7.4. Somatotropin ................................................................................. 175 7.5. Somatostatin ................................................................................... 176 7.6. Human Interferon Gene (HIG) ........................................................ 177 7.7. Vaccines For Hepatitis B Virus ........................................................ 177 7.8. Rabies Virus (RV)............................................................................. 177 7.9. Vaccines For Poliovirus ................................................................... 178 7.10. Vaccines For Foot And Mouth Disease Virus (FMDV) ..................... 178 7.11. Vaccines For Smallpox Virus.......................................................... 178 7.12. Malaria Vaccines........................................................................... 179 7.13. DNA Vaccines .............................................................................. 179 7.14. Genes Associated With Genetic Diseases ..................................... 179 7.15. Phenylketonuria Genes ................................................................ 180 7.16. Urokinase Genes .......................................................................... 180 7.17. Thalassemia Genes ...................................................................... 180 7.18. Hemophilia Genes ....................................................................... 180 7.19. Enzyme Engineering ..................................................................... 180 7.20. Commercial Chemicals................................................................. 181 7.21. Prevention, Diagnosis And Cure Of Disease ................................ 181 7.22. Parasitic Disease ........................................................................... 181 7.23. Monoclonal Antibodies ............................................................... 182 7.24. Gene Therapy .............................................................................. 182 7.25. Types Of G ene Therapy ................................................................. 182 7.26. Methods Of Gene Transfer ............................................................ 183 7.27. Gene Therapy Success ................................................................. 183 7.28. DNA Profiling ............................................................................... 183 7.29. Method Of DNA Fingerprinting ................................................... 184 7.30. Application Of DNA Profiling ....................................................... 184 7.31. Immigrant Dispute ........................................................................ 185 7.32. Animal And Plant Improvement ................................................... 185

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7.33. Pollution Of Abatement ............................................................... 186 Chapter 8

Animal Biotechnology ........................................................................... 187 8.1. Introduction ................................................................................... 187 8.2. History Of Animal Cell Culture ....................................................... 187 8.3. Requirements For Animal Cell And Tissue Culture .......................... 188 8.4. Animal Cell Growth In Culture ...................................................... 188 8.5. Substrates For Cell Growth ............................................................. 189 8.6. Culture Media ................................................................................ 189 8.7. Glassware, Equipments, And Culture Media ................................... 189 8.8. Equipment Required For Animal Cell Culture .................................. 190 8.9. Laminar Air Flow (LAF) .................................................................. 190 8.10. Co2 Incubators .............................................................................. 190 8.11. Centrifuges ................................................................................... 191 8.12. Inverted Microscope .................................................................... 191 8.13. Culture Room ............................................................................... 191 8.14. Data Collection (Observation) ...................................................... 191 8.15. Isolation Of Animal Material (Tissue) ............................................ 191 8.16. Establishment Of Cell Cultures...................................................... 192 8.17. Cell Lines...................................................................................... 192 8.18. Hybridoma Technology ................................................................ 193 8.19. Monocional Antibodies Production Of Monoclonal Antibodies (MOAB) ...................................................................... 193 8.20. Applications Of Monoclonal Antibodies ...................................... 193 8.21. Disease Diagnosis......................................................................... 194

Chapter 9

Plant Biotechnology .............................................................................. 195 9.1. Introduction .................................................................................... 195 9.2. History............................................................................................ 195 9.3. Requirements For In-Vitro Cultures ................................................. 197 9.4. Tissue Culture Laboratory ................................................................ 197 9.5. Washing And Storage Facilities ....................................................... 197 9.6. Media Preparation Room ................................................................ 198 9.7. Transfer Area ................................................................................... 198 9.8. Nutrient Media ............................................................................... 198 9.9. Inorganic Chemicals ....................................................................... 198 xv

9.10. Growth Hormones ........................................................................ 198 9.11. Organic Constituents .................................................................... 199 9.12. Vitamins........................................................................................ 199 9.13. Amino Acids ................................................................................. 199 9.14. Solidifying Agents ......................................................................... 200 9.15. pH ................................................................................................ 200 9.16. Maintenance Of Aseptic Environment ........................................... 200 9.17. Sterilization Of Glassware ............................................................ 200 9.18. Sterilization Of Instruments........................................................... 200 9.19. Sterilization Of Culture Rooms ..................................................... 200 9.20. Sterilization Of Nutrient Media ..................................................... 201 9.21. Sterilization Of Plant Materials ..................................................... 201 9.22. Methods Of Plant Cell, Tissue Organ Culture Basic Steps .............. 201 9.23. Types Of Culture ........................................................................... 201 9.24. Explant Culture ............................................................................. 202 9.25. Callus Formation........................................................................... 202 9.26. Organogenesis .............................................................................. 203 9.27. Root Culture ................................................................................. 203 9.28. Shoot Culture................................................................................ 203 9.29. Micropropagation ......................................................................... 204 9.30. Cell-Suspension Culture................................................................ 204 9.31. Benefits Of Cell Culture ................................................................ 204 9.32. Somatic Embryogenesis ................................................................ 204 9.33. Somaclonal Variation .................................................................... 205 9.34. Protoplast Culture ......................................................................... 205 9.35. Isolation Of Protoplasts ................................................................. 206 9.36. Regenration .................................................................................. 206 9.37. Protoplast Fusion And Somatic Hybridization ............................... 207 9.38. Fusion Product.............................................................................. 208 9.39. Methods Of Somatic Hybridization............................................... 208 9.40. Selection Of Somatic Hybrids And Cybrids ................................... 209 9.41. Anther And Pollen Culture ............................................................ 209 9.42. Culturing Techniques .................................................................... 210 9.43. In-Vitro Androgenesis ................................................................... 211 9.44. Direct Androgenesis...................................................................... 211 xvi

9.45. Indirect Androgenesis ................................................................... 211 9.46. Mentor Pollen Technology ............................................................ 211 9.47. Embryo Culture............................................................................. 212 9.48. Embryo Rescue ............................................................................. 212 9.49. Triploid Production ....................................................................... 213 9.50. Protoplast Fusion In Fungi............................................................. 213 9.51. Application In Agriculture ............................................................. 214 9.52. Bioethics In Plant Genetic Engineering ......................................... 214 Chapter 10 Industrial Biotechnology ....................................................................... 215 10.1. Technique Of Microbial Culture ................................................... 215 10.2. Growth Media ............................................................................. 215 10.3. Sources Of Nutrition .................................................................... 216 10.4. Procedures Of Microbial Culture .................................................. 217 10.5. Sterilization .................................................................................. 217 10.6. Control Of Environmental Conditions For Microbial Growth ........ 217 10.7. Aeration And Mixing .................................................................... 217 10.8. Vessels For Microbial Cultures ...................................................... 218 10.9. Types Of Microbial Cultures.......................................................... 218 10.10. Measurement Of Microbial Growth ............................................ 219 10.11. Metabolic Pathways In Micro-Organisms ................................... 220 10.12. Glycolysis Or EMP Pathway ....................................................... 221 10.13. Entner-Doudoroff Pathway .......................................................... 221 10.14. Pentose Phosphate Pathway ........................................................ 221 10.15. Microbial Products .................................................................... 222 10.16. Primary Metabolites ................................................................... 222 10.17. Secondary Metabolites ............................................................... 222 10.18. Enzymes ..................................................................................... 222 10.19. Microbial Biomass ...................................................................... 223 10.20. Scale-Up Microblial Process ...................................................... 223 10.21. Downstream Processing ............................................................. 223 10.22. Separation Of Biomass................................................................ 224 10.23. Cell Disruption ........................................................................... 224 10.24. Concentration Of Broth .............................................................. 224 10.25. Initial Purification Of Metabolites ............................................... 224 xvii

10.26. Metabolite-Specific Purification .................................................. 224 10. 27. Isolation And Improvement In Microbial Strains ........................ 225 10.28. Strain Improvement Or Microorganism ....................................... 225 10.29. Recombination ........................................................................... 226 10.30. Recombinant DNA Technology (= Genetic Engineering Technique) ................................................................................... 226 Chapter 11 Environmental Biotechnology ............................................................... 227 11.1. Energy Source ............................................................................... 227 11.2. Nuclear Energy ............................................................................ 227 11.3. Fossil Fuel Energy ........................................................................ 228 11.4. Non-Fossil And Non-Nuclear Energy ........................................... 228 11.5. Biomass As A Source Of Energy .................................................... 228 11.6. Composition Of Biomass ............................................................. 228 11.7. Terrestrial Biomass ........................................................................ 229 11.8. Aquatic Biomass ........................................................................... 229 11.9. Salvinia......................................................................................... 229 11.10. Water Hyacinth (Eicchornia Crassipes) ....................................... 230 11.11. Waste As A Renewable Resources .............................................. 230 11.12. Composition Of Wastes .............................................................. 230 11.13. Sources Of Wastes ...................................................................... 230 11.14. Non-Biological Process (Thermo-Chemical Process) .................. 231 11.15. Direct Combustion ..................................................................... 231 11.16. Pyrolysis ..................................................................................... 232 11.17. Gasification ................................................................................ 232 11.18. Liquefaction................................................................................ 232 11.19. The Biological Process (Bioconversion) ...................................... 232 11.20. Enzymatic Digestion ................................................................... 233 11.21. Anaerobic Digestion ................................................................... 233 11.22. Aerobic Digestion ....................................................................... 233 11.23. Bioremediation ........................................................................... 234 11.24. Phytoremdiation ......................................................................... 234 11.25. Vermicomposting ........................................................................ 240 Chapter 12 Biosafety Guidelines Intellectual Property Rights And Entrepreneurship Development............................................................. 243 xviii

12.1. Biosafety ....................................................................................... 243 12.2. Hazards Of Environmental Engineering ........................................ 243 12.3. Biosafety Guidelines And Regulation ............................................ 244 12.4. Intellectual Property Rights ........................................................... 245 12.5. Patents .......................................................................................... 246 12.6. Copyrights .................................................................................... 247 12.7. Trademarks ................................................................................... 247 12.8. World Intellectual Property Organization (WIPO) ......................... 248 12.9. General Agreement Of Tariffs And Trade (Gatt) And Trade Related IPRS (Trips) ...................................................................... 248 12.10. Biodiversity Bill 2002 ................................................................. 248 12.11. Geographic Indicator Bill............................................................ 249 Chapter 13 Biotechnology Practical ........................................................................ 251 13.1. Sterilization Techniques (Experiment 1) ......................................... 251 13.2. Media Preparation (Experiment 2) ................................................. 254 13.3. Isolation And Characterization Of Unknown Bacteria (Experiment 3) .............................................................................. 257 13.4. Bacterial Growth Kinetics (Experiment 4) ...................................... 260 13.5. Cell Viability Assay (Experiment 5) ................................................ 262 13.6. Isolation Of Genomic DNA From Bacteria (Experiment 6) ............ 263 13.7. Plasmid DNA Isolate From Bacteria (Experiment 7) ....................... 265 13.8. Rna Extraction By Kit (Experiment 8) ............................................. 268 13.9. Identification Of Bacterial Species Based On 16S RDNA Sequences (Experiment 9) ............................................................ 271 13.10. Isolation Of Milk Protein (Experiment 10) ................................... 275 13.11. Protein Estimation (Experiment 11) ............................................. 276 13.12. Assay Of Acid Phosphatase (Experiment 12)................................ 279 13.13. Identification N-Terminal Amino Acid Of A Protein (Experiment 13) ............................................................................ 281 13.14. Sds-Page Analysis Of Proteins (Experiment 14) ............................ 284 13.15. Restriction Digestion Of DNA (Experiment 15) ........................... 288 13.16. DNA Sequencing (Experiment 16) .............................................. 290 13.17. Protoplast Preparation And Fusion (Experiment 17) ..................... 291 Chapter 14 Biostatistics ........................................................................................... 295 14.1. Introduction .................................................................................. 295 xix

14.2. Biostatistics Concept ..................................................................... 295 14.3 Frequency Distribution .................................................................. 297 14.4. Variables ....................................................................................... 308 14.5. Qualitative Variable ...................................................................... 308 14.6. Quantitative Variable .................................................................... 308 14.7. Discrete Variable........................................................................... 309 14.8. Continuous Variable .................................................................... 309 14.9. Random Variable .......................................................................... 309 14.10. Non-Random Variable ................................................................ 309 14.11. Sample Survey Method ............................................................... 310 14.12. Census Method ........................................................................... 310 14.13. Graphical Representation Of Data .............................................. 310 14.14. Primary Data .............................................................................. 310 14.15. Secondary Data .......................................................................... 311 14.16. Biostatistics ................................................................................ 312 14.17. Presentation Of Data .................................................................. 312 14.18. Different Graphs And Diagram ................................................... 313 14.19. Line Diagram .............................................................................. 314 14.20. Bar Diagram ............................................................................... 319 14.21. Pie Diagram................................................................................ 320 14.22. Stem And Leaf Plate .................................................................... 321 14.23 Histogram .................................................................................... 321 14.24 Ogives ......................................................................................... 321 14.25. Scatter Diagram .......................................................................... 321 14.26. Some Important Facts.................................................................. 325 14.27. Descriptive Statistics ................................................................... 325 14.28. Mean .......................................................................................... 325 14.29. Calculate Average ....................................................................... 335 14.30. Median ....................................................................................... 335 14.31. Mode .......................................................................................... 341 14.32. Standard Deviation .................................................................... 349 14.33. Short Cut Method ....................................................................... 350 14.34. Calculate Standard Deviation On Excel ...................................... 357 14.35 Correlation (R) ............................................................................. 361 14.36. Regression .................................................................................. 363 xx

14.37 Null Hypothesis ........................................................................... 365 14.38. Alternative Hypothesis ................................................................ 365 14.39. Central Tendency ........................................................................ 369 14.40 Measures Of Variation.................................................................. 369 14.41 Binomial Distribution .................................................................. 377 14.42. Skewness .................................................................................... 387 14.43. Kurtosis And Moment ................................................................. 388 14.44 Set Theory And Probability ........................................................... 390 14.45. Comparative Statistics ................................................................. 391 14.46. Chi Square Test ........................................................................... 391 14.47. Student “T” Distribution Test....................................................... 392 14.48. Z-Test.......................................................................................... 395 14.49. F-Test Or Fisher’s F Test ............................................................... 395 14.50. T Test .......................................................................................... 396 14.51. Anova (Analysis Of Variances) .................................................... 397 14.52. Non-Parametric Statistics ............................................................ 398 14.53. Important Formulas ..................................................................... 399 Chapter 15 Software Used For The Analysis Of Observations And Data ................. 401 15.1. Sigma Plot .................................................................................... 401 15.2. Spss (Statistical Package For The Social Sciences) .......................... 401 15.3. Origin Pro Software ...................................................................... 401 Index ..................................................................................................... 417

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GLOSSARY

Agarose Gel Electrophoresis: It is a process in which a matrix composed of a highly purified form of agar is used to separate large DNA and RNA molecules. Allele: One of pair of variant forms of a gene occurring at a given locus of a gene. Amplification: There are formation of many copies of DNA segment of PCR. Annealing: The process of healing and slowly cooling of double stranded DNA to allow the formation of hybrid DNA or DNA-RNA molecules. Antibiotic: A biological substances produced by one organism inhibiting the growth of another organism. Antibody: A protein synthesized by B lymphocytes that recognizes a specific site on an antigen. Antigen: A compound that induces the production of antibodies. Antiparallel orientation: Arrangement of two strands of DNA molecule oriented in opposite directions so that the 5’ PO4 end of one strand is aligned with 3’OH end of the complementary strand. Antisense DNA: The sequence of Chromosomal DNA that is transcribed. Antisense RNA: An RNA sequence complementary to all or part of a functional RNA. Antisense therapy: In vitro treatment of genetic disease by blocking translation of a protein with DNA or RNA sequences that is complementary to a specific mRNA. Artificial seeds: A synthetic seed consisting of a somatic embryo surrounded by nutrient medium which is protected by a thin membrane of chemical. Autoclave: An instrument used for sterilization of glassware and culture media. Auxins: A class of plant growth regulators that stimulate cell division, cell elongation, apical dominance and root initiation. They are used in plant cell and tissue culture, e.g., 2,4-D, IAA, NAA, etc.

Axenic culture: A pure of culture single propagule of an organism not contaminated by any other microorganism. Axillary bud: A bud produced in the axis of leaves. Bioaccumulation: The concentration of a chemical agent, e.g., DDT in the increasing amount in the organisms of a food chain. Biodegradation: The gradual breakdown of a compound to its constituents by a living organism. Biodiversity: The variability among the living organism from all sources, soil, water, air, extreme habitat or associated with organisms. Biofertilizers: The commercial preparation of microorganisms by using which nitrogen and phosphorus level and growth of plants increased. Bioremediation: The process of using living organisms to remove contaminants, pollutants or unwanted substances from soil or water. Biosensor: An electronic device that uses biological molecules or cells to detect specific compounds. Biotic stress: Stress resulting from living organisms which can harm the other plants such as virus, bacteria, fungi, weeds, harmful insects. Callus: An organized mass of plant cells capable of cell division and growth in vitro. cDNA Clone: A double stranded DNA complement of mRNA synthesized in vitro by using reverse transcriptase and DNA polymerase. cDNA library: A collection of cDNA clones generated in vitro from the mRNA sequences of a single tissue or cell population. Cell culture: The cell culture of growing cells in vitro in liquid medium. Cell line: The cell line is derived from a primary culture and selected at the time of the first subculture or derived from cell population with particular attributes. Chimera: Recombinat DNA molecules containing sequences from different organisms. Clone: A collection of genetically identical cells or organisms derived from a common ancestor where all members have similar genetic composition. Consensus sequences: The nucleotide sequences that is present in majority of genetic signals that perform a specific function. Cosmid: A plasmid vector which consists of the COS site of phage lambda (λ) and one or more selectable markers such as an antibiotic resistance gene. Crown gall: A tumor occurring at the base of certain plants due to infection of xxiv

the plant by Agrobacterium tumifaciens. Cytokinins: The class of plant growth regulators which causes cell division, cell differentiation, shoot differentiation and breaking of apical dominance, e.g., BAP, Kinetin and zeatin. Denaturation: The separation of DNA double stranded molecule into single stands. Differentiation: The process of biochemical and structural changes by which cells become specialized in form and function. DNA construct: A suitable DNA which has been prepared for cloning purpose. DNA polymerase: A n enzyme that catalyzes the phosphodiester bond in the formation of DNA. DNA probe: A radiolabeled (32P) DNA segment used to hybridize the base between the probe and a complementary base sequences in a DNA sample. Electrophoresis: The method of separation of molecule such as DNA, RNA or protein based on their relative migration applying a strong electric field. Embryo transfer: Implantation of embryos from donor animals or generated by in vitro fertilization into the uterus of the recipient animals. Excision: Enzymatic removal of DNA segment from a chromosome. Exonucleases: An enzyme the digest DNA or RNA from the ends of strand; 5’ exonuclease requires a free 5’ end and degrades the molecule in 5’-3 direction; 3’ exonuclease requires a free 3’ end and degrades the DNA IN 3’-5 direction. Explant: The tissue taken from a plant or seed and transferred to a culture medium to establish a tissue culture system or regenerate the plant. Fermentation: The process by which microorganism turn raw materials such as glucose into products such as alcohol. Fusion product: A single protein molecule encoded by parts of two or more genes combined together as one unit. Gene: A hereditary unit or the segment of DNA coding a specific protein. Genetic fingerprinting: A technique of analyzing DNA of an individual to reveal the pattern of repetition of particular nucleotide sequences through the genome. Hybridoma: A unique fused cells or protoplasts into a matrix. In vitro: A ny process carried out in sterile cultures or measurement of biological processes outside the intact organism such as enzyme reaction. In vivo: The natural condition in which organism live. xxv

Leader sequences: It is the non-translated sequence at the 5’ end of mRNA that precedes the initiation codon. Lysogeny: The phenomenon in which prophage survives within a host bacterium as a part of host genome or as extrachromosomal element and does not cause lysis. Micropropagation: The small piece of tissue such as meristem grown in culture to produce large number of plants. Monoclonal antibodies: It is derived from a single clone cells which recognize only one kind of antigen. Nod genes: Genens encode nodulins. Oncogene: A gene that cause cancer. Operon: A cluster of gene that are coordinately regulated. Palindromic sequences: The complementary DNA sequences that are the same when each stand is read in the same direction, e.g., (5’-3). These sequences act as recognition sites for type II restriction endonuclease. Patent: A legal document issued by a government that permits the holder the exclusive right to manufacture, use of sell an invention for a defined period, which varies country to country. Plasmid: Extrachromosomal, self-replicating circular double stranded DNA molecules containing some non-essential genes. Primer: A short oligonucleotide that hybridizes the template strand and give 3’ OH end for the initiation of nucleic acid synthesis. Recombinant: An individual whose genes on a chromosomes results from one or more cross over events. Secondary metabolites: The metabolites produced as end products of primary metabolism and not involved directly in metabolic activity. Splicing: The gene manipulation where the one DNA molecule is attached to one another. Totipotency: A property of normal cells that they have the genetic potential to give rise to a complete individuals except terminally specialized cells. Transfection: DNA transfer into a eukaryotic cell. Transposon: A DNA segment that can Jump from locus and join a new locus on the DNA molecule by involving the enzyme transposase. Vectors: The vehicles for transferring DNA from one cell to another or DNA molecule that can carry inserted DNA and be perpetuated in a host cell. xxvi

Western blot: A technique in which protein is transfer from an electrophoresis gen to a cellulose or nylon support membrane following electrophoresis. Yeast artificial chromosomes: A vector is used for hundreds to kilobases long used for cloning of DNA fragment. Zooblot: The cloned DNA from one species to DNA from the other organism to determine the extent to which the cloned DNA is evolutionary conserved.

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LIST OF FIGURES Figure 1.1. Gram-positive and gram-negative bacteria membrane Figure 1.2. Cell shapes in bacteria and archaea Figure 1.3. Morphology of fungi Figure 1.4. Viruses-bacteriophage, tobacco mosaic virus and human immunodeficiency virus (HIV), single stranded RNA virus with helical symmetry Figure 1.5. Prion replication cycle Figure 1.6. A tree of biotechnology Figure 2.1. Nucleotides Figure 2.2. Nirogeneous base. Figure 2.3. Watson Crick’s model of DNA Figure 2.4. Cistron (A) Recon (B) Muton (C) Genes as a unit of function. Figure 2.5. Presentation of Different regions in and around a gene in a genomic sequence. Figure 2.6. Central Dogma Figure 2.7. Gene machine Figure 2.8. Polymerase chain reaction Figure 3.1. Gel permeation chromatography (a) spatial accessibility of molecules during gel filtration (b) movement of molecules of different sizes through microporus gel forming stationary phase. Figure 3.2. Ion exchange chromatography (a) cation exchange (b) anion exchanger. Figure 3.3. Pulse filed gel electrophoresis Figure 3.4. Isoelectric focusing Figure 3.5 Electrospray ionization source Figure 4.1. Action of exonuclease (a) endonuclease (b). Figure 4.2: Host controlled restriction and modification of phage I in E. coli strain K and strain B.

Figure 4.3 Alkaline phosphates Figure 4.4. Linker and ADAPTOR molecules Figure 5.1. pBR322 vector. Figure 5.2. puc19 cloning vector. Figure 5.3. Octopine plasmid (a) Nopaline plasmid (b). Figure 5.4. Shuttle vector Figure 6.1. Gene cloning Figure 8.1 ELISA testing Figure 9.1. Structure of Growth Hormones (a) auxins (b) cytokinin (c) Gibberellin A3.

Figure 9.2. Regeneration of plant through plant tissue culture Figure 9.3 Isolation of protoplasts

Figure 9.4. Purification, culture and regeneration of protoplasts Figure 9.5. Cybrid/Hybrid production Figure 9.6. Anther culture Figure 11.1. Enzymes secretion by microbial cells Figure 11.2 Types of phytoremdiation process. Figure 11.3. Phytochelatins (PCs) genes expressed in various plants under heavy metal stress. Figure 11.4 Diagram of detoxification, conjugation, and sequestration in the vacuole where the pollutant can do harm to the cell Figure 11.5. Heavy metal ATPase (HMA) gene contributes in hyperaccumulation of heavy metals Figure 14.1. Screenshot represents the data input in excel sheet and form Line diagram. Figure 14.2. Screenshot represents the data input in excel sheet and form Bar diagram. Figure 14.3. Screenshot represents the data input in excel sheet and form Pie diagram. Figure 14.4. Screenshot represents the data input in excel sheet and form scatter diagram Figure 14.5. Screenshot represents the average values of data Figure 14.6. Screenshot represents the median values of data xxx

Figure 14.7. Screenshot represents the mode values of data Figure 14.8. Screenshot represents the standard values of data Figure 14.9. Screenshot represents the Variance of data Figure 14.10. Screenshot represents the correlation values of data Figure 15.1. Screenshot represents the Origin Pro software

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LIST OF TABLES Table 1.1. Fermented Foods Prepared in Different Parts of India Table 14.1 Geo-Statistics of Individual Layers of Groundwater Quality Parameters Table 14.2. Simple Frequency Distribution Table 14.3. Group Frequency Distribution Table 14.4. Class Interval, Class Frequency, Class Limits, Class Boundaries, Class Marks, Class Width, Frequency Density, Relative Frequency Table 14.5. Cumulative Frequencies

LIST OF ABBREVIATIONS

AAF

Accidents and Fire

AIDS

Acquired Immuno Deficiency Syndrome

APS

Ammonium Per Sulfate

AVL

Anton Van Leewenohoek

BB

Bunsen Burner

BFM

Bright Field Microscope

BS

Bacterial Staining

BSA

Bovine Serum Albumin

BSL

Bio Safety Level

BSML

Biosafety in Microbiology Lab

CH

Chemical Handling

CDCAP

Centers for Disease Control and Preventation

CGH

Cleaning of Glassware and Handling

CJD

Creutzfelt Jacob Disease

CSPD

Chemiluminescent Substrate for Alkaline Phosphatase

CT

Catalase Test

CTAB

Cetyl Trimethyl Ammonium Bromide

DAM

Di-Acetyl Monoxime

DFM

Dark Field Microscopy

DNA

De-Oxyribo Nucleic Acid

DNS

Di Nitro Salicylic acid

DAPI

Diamidino-2-Phenyl Inodole

dNTP

De-Oxy Nucleotide Tri Phosphate

DT

Decarboxylase Test

DTSFT

Durham Tube Sugar Fermentation Test

EDTA

Ethylene Di-Amine Tetra-Acetic Acid

ELISA

Enzyme-Linked Immuno Sorbent Assay

EtBr

Ethidium Bromide

FAD

Flavin Adenine Di-nucleotide

FMN

Flavin Mono Nucleotide

GHT

Gelatin Hydrolysis Test

GC

Gas Chromatography

HAO

Hot Air Oven

Hb

Heamoglobin

HEPA

High Efficiency Particulate Arrestance

HgCl2

Mercuric Chloride

HIV

Human Immunodeficiency Virus

IR

Infrared Spectroscopy

IRRN

Isolation Rhizobium Root Nodules

ISE

Ion Selective Electrode

IT

Indole Test

MCD

Mad Cow Disease

MLSRR

Microbiology Laboratory Safety Rules and Regulations

MRT

Methyl Red Test

MS

Mass Spectrometry

NAD

Nicotinamide Adenine Di-Nucleotide

NADPH

Nicotinamide Adenine Di-Nucleotide Phosphate-Oxidase

NBT

Nitro Blue Tetrazolium

NMS

Normal Microbiota Skin

NRT

Nitrate Reduction Test

NTP

Normal Temperature and Pressure

OD

Optical Density

OFT

Oxidation Fermentation Test

OT

Oxidase Test

PAGE

Poly Acrylamide Gel Electrophoresis

PCR

Polymerase Chain Reaction

pNPP

para-Nitro Phenyl Phosphate

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PPM

Part Per Million

PVP

Poly Vinyl Pyrrolidone

q-PCR

quantitative Polymerase Chain Reaction

RBC

Red Blood Cell

R&D

Research & Development

RH

Relative Humidity

RNA

Ribo Nucleic Acid

RPW

Recording Practical Work

RPM

Rotation Per Minute

SARS

Severe Acute Respiratory Syndrome

SC

Safe Clothing

SD

Standard Deviation

SDS

Sodium Dodecyl Sulfate

SEM

Scanning Electron Microscope

SG

Safety Guidelines

SHT

Starch Hydrolysis Test

SPT

Streak Plate Technique

SPM

Spread Plate Method

SSC

Sodium Saline Citrate

STDEV

Standard Deviation

TAE

Tris Base, Acetic Acid and EDTA

TBC

Total Blood Cell

TBE

Tris Base, Boric Acid and EDTA

TCA

Tri Chloro-Acetic Acid

TE

Tris-EDTA

US

United States

UT

Urease Test

UV

Ultra Violet

Vis

Visible

VPPT

Voges Proskaur Test

WBC

White Blood Cell

WD

Waste Disposal xxxvii

PREFACE

The book “Introduction to Biotechnology and Biostatistics” has been written to serve as a textbook for undergraduate, postgraduate and research scholar of Indian and other country universities. There are many books on the subject of Biotechnology, but the combination of Biotechnology and Biostatistics is not available. This book has covered a range of syllabus, and the student is an easy way to go through this book content. Some books are available on the subject of Biotechnology but still not up to date. While this book has prepared for students that they have acquired or gain fair knowledge of “Biotechnology and Biostatistics” and easy presentation of language has written for proper understand each and every topic. The chapters have been so arranged to give a sequential knowledge of the introduction of Biotechnology, Genes, Genomes, Genetic Engineering, Recombinant DNA Technology and Tools of Genetic Engineering, Tools of recombinant DNA technology and very vast knowledge of biostatistics, etc., in this book with practical approaches. The examples to illustrate different points up to date have been chosen from different universities and institution of the country. This book would serve not only for undergraduate but postgraduate students of biotechnology and also research scholar of various universities. The numbers of scientists and authors have been quoted or fully acknowledge and referenced at the end of the book. The preparing of this book, I have been greatly helped by several books which I express my acknowledgment in the references, and if I forget any references in my notice, please ignore these mistakes. Comments and suggestions are most welcome from students as well as from many teachers’ friends for corrections and improvement of this book will be greatly acknowledged. I am particularly thankful to Dr. Naveen Kumar (Principal Scientist, Hisar), Dr. Suphiya Khan (Associate Professor, Banasthali University), and Dr. Pankaj Kumar Saraswat (Scientist) have given constant inspiration and fullest cooperation. My family members and my husband, Mr. Manoj Chaudhary (CA) have given constant cooperation to complete this book. He deserves all appreciation for the help and moral support at every moment. Last but not least, I would be thankful to readers for pointing out errors and most welcome for suggestion in the next book.

ACKNOWLEDGMENT

First and foremost, we are grateful to the almighty God. We could never have done this without his blessings. We would also like to acknowledge the major we are sincerely thankful to Dr. Naveen Kumar (Principal Scientist-NRCE Hisar, Haryana, India), Dr. Suphiya Khan (Associate Professor-Banasthali University, Tonk Rajasthan, India) and Dr. T. Duhan (Director General HIMT, Greater Noida), for providing us research facilities and the Bioinformatics center, for extensive use of computational facilities. We are deeply indebted to many people who gave their valuable support in the writing of this book; to all those who provided encouragement, support, discussed things over, offered comments assisted in the editing. Above all, we want to thank our family, friends, and lab mates for their passionate encouragement and support throughout the writing of this book. We are also thankful to those entire people who directly and indirectly support in the writing journey of this book. Otherwise, a huge textbook could not be prepared.

1 Introduction of Biotechnology

1.1. SCOPE The biotechnology term was coined in 1917 by a Hungarian Engineer, Karl Ereky, to describe a process for large-scale production of pigs. According to him all types of work are biotechnology by which products are produced from raw materials using living organisms. During the end of 20th century biotechnology emerged as a new discipline of biology integrating with technology, but the route of biotechnology lies in biology. There was no sudden sprout of this discipline, but some of the methods for production of products were developed centuries back. Therefore, biotechnology is concerned with exploitation of biological components for production of useful products. •

•

•

• •

Biotechnology is defined by different organizations in different ways. It has been broadly defined as “the development and utilization of biological processes, forms and systems for obtaining maximum benefits to man and other forms of life.” Biotechnology is “the science of applied biological process” (Biotechnology A Dutch Perspective, 1981). Following are some of the definitions given by other organizations. Biotechnology is the “controlled use of biological agents such as microorganisms or cellular components for beneficial use” (U.S. National Science Federation). The definition was given by OECD “scientific and engineering principles” refer to microbiology, biochemistry and genetics, etc. Biological agents: we can call biological agents such as microorganisms, enzymes, plant and animal cells. The meaning of

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these three definitions and other given by many organizations are more or less similar. The definition of genetic engineering has been given by Smith (1996) as “the formation of new combinations of heritable material by the insertion of nucleic acid molecules produced by whatever means outside the cell, into any virus bacterial plasmid or other vector system so as to allow their incorporation into host organisms in which they do not naturally occur but in which they are capable of continued propagation.”

1.2. IMPORTANCE Biotechnology is widely used in pharmacy to create more efficient and less expensive drugs. Recombinant DNA technology is used for production of specific enzymes, which enhance the rate of production of particular range of antibodies in the organism. The hormones such as somatostatin, insulin and the human growth hormone can be synthesized easily and cheaply. The first human hormone to be synthesized by genetic engineering was somatostatin. Somatostatin is brain hormone originating from hypothalamus. It acts to inhabit the release of human growth hormone and insulin is related to treatment of diabetes, pancreatis and few other conditions. Genetech, a California based company, has produced human growth hormone (hGH) from genetically engineered bacteria. Human insulin is the first genetically engineered pharmaceutical product, developed by Eli Lilly and company in 1982. Bovine Somatotropin (BST) is produced for a large quantity of milk production in cows. Antibiotics are chemical substances produced by several microorganisms. Recombinant DNA technology has helped in increased production of antibiotics for example; the rate of penicillin produced at present is about 150,000 unit/ml against about 10 unit/ml in 1950s. Antibiotics produced using such technology have very specific effects and causes fewer side effects. Currently, scientists are working on vaccines for fatal illnesses such as AIDS, hepatitis, malaria, flu, and even some forms of cancer. Interferon, an anti-viral protein, is prepared from the mammalian cells by recombinant DNA technology. By cloning cDNA to genes for human interferon, it has been found that there are large number of interferon differing in amino acid sequences and properties. A large number of interferon is prepared in yeast cells by fermentation process. Expects that in the near future vaccines will come in more convenient ways “some will come in the form of mouthwash; others will be swallowed in time-release capsules, avoiding the need for boosters.”

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1.3. BIOTECHNOLOGY CENTURY Biotechnology is the technologies applied to biology, molecular biology, genetics, and many other subfields of biology. Biotechnology utilizes cellular and biomolecular processes to create technologies and products that help improve our lives and the nature. By making useful food, such as bread and cheese, and preserving dairy products, we have done these for many years by now. Recent biotechnology develops breakthrough products and technologies to fight diseases, reduce our environmental harm, feed the hungry, and use less and cleaner energy, and have safer, cleaner and more efficient industrial manufacturing processes. So far, more than 250 biotechnology health care products and vaccines have been made available to patients, many for previously untreatable diseases. More than 13.3 million farmers around the world use agricultural biotechnology to increase yields, prevent damage from insects and pests and reduce damage done on environment due to farming. And more than 50 biorefineries are being built across North America to test and refine technologies to produce biofuels and chemicals from renewable biomass, which can help reduce greenhouse gas emissions. Biotechnology is the third wave in biological science and represents such an interface of basic and applied sciences, where gradual and subtle transformation of science into technology can be witnessed. Biotechnology is defined as the application of scientific and engineering principals to the processing of material by biological agents to provide goods and services. Biotechnology comprises a number of technologies based upon increasing understanding of biology at the cellular and molecular level. The Bible already provides numerous examples of biotechnology. Namely, it deals with the conversion of grapes to wine, of dough to bread and of milk to cheese. The oldest biotechnological processes are found in microbial fermentations, as born out by the Babylonian tablet dated circa 6000 B.C explaining the preparation of beer. The Sumerians were able to brew as many as twenty types of beer in the third millennium B.C. In about 4000 B.C. leavened bread was produced with the aid of yeast. During Vedic period (5000–7000 B.C) Aryans had been performing daily Agnihotra or Yajna. In Ayurved, production of ‘Asava’ and ‘Arista’ using different substrates and flowers of mahua (Madhuca indica) or dhataki (Wodfordiafructicosa) has been well characterized till today since Vedic period. One of the materials used in Yajna is animal fat (i.e., ghee), which is fermented product of milk. The term ‘biotechnology’ was described in a Bulletin of the Bureau of Biotechnology published in July, 1920 from the office of the same name in Leeds in Yorkshire. The articles in this

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bulletin described the varied roles of microbes in leather industry to pest control. There are numerous sub-fields of biotechnology: •

Red biotechnology is biotechnology applied to medical processes. Some examples are the designing of organisms to produce antibiotics, and the engineering of genetic cures to cure diseases through genomic manipulation. • White biotechnology, also known as grey biotechnology, is biotechnology applied to industrial processes. An example is the designing of an organism to produce a useful chemical. White biotechnology tends to consume less in resources than traditional processes when used to produce industrial goods. • Green biotechnology is biotechnology applied to agricultural processes. An example is the designing of an organism to grow under specific environmental conditions or in the presence (or absence) of certain agricultural chemicals. One hope is that green biotechnology might produce more environmentally friendly solutions than traditional industrial agriculture. An example of this is the engineering of a plant to express a pesticide, thereby eliminating the need for external application of pesticides. Whether or not green biotechnology products such as this are ultimately more environmentally friendly is a topic of considerable debate. • The term blue biotechnology has also been used to describe the marine and aquatic applications of biotechnology, but its use is relatively rare. Broadly biotechnology can be divided into two major branches: •

Non-gene biotechnology – deals with whole cell, tissues or even individual organisms • Gene biotechnology – involves gene manipulation, cloning, etc. Non-gene biotechnology is a more popular practice, and plant tissue culture, hybrid seed production, microbial fermentation, production of hybridoma antibodies or immunochemicals are wide spread biotechnology practices. For centuries humans have used microorganisms to produce foods and drinks without understanding the microbial processes underlying their production. In recent years the understanding of the biosynthetic pathways and regulatory control mechanisms used by microorganisms for production of several metabolites has been increased by developing the knowledge of biochemistry of industrially important organisms. Notable biotechnologies for food processing include fermentation technology, enzyme technology and monoclonal antibody technology.

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Beneficial microbes participate in fermentation processes, producing many useful metabolites such as enzymes, organic acids, solvents, vitamins, amino acids, antibiotics, growth regulators, flavors and nutritious foods. Some leading food bioprocessing technologies are dairy processing, alcohol and beverage processing. Production of alcoholic beverages include: wine, beer, whiskey, rum, shake, etc. utilizing microorganisms like Clostridium acetobutylicum, Lecuonostoc mesenteroides, Aspergillus oryzae, Saccharomyces cerevisiae, Rizopus sp. and Mucor sp. Biotechnologically produced organic acids like citric acid, acetic acid, gluconic acid, D-Lactic acid, fumaric acid also has very high market value. The application of biotechnology can result in (a) new ways of producing existing products with the use of new inputs, and (b) new ways of producing new products. Examples of the former include the production of gasoline from ethanol which in turn is produced from sugar; the production of insulin using recombinant DNA technology; the production of hepatitis B vaccine using recombinant DNA technology and the extraction of copper using mineral leaching bacteria. The alternative inputs are oil for gasoline, porcine pancreases for insulin, human blood for hepatitis vaccine, and the conventional mining techniques for copper. Examples of the latter include possible medicinal substances which are produced in minute quantity in the human body and which cannot be synthesized such as insulin, interleukin or Tissue Plasminogen Activator (TPA). A wide variety of microorganisms are now being employed as tools in biotechnology to produce useful products or services. Raw materials can be converted to useful finished products both by ordinary chemical processes and by biological means. Generally, the costs of chemical conversion are quite high as the reactions require high temperature or pressure. In contrast, biological alternatives, using microbes or cultured animal or plant cells, operate at physiologically normal conditions of temperature, pressure, pH, etc. During the next few decades biotechnology would have overtaken chemical technology and many such chemicals which are today produced chemically would be made through biotechnology. Enzyme technology is an area of considerable current interest and development. Enzymes are biological catalysts and have been used for many years as isolated agents particularly in food, e.g., rennin, papain and invertase. These enzymes have increasingly replaced plants and animal enzymes thus amylase from Bacillus and Aspergillus has substituted those of malted wheat and barley in brewing, baking and biscuit-making and also in the textile industry, etc. Today, enzyme technologies have four distinct areas of application in cosmetics, therapy, the food and feed industry, and for diagnostic purposes.

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One very important recent application is the production of foodstuffs from nontraditional raw materials for instance, the development of the sweetener, high fructose corn syrup (HFCS), also called isoglucose. Another recent application is the use of phytase in animal feed. Nowadays, interest in the traditional fermentation technology for food processing has greatly increased because of emphasis placed upon plant materials as human foods. Single-cell protein (SCP) is term generally accepted to mean the microbial cells (algae, bacteria, actinomycetes and fungi) grown and harvested for animal or human food. During World War II, when there were shortages in proteins and vitamins in the diet, the Germans produced yeasts and a mold (Geotrichum candidum) in some quantity for food. Research on SCP has been stimulated by a concern over the eventual food crisis or food shortages that will occur if the world’s population is not controlled. Many scientists believe that the use of microbial fermentations and the development of an industry to produce and supply SCP are possible solutions to meet a shortage of protein if and when the amount of protein produced or obtained by agriculture and fishing becomes insufficient. The roots of molecular biology were established only after the British biophysicist Francis Crick and the American Biochemist James Watson, in 1953, proposed the structure of DNA (deoxyribonucleic acid) molecule which is well known as the chemical bearer of genetic information of most of the organisms. We really began understanding and utilizing molecular biotechnology (or gene biotechnology) only after recombinant DNA technology was developed in 1970’s. Daniel Nathans (in 1971) of John Hopkins University utilized the restriction enzyme to split DNA of monkey tumor virus, Simian Virus (SV40). Recombinant DNA technology, often referred to as genetic engineering or gene manipulation, involves extraction of a particular gene of interest form one organism and then insertion of the gene into other organisms. Genetic manipulation may be defined as the extracellular (i.e., in vitro) creation of new forms of arrangements of DNA in such a way as to allow the incorporation or continued propagation of altered genetic condition in nature. Among the first scientist to attempt genetic manipulation was Paul Berg of Stanford University who in 1971 along with his co-workers opened the DNA molecule of SV40 and spliced it into a bacterial chromosome and constructed the first recombinant DNA molecule. The genetic engineering techniques are useful tools for genetic research. They can help to gain in the structure, function and regulation of genes. They also help to prepare the physical maps of viral genome. Maps of several viruses

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have been made available like SV40, Polyoma virus and adenovirus. Another goal in genetic engineering is to design super bug, which can degrade most of the major hydrocarbon components of petroleum. The different strains of Pseudomonas putida contain a plasmid which has genes coding for enzymes that digest a single family of hydrocarbons. By crossing the various strains of this bacterium, a super bug has been created. The multiplasmid bacterium is able to grow on a diet of crude oil. The super bug has potential for clearing up oil spills. One of the best-known applications of genetic engineering is that of the creation of genetically modified organisms (GMOs). There are potentially momentous biotechnological applications of GM, for example oral vaccines produced naturally in fruit, at very low cost. This represents, however, a spread of genetic modification to medical purposes and opens an ethical door to other uses of the technology to directly modify human genomes. A genetically modified food is a food product derived in whole or part from a genetically modified organism (GMO) such as a crop plant, animal or microbe such as yeast. Genetically modified foods have been available since the 1990s. The principal ingredients of GM foods currently available are derived from genetically modified soybean, maize and canola. Between 1996 and 2001, the total surface area of land cultivated with GMOs had increased by a factor of 30, from 17,000 km² (4.2 million acres) to 520,000 km² (128 million acres). The value for 2002 was 145 million acres (587,000 km²) and for 2003 was 167 million acres (676,000 km²). Future applications of GMOs include bananas that produce human vaccines against infectious diseases such as Hepatitis B, fish that mature more quickly, fruit and nut trees that yield years earlier, and plants that produce new plastics with unique properties. Now scientists have transformed Tobacco Mosaic Virus (TMV) to infect host plants and produce immunizing proteins rather than debilitating leaf shrivel, turning greenhouse tobacco into a biofactory for plague vaccine. Genetic diseases could be treated through the use of genetic engineering. Defective genes in an organism cause genetic disorders. If a defective gene could be identified and located in a particular group of cells – it could be replaced with a functional one. The transgenic cells are then planted into the organism, resulting in a cure of the disorder. Cloning is a relatively new sector of biotechnology, but it promises answers to very important problems related to surgery. Tissues and organs could be cloned for surgical purposes. If scientists could isolate stem cells and then direct their development, they would be able to create any kind of a tissue, organ or even a whole part of a body. Another revolutionizing tool of biotechnology is DNA fingerprinting. DNA fingerprints are useful in several applications of human health care research, as

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well as in the justice system. DNA fingerprinting is used to diagnose inherited disorders in both prenatal and newborn babies in hospitals around the world. These disorders may include cystic fibrosis, hemophilia, Huntington’s disease, familial Alzheimer’s, sickle cell anemia, thalassemia, and many others. Early detection of such disorders enables the medical staff to prepare themselves and the parents for proper treatment of the child. In some programs, genetic counselors use DNA fingerprint information to help prospective parents understand the risk of having an affected child. DNA fingerprint information can also help in developing cures for inherited disorders. DNA fingerprints helps to link suspects to biological evidence blood or semen stains, hair, or items of clothing – found at the scene of a crime and help in solving crime. Another important use of DNA fingerprints in the court system is to establish paternity in custody and child support litigation. The U.S. armed services have just begun a program to collect DNA fingerprints from all personnel for use later, in case they are needed to identify casualties or persons missing in action or for suspect verification. Due to the revolutionary development of biotechnology during last couple of decades agriculture has drastically advanced. Sensational achievements were made in both plant cultivation and animal husbandry. Plants have been improved in four different ways: • •

Enhanced potential for more vigorous growth and increasing yields. Increased resistance to natural predators and pests, including insects and disease-causing microorganisms. • Production of hybrids exhibiting a combination of superior traits derived from two different strains or even different species. • Selection of genetic variants with desirable qualities such as increased protein value, increased content of limiting amino acids, which are essential in the human diet, or smaller plant size, reducing vulnerability to adverse weather condition. Another important area of biotechnology is improvement of livestock. Improvement in disease control, efficiency of reproduction, yields of livestock products, i.e., meat, milk, wool, eggs, composition of livestock products, i.e., leaner meat, feed value of low quality feeds, i.e., straw, are some of the applications of biotechnology. One of the major scientific revolutions of the twentieth century was the breaking of the genetic code and the development of tools that enable scientists to probe the molecules of life with incredible precision. Now, in the twenty-first century, these developments in biology are being married with the use of everincreasing computer power to help us face the challenges that the new century brings.

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Bioinformatics is the name given to the new discipline that has emerged at the interface of biology and computing. Huge amount of genetic data (DNA, RNA, amino acid and protein sequences) of various organisms, form bacteria to humans, being generated worldwide is stored in a computer database. Specialized software programs are used to find, visualize, and analyze the information, and most importantly, communicate it to other people. Various computer tools are used to predict protein structure which is valuable information for development of vaccines, diagnostic tools as well as more effective drugs. Bioinformatics can help in easy and early detection of various diseases like cancer, diabetes and many more with the help of microarray chips (microarrays are miniature arrays of gene fragments attached to glass slides). Bioinformatics also helps scientists to construct phylogenetic tree based on molecular biology and ultimately contribute in the study of evolution. Computer simulations model such things as population dynamics, or calculate the cumulative genetic health of a breeding pool (in agriculture) or endangered population. One very exciting potential of this field is that entire DNA sequences or genomes of endangered species can be preserved. Biotechnology has a promising future. In future biotechnology will be accredited for some revolutionary technology. Recent advances in bioenergy, bioremediation, synthetic biology, DNA computers, virtual cell, genomics, proteomics, bioinformatics and bio-nanotechnology have made biotechnology even more powerful. Recent discovery of conduction of electricity by DNA and its behavior as a superconductor has opened a new realm in modern science. In future biotechnology will have profound impact in world economy. Biotechnology is a golden tool to solve some of the key global problems like global epidemic, fatal diseases, global warming, rising petroleum fuel crisis and above all poverty. For all the positive effects of biotechnology there are some possible side effects. Nobody knows what ecological hazards could be caused by transgenic organisms. Some even speculate that some transgenic organisms could fall into wrong hands to develop bioweapons. The opposition of genetic engineering says that “the science is very young and needs a lot more research.” The path from a test tube to the field is not a straight highway. Both intellectual and financial resources should be realized before new discoveries pave their way to industrial applications. In conclusion, biotechnology has also proved to be extremely productive and innovative and 21st century should be the century of biotechnology.

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1.4. HISTORY OF BIOTECHNOLOGY The origin of biotechnology, it is as old as human civilization. The development of biotechnology can be studied considering its growth that occurred in two phases: • •

traditional biotechnology (old); and new biotechnology (modern).

Origin and Definition: Old vs. New Biotechnology The origin of biotechnology can be traced back to prehistoric times, when microorganisms were already used for processes like fermentation. Although a molecular biologist may consider cloning of DNA to be the most important event in the history of biotechnology, later has actually been rediscovered in 1970’s for the third time during the present century. In 1920’s Clostridium acetobutylicum was used by Chaim Weizmann for converting starch into butanol and acetone, the latter was an essential component of explosives during World War I. This raised hopes for commercial production of useful chemicals through biological processes, and may be considered as the first rediscovery of biotechnology in the present century. Similarly, during World War II (in 1940’s), the production of penicillin (as an antibiotic discovered by Alexandar Fleming in 1929 on a large scale from cultures of Pencillium notatum, the second rediscovery of biotechnology. This is the beginning of an era of antibiotic research. The third rediscovery of biotechnology is its recent reincarnation in the form of recombinant-DNA technology, which lead to the development of a variety of gene technologies and is considered to be the greatest scientific revolution of the century. Thus, even though biotechnology had its origin in the past, it has assumed special significance only in 1970’s and 1980’s. Biotechnology is the product of interaction between the science of biology and technology. This relationship between science and technology has been observed to be complex, so that not only the science has influenced the technology, but the technology has also influenced science. Because of this complex relationship and its major impact on human welfare, it is believed that biotechnology in future may become a major force for human existence. Already, the products of biotechnology (including, diagnosis, prevention and cure of diseases; new and cheaper chemical products, e.g., pharmaceutical drugs and the new food sources; devices for environment protection and energy conservation, etc. are playing a very important role in employment, productivity, trade, economics and the quality of human life throughout the world. Biotechnology is being such an important area of study; attempts have been made to define it. It has been recognized that a complete definition of biotechnology is difficult

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due to such a wide range of its usage. Fermentation, by some microorganisms, formation of yoghurt (curd) and cheese from penicillin from certain fungi and the processes of baking and brewing are often included in describing what is called old biotechnology. The other example of biological processes involving the techniques of recombinant DNA and polymerase chain reaction (PCR) and bioprocessing which become possible only through the researches in molecular biology have been described as new biotechnology. However, in recent literature, no effort as made to distinguish between old and new biotechnology. Following are the some available definitions of biotechnology, and one of them may be really adequate. •

•

• •

Biotechnology is “the application of biological organism, system or processes to manufacturing and service industries” (British biotechnology). Biotechnology is the “ the integrated use of biochemistry, microbiology, and engineering sciences in order to achieve technological application of the capabilities of micro-organisms, cultured tissue cells and parts thereof.” Biotechnology is “a technology using biological phenomena for copying and manufacturing various kinds of useful substances.” Biotechnology is “the controlled use of biological agents, such as micro-organisms or cellular components, for beneficial use.”

1.5. TRADITIONAL BIOTECHNOLOGY It is kitchen technology developed by our ancestors using the fermenting bacteria. It is as old as human civilization. The Aryans are performing daily Agnihotra or Yojna during the Vedic period (5000–7000 BC). The material used in Yajna is animal fat (ghee), which is fermented product of milk. Similarly, the divine “soma” had been offered to God (a fermented a microbial product used as beverage). Summarians and Babylonians (6000 BC) are drinking the beer. Egyptians were baking leavened bread by 4000 BC. The preparation of curd, ghee, wine, beer and vinegar, etc. were the kitchen technology. In spite of all development, preparation of curd, ghee, vinegar and alcoholic beverages, idli, jalebi, dosa (Table 1.1) have become an art of the kitchen of all Indians. The traditional biotechnology refers to the conventional biotechnology which have ben used for many centuries. Beer, cheese, bine and other many foods have been produced using traditional biotechnology. Thus the

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traditional biotechnology includes the processes that are based using traditional biotechnology. It includes the process that is based on the natural capabilities of microorganisms. It has established a huge and expanding world market. In monetary term, it represents a major part of all biotechnology financial profits. There is one story related to the butter “Makhan Chor” (butter stealing) by Lord Sri Krishna during Mahabharat period. The butter has been produced following the same kitchen method or art. Table 1.1: Fermented Foods Prepared in Different Parts of India Types of foods Wari

Substrate Black gram

Regions North

Vadai (wada)

Black Gram

North

Tharra

Mahua

North

Tari

Date Palm

East

Sinki

Radish tap root

Himalaya

Srikhand

Milk

West

Rabdi

Mixture of butter-milk/ wheat/ barley/ peralmillet Milk

West

Black gram flour (besan) Wheat Flour (Maida)

North

Milk Bamboo shot Cucumber

East Himalaya Himalaya

Paneer Papad Nan

Mishti dahi Mesu Khalpi

North

North

Quality and Uses Spongy cake used as snack Deep fired cake used as snack Sweet alcoholic beverage obtained through distillation Sweet alcoholic beverage Sun-dried sour soup pickles Concentrated sweetened preparation Cooked paste used as Staple food

Soft cheese used as fried curry Circular wafers used as snack Leavened flat baked bread used as staple food Thick sweet gel Sour pickles Sour pickles

Introduction of Biotechnology Khaman

Bengal gram

West

Jalebi

North

Dhokla

Wheat Flour (Maida) Rice and Black gram Leafy Vegetables Bengal-gram

Dahi

Milk

North

Dosa

South

Chhurpi

Rice and black gram Milk

Ambali

Millet and rice

South

Bhatura

Wheat flour (Maida)

North

Idli Gundrum

South Himalaya West

Himalaya

13

Spongy cake, used in breakfast Crispy, deep-fried used as sweet confectionery Steamed Spongy cake used in Breakfast Sun-dried, sour taste, used as-soup or pickles Spongy cake used as a snack Sour, thick gel with Whey Spongy, shallow fried used as staple food Cheese like mild sour, soft mass used as curry Steamed cake used in snack Flat fried, leavened bread used with chhola

The Egyptians during the 2000 BC period used to prepare vinegar from crushed dates by keeping for longer time. But the crushed dates produce intoxicants at first. In Egypt, Mesopotamia and Palestine during 1500 BC the art of production of wine from crushed grapes, and beer from the germinated cereals (malt) using a bread leaven (a mass of yeast) was established. In Indian Ayurved, production of “Asava” using different substrates and flowers of mahua (Madhuca indica) or dhataki (Wodfordia fructicosa) has been well characterized till today since Vedic period. In these methods various substrates are transformed into a number of products. Hence the odor, color, and taste of final products are changed. Moreover use of salts for preservations of different foods has been earlier developed even in Europe. The preservation methodology is still of the mummies of Egypt are noteworthy. The possible mummification have been done through dehydration of dead body followed by use of mixture of salts largely sodium carbonate. The traditional technology is an art rather than a science. The cause of fermentation could be discovered after observing microorganisms using a microscope by Antony van Leeuwenhoek (1673–1723) at Delt (Holland). The significant contribution was done by chemists on the process and the products of fermentation during the 18th century. The milky

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precipitate was obtained when the gas evolved from fermentation was passed through the limewater in 1757. It was called limewater test. A similar gas was comes out from burning of charcoal. Henry Cavendish demonstrated that the gas evolved from brown sugar in water treated with yeast was absorbed by sodium hydroxide solution. Schele and J. Priestley in 1772–1774 confirmed the identity of oxygen gas present in air. Using analytical technique for carbon estimation, Antoine Lavoisier gave the chemical basis of alcoholic fermentation. A French Man, Nichola Appert in 1810 was described the method of food preservation. In the same year Peter Durand also gave the use of tin container for food preservation. It was done by putting an air-tight vessel containing food material in the boiling water. It increased the importance of canning industry. Lack of oxygen in such a closed and heated vessel was reported by Gay Lussac. He concluded the oxygen required for initiation of alcoholic fermentation, but not for further progress of fermentation. Charles B Astier gave the concept that air is the carrier of all kinds of germs after 1830. Theodore Schwann after a series of experimentation demonstrated that the development of the fungus (sugar fungus) on fruit juice causes fermentation. He was the first to observed and describe the yeast in growing process. Charles Cagiard-Latour (1838) observed yeast budding using a microscope allowing 300–400 power magnification. Justus von Liebig in 1839 a well known chemist proclaimed that all the activity of yeast cells was the result of chemical and physical reactions going on in the medium. Microbes also called microorganisms are invisible creatures too small to be seen with the unaided eye and a microscope is needed to study them. They are diverse, present everywhere and exist in a range of environments from mountains and volcanoes to deepsea vents and hot spring. Thermus aquaticus is an example of unusually heat stable bacterium isolated from hot springs from which heat resistant enzyme Taq polymerase was isolated. Microorganisms can be finding everywhere like in the air, water, in the food we eat and even in our body. In fact we come in contact with countless numbers of microorganisms every day. It plays a vital role in ecology of earth. They degraded dead bodies of plants and animals and recycle chemical elements to be used by living plants and animals. Some are used commercially in the production of industrially important chemicals such as alcohol, organic acids and antibiotics. It is also responsible for food fermentations thus enhancing their quality and shelf life. Using microbes to make products such as different foods and chemical is called biotechnology. Using recombinant DNA, microbes can produce important substances such as proteins, vaccines and enzymes.

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Microbes include bacteria, archaea, fungi, algae, protozoa, virus, viroids, and prions. While bacteria and archaea are classed as prokaryotes and the term introduced by Edorand Chatton for the cells in which the nuclear material is not surrounded by a haploid. The fungi, algae and protozoa are eukaryotic whose genomic DNA contains in a nucleus surrounded by a membrane, others don’t have regular cell structure and classified separately (Aneja, 2014). Microbes range in size from ultramicroscopic viruses 20 nm diameter to large protozoans 5 mm or more in a diameter. In simple words, the largest microbes are as much as 2,50,000 times the size of the smallest ones. The study of all organisms in the microscopic range is called microbiology. The naming of organisms called the binomial system of nomenclature was devised by Caroleus Linnaeus in 1735. In this system each living organism is assigned two names- the genus and the specific epithlet, both of which are underlined or italicized, e.g., Escherichia coli. Microorganisms are relevant of all of us in a different ways. Sometimes the influences of microorganisms in human life are beneficial apart from those other times it is detrimental or harmful to human beings. For example microorganisms are required for the production of cheese, beer, bread, yogurt, alcohol, wine, antibiotics, vitamins and vaccines and many ore important products which are useful in human life. Microorganisms are essential components of our ecosystem. Fungi and bacteria play an important role in the recycling or organic and inorganic material through their role in the carbon, nitrogen and sulfur cycles. They also are a source of nutrients at the base of all ecotropical food chains and webs. Recent study discovered by Professor Jonathan Rhodes with his colleagues at the University of Liverpool, North West England, says that common edible mushrooms contain a protein lectin that can stop cancer cell multiplication. This discovery finally could lead to new targets for therapy. Taxomyces andreanae, an endophytic fungus known to occur in the U.S. A is being used to produce taxol, an antitumor diterpinoid used in the treatment of some cancers. Taxol was originally obtained from the bark of pacific yew (Taxus brevifolia). It was in the year 1347 when plaque or Black Death struck Europe and within 4 years killed 25 million people that is 1/3 of the population. Over the years the disease struck again and again, wiping out 75% of the European population by 1431. This disease is believed to have changed European culture. It was Robert Koch 1843–1910, a German bacteriologist, who in 1876 first of all proved that anthrax is caused by a microbe Bacillus anthracis. The past 30 years have witnessed an increase in new and emerging infectious agents such as AIDS virus (HIVhuman immunodeficiency virus) and Escherichia coli and several other older diseases regularly appear to be on the increase. Recently correlation has been shown between gastric ulcers and the bacterium (Helicobactor) that invades the

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stomach, between type 1 diabetes and certain coxsackie viruses and between coronary artery disease and cytomegalovirus infection. Microbes play a role in decomposition of waste material and help to other animals for digest grass such as cattle, sheep and termites, so they can also balance nature. Microbe’s affects on human health. Environment provides development of biodegradable products and safe drinking water, which use bacteria to clean up oil spills through bioremediation process. Microbes participate in agriculture research has led to healthier live-stocks and crops free from disease. Microbes play a role in industries such as (foodstuffs, beer, wine, cheese and bread), antibiotic, genetic engineering and insulin. It is specialized area of biology that deals with the study of microorganisms or microbes. The roughly speaking, organisms with a diameter of one mm or less are microorganisms and fit into the broad domain of microbiology (Aneja, 2014; Garg et al., 2013). The branches of microbiology are categorized into pure and applied sciences such as: • • • • • • • • • • • •

•

•

Bacteriology: It is the study of bacteria. Mycology: It is study of fungi. Protozoology: It is the study of protozoans. Virology: It is the study of viruses. Algology or Phycology: It is the study of algae. Parasitology: It is the study of parasitism or parasites. Microbial Ecology: It is the study of interrelationship between the environment and microbes. Microbial Morphology: It is the study of detailed structure of microbes. Microbial Taxonomy: It is concerned with the classification, naming and identification of different microbes. Microbial Physiology: It is the study of metabolism of microbes at the cellular and molecular levels. Microbial Genetics: It is the study of genetic material of microbes. Molecular Microbiology: It is the study of structure and function and the biochemical reactions of microbial cells involved in metabolism and growth. Industrial Microbiology: It is concerned with the industrial uses of microbes in the production of vitamins, alcoholic beverages, amino acids, enzymes, antibiotics and other drugs. Food Microbiology: It is the study of interaction between food and microbes in relation to food bioprocessing, food spoilage and their

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prevention. • Agricultural Microbiology: It is the study of interrelationship of crops and microbes with an emphasis on control of plant diseases and improvement of yield. • Dairy Microbiology: It deals with the production of and maintenance in quality control of dairy products. • Aquatic Microbiology: It is the study of microorganisms and their activity concerning human and animal health in fresh, estuarine and marine waters. • Air Microbiology: It deals with the role of aerospora in contamination and spoilage of food and discrimination of plant and animal disease by air. • Exomicrobiology: It is the study of exploration for microbial life in outer space. • Medical Microbiology: It deals with the fundamental principles and techniques involved in study of pathogenic organisms as well as their application in the diagnosis of infectious diseases. • Immunology: It deals with the immune system that protects against the infections and attempts to understand the many phenomena that are responsible for both acquired and innate immunity. • Biotechnology: It is the scientific manipulation of living organisms especially at the molecular and genetic level to produce useful products. • Public Health Microbiology: It is concerns with monitoring, control and spread of diseases in communities. The first living things to appear on earth and the study of fossil remains indicate that microbial infections and epidemic disease existed thousands of year’s ago. It was Rogen Bacon (1220–125) who in the 13th century postulated that disease is produced by invisible living creatures. This idea was made again in 1546 by a physician “Girolamo Fracastoro (1478- 1553) of north Itlay. Fracastoro wrote a treatise-De Contagione in which he said disease was caused by minute “seed” or “germ” and was spread from person to person. As early as 1658, A monk named Athanasius Kircher (1601–1680) referred to “Worms” invisible to the naked eye in decaying bodies, meat, milk and diarrheal secretions. He examine thin cork slices, the bark of oak tree, and found that cork was made of tiny boxes that Hooke referred to as “Cells” The work of Hook was followed by Matthias Schleiden and Theodore Schwann who examined a variety of organisms and in 1838–1839 reported that all forms of life are composed of cells. An observation later became foundation of “Cell Theory.”

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The cell theory since been modified to all life is composed of cells that originate from other cells. The discovery of microbiology as discipline could be traced along the following historical eras. It is the period concerns with the discovery of microbial world that has been dominated by Antony van Leeuwenhoek. He was the first person to observe and accurately describe microorganisms in 1676. He also examines blood and other human tissues including his own tooth scrapping, insects, minerals and plant materials. He was the first person to describe about bacteria and protozoa using microscope he made himself, due to this extraordinary contribution to microbiology he considered as the father of bacteriology and protozoology. Transition Period Later by Leeuwenhoek time, there are two notes worthy contributions such as (1) controversy over spontaneous generations that said that living organisms could develop from non-living matter called as abiogenesis and (2) disease transmission Francesco Redi (1626–1697) In 1665, Redi put theory of spontaneous generation to rest by conducting experiment in which he placed meat in three jars. One jar covered with fine gauze, second was covered with paper and third was not covered. Flies entered the jars that was open to air, i.e., left uncovered and landed on meet where they laid their eggs that later developed into maggots. The other two pieces of meat did not produce maggots spontaneously. However flies were attached to the gauze covered jars and laid their eggs on the gauze and maggots developed without access to the meat and indicating that maggots were the offspring of the flies and did not arise from some vital source in the meat as previously believed. John Needham (1713– 1781) He was greater supporter of the theory of spontaneous generation. In 1749, He proposed that tiny organisms the animalcules arose spontaneously on his mutton gravy. He had covered the flasks with cork as done by Redi and even heated some flasks. Still the microbes appeared on the mutton broth. Lazzaro Spallanzani (1729–1799). In 1775, he attempted to refute Needham’s work by performing experiments. He boiled beef broth for longer period and removed the air from the flask and then sealed the container. After the incubation time which he was followed, no growth was observed by him in these flasks. When he was accused of destroying the vegetative force of the nutrients by over heating, showed that the heated nutrients could still grow animalcules when exposed to air by simply making a small crack in the neck so he was disproved the spontaneous generation. Georg Schroeder and Theodor von Dusch In 1854, he was convincing experiments to disprove the theory of spontaneous generation by simply passing air through cotton into flasks containing heated broth. No growth was observed on the infusions due to filtering out of microscopic organisms by cotton. He was the first to introduce the idea of using cotton plugs for plugging microbial culture tubes in 1854.

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He was finally resolved the controversy of spontaneous generation versus biogenesis and proved that microorganisms are not generated spontaneously; it is from inanimate matter but a ride from other microorganisms. John Tyndall (1820–1893) He was final blow to spontaneous generation in 1877. He was conducted experiments in an aseptically designed box to prove that dust indeed carried the germs. He was demonstrated that if no dust was present, sterile broth remained free of microbial growth for indefinite period even if it was directly exposed to air. The diversity of our life is present in our planet with new species of all types being constantly discovered. The formal system of organizing classification and naming living things is called taxonomy. Caroleus Linnaeus also gave the binomial system of nomenclature, i.e., naming of an organism by two names such as genus and species. Therefore Linneaus system provides each organism names and arranges them into groups called taxa with other similar organisms to reflect their phylogeny and evolutionary relatedness. The main taxa or groups in a classification scheme are organized in several descending ranks beginning with the kingdom the largest and followed by “Phylum or Division, Class, Order, Family, Genus and Species. The classification of microorganisms began in 1674 with the invention of light microscope and today is discipline based on increasingly complex criteria. The first phylogenetic trees of life were constructed on the basis of just two kingdoms such as Plantae and Animalia. In 1886, a German scientist Ernest Haekel proposed that the bacteria, algae, fungi and protozoa that lacked tissue differentiation be removed from the plant and animal kingdoms and be separated in a third kingdom Protista. In 1940, some microorganisms, e.g., typical bacteria the genetic material was not enclosed by a nuclear membrane. This remarkable discovery, the absence of nuclear membrane bound internal structure in one group of Protista (bacteria) and the presence of bound structure in majority of others such as algae, fungi and protozoa was discovered with significantly. This results in the division of these organisms into two groups Prokaryotes and Eukaryotes. Therefore several systems of classification of living organisms have been developed between 1940 and 1969 but the one that is most widely accepted was developed in 1960 by Robert Whittaker of Cornell University. This system places all living things except viruses into five kingdoms based on cellular organization and nutritional patterns such as (1) prokaryotic or Monera; (2) Protista; (3) Myceteae or Fungi; (4) Plantae and; (5) Animalia. There three kingdom domain which was discover by the scientist Carl Woese and George Fox have proposed a new system of classification that assign all organisms to one of the three super kingdoms (Figure 1.1) called domain such as (1) Archae; (2) Bacteria; and (3) Eukaryotic. It is believed that these three kingdoms arose from an ancestor most similar to the archaebacteria.

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There are several classification have been proposed for bacteria which Bergey’s Manual of Systematic Bacteriology has wide acceptance. The Bergey’s Manual of Determinative Bacteriology published by Williams and Wilkins Co. Baltimore, USA (1974), is the international standard for bacterial taxonomy. It deals with the classification of bacteria and contains description of all the species known at the time of its publication. In this bacteria are assigned 19 parts on based primarily on the following five features such as (1) Morphology and Gram stain Reaction; (2) Gaseous requirements; (3) Energy and carbon sources; (4) Mode of locomotion; and (5) ability to produce endospores. It serves as a practical guide for the identification of bacteria. After sometime manual was revised between 1984 -1989 and appeared volumes with a new name is Bergey’s Manual of Systematic Bacteriology. In this four volumes bacteria have been classified in 33 sections based on the following characteristics such as general shape and morophology, gram staining properties, presence of endospores, oxygen relationship, motility and mode of energy production. Therefore in the four volumes the bacterial groups are divided as follows (1) gram-negative bacteria of general, medical, and industrial importance; (ii) Gram-positive bacteria other than Actinomycetes; (iii) Gram-negative bacteria with distinctive properties, cyanobacteria and archaeobacteria; and (iv) Actinomycetes (gram-positive filamentous bacteria). The Bergey’s Manual of Systematic Bacteriology places all bacteria in the kingdom of prokaryotic which in turn is divided into four divisions as follows. The Gracilicutes (thin skin): Prokaryotes with a complex cell wall structure characteristics of gram-negative bacteria, e.g., Nonphotosynthetic bacteria. Firmicutes (Strong skin): Prokaryotes with a cell wall structure characteristics of gram-positive bacteria (Rods and cocci, Actinomycetes and related organisms). Tenericutes (soft or tender skin): Prokaryotes that lack a cell wall (e.g., mycoplasms). Mendosicutes (Skin with faults): Prokaryotes with unusual cell walls, e.g., Archaeobacteria. There are groups which do not reflect phylogenetic relationship between the organisms; each division is separated into classes for total 7 classes of bacteria. The system used in Bergey’s manual recognizes 7 classes under the 4 divisions. Groups of Bacteria as per Bergey’s Manual: Division I Gracilicutes Gramnegative bacteria Class I Scotobacteria Gram-negative non-photosynthetic bacteria Class II Anoxyphotobacteria Gram-negative photosynthetic bacteria that do not produce oxygen (green or purple bacteria) Class III Oxyphotobacteria Gram-negative photosynthetic bacteria that evolve oxygen (cyanobateria) Division II Firmicutes Gram-positive bacteria Class 1 Firmibacteria Grampositive rods or cocci Class II Thallobacteria Grams positive branching cells

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(actinomycetes) Division III Tenericutes Class I Mollicutes Bacteria lacking a cell wall (mycoplasmas) Division IV Mendosicutes Class I Archaeobacteria Bacteria with typical compounds in the cell wall and membranes. A Bacteria (singular bacterium), the microscope, single-celled (unicellular), prokaryotic organisms, reproduce asexually, are haploid and do not have their DNA localized with in a nucleus surrounded by unit membrane (i.e., without nucleus). It is two types’ of bacteria gram-positive and gram-negative and. They lack the membrane-enclosed intracellular structures found in most other cellular organisms. The shapes of bacteria are spherical or ovoid (coccus), rod like (bacillus), corkscrew or curved (spiral), comma, helical and filaments. Individual bacteria may form pairs, chains, clusters or others groupings. Such arrangements are usually characteristics of a particular genus or species of bacteria. All bacteria have a cell wall composed of a carbohydrate and proteins complex called peptidoglycan. Bacteria reproduce asexually by a process called binary fission, i.e., by dividing into two equal cells. They possess flagella moving appendages for their motility. For nutrition, most bacteria absorb nutrients from their environment from the dead or living organisms, some can manufacture their own food by photosynthesis, and some can derive nutrition from inorganic substances. The study of bacteria is called bacteriology.

Archaea bacteria It is like bacteria, consists of unicellular prokaryotic cells, but do not have peptidoglycan in their cell walls. They often live in extreme environments and carry out unusual metabolic processes. These are found in hot springs, geothermally heated marine sediments and submarine hydrothermal vents. They have no nucleus and any other organelles. It is classified as bacteria and called as archaebacteria. They are further divided into phyla. It has same structure size as bacteria such as flat and square shaped cells. They use more energy sources than eukaryotic such as sugar, metal ions and ammonia or even hydrogen gas. They reproduce asexually by budding, fragmentation or binary fission (Figures 1.1 and 1.2). It has range from 0.1 µm to 15 µm in diameter and various shapes such as rods, spheres and spirals or plates.

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Figure 1.1: Gram-positive and gram-negative bacteria membrane.

Figure 1.2: Cell shapes in bacteria and archaea.

Fungi Fungi are eukaryotic with different nucleus containing the genetic material (DNA) surrounded by an envelope, the nuclear membrane. True fungi have cell walls composed of chitin, a polysaccharide also found in the exoskeleton of arthropods such as ticks and spiders. It reproduces both methods asexually and sexually and also separately. Fungi have generally a mycelium, a loosely organized mass consisting of thread like hyphae. It consists of three groups of organisms- yeasts, molds and mushrooms (Figure 1.3). The study of fungi is called mycology.

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Figure 1.3: Morphology of fungi.

Protozoa Protozoa are unicellular eukaryotic organisms which have no cell wall, no photosynthesis, no fruiting bodies, reproduce sexually and asexually having various ways of locomotion such as pseudopodia, flagella or cilia. Protozoa are classified into four groups on the basis of their motility. Protozoa are found in soil and water and as normal microbiota of animals.

Algae Algae are photosynthetic eukaryotes containing chloroplast an organelle carrying out oxygenic photosynthesis. The body of algae is called thallus. Most algae are single-celled, simple microscopic organisms. But some are multicellular which can attain an enormous size. The cell walls of most algae are composed of cellulose. They reproduce both sexually and asexually. It is abundant in fresh water and salt water, in soil and in symbiotic association with fungi called lichens. The study of algae is called algology (phycology).

Viruses Viruses are acellular (non-cellular) entities, ultramicroscopic, ranging in size from 30 nm to 300 nm. Hence they can be seen only with an electron microscope. They lack the characteristics of life because of that viruses are considered to be non-loving entities. A virus particle (called virion) has either DNA or RNA, but never both. The DNA and RNA found in viruses may be single stranded or double stranded. Indeed, viruses can be crystalized and stored in a container on

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a shelf for years, but they retain the ability to invade cells. Virus can reproduce only by using the cellular machinery of their host. Viruses are obligate intracellular parasites of bacteria (bacteriophages), protozoa, fungi, algae, plants and animals and responsible for diseases of humans (AIDS, polio, smallpox, swine flu, H1N1 influenza, bird flu) and plants (tobacco mosaic, cauliflower mosaic) (Figure 1.4). The study of viruses is called virology. Viroids are small circular pieces of naked single-stranded RNA, only 300 to 400 nucleotides long, with no protein coat. They are the smallest known infective agent causing diseases in plants only. They retard the growth of plant on which they multiply interfering with the mRNA processing. One of the worst diseases caused by viroids is potato spindle tuber viroid causing losses of millions of dollars from crop damage.

Figure 1.4: Viruses-bacteriophage, tobacco mosaic virus and human immunodeficiency virus (HIV), single stranded RNA virus with helical symmetry.

Prions Prions also called virions are proteinaceous infections particles having no nucleic acid. They cause infectious diseases the most deadly disease in cattle is the “mad-cow disease” also called bovine spongiform encephalopathy (BSE) and Creutzfeldt Jacob disease (CJD) of humans which involves the degeneration of brain tissue. Prion diseases are the result of an altered proteins; the cause can be a mutation in the normal gene for PrPc (normal host glycoprotein) or contact with an altered protein (PrPsc) (Figure 1.5).

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Figure 1.5: Prion replication cycle. The study of microbiology was started since the first report of Louis Pasteur (1857) on lactic acid fermentation from sugar. He isolated the microorganisms (lactic yeast) that were associated with lactic acid and formed curd. The cells of lactic yeast were smaller than that of beer yeast. Lactic acid production got increased when he added chalk powder to fermentation medium. Pasteur showed the presence of lactic acid in curd by using a polarimeter. In 1860, Pasteur provided a detailed report on the use of synthetic medium for microbiological studies. He concluded that there are many steps for fermentation process such as • •

Fermentation is carried out anaerobically by the living cells. During the alcoholic fermentation in synthetic medium the yeast increased the weight with increase in carbon and nitrogen contents of overall batch. • The increase in yeast protein in synthetic medium was accompanied by a related decrease in the ammonium nitrogen in the medium. • Fermentation sugar was required for multiplication of yeast cells. • Similar phenomenon occurred in fermentation of lactic acid, tartaric acid and butyric acid, etc. • The pure culture is not using; some of the fermentation process were stopped. • Growth and physiology of yeast differ when they are grown under anaerobic growth conditions. A large amount of sugar was converted into alcohol under anaerobic growth conditions, while the aerobic conditions large amount of sugar was converted into yeast cell mass. Pasteur suggested that the high percentage of microbial population is killed

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by heating the juice at 62.8̊ C which is now called as Pasteurization. Robert Koch in 1881 gave the method for establishing relationship of pathogen with a disease which is known as “Koch’s” postulates or “pathogenicity test.” He proved that the anthrax disease is caused by “Bacillus anthracis.” The first demonstrate enzymatically mediated fermentation reaction by Edward Buchner in 1897. He showed that the cell free yeast juice mixed with concentrated sugar solution evolved upon incubation the carbon dioxide and produced ethyl alcohol. A similar product was also produced in aqueous solution of the other sugars such as glucose, sucrose, fructose and maltose. Buchner called the dissolved substance in juice responsible for sugar fermentation as “zymase.” This work showed improved techniques of fermentation. Fermentation could also be demonstrated in test tubes without a living organism. The work of chemistry had more roles in understanding the fermentation phenomenon than the microbiology. Hans Buchner and Martin developed more effective method of getting cell free extracts after disrupting the microbial cells. They ground the microbial cells with quartz and adding Kieselguhr. Thus sucrose fermentation using cellfree extracts from yeast cells obtained by the above method was demonstrated. Discovery of viruses and their role in disease was possible when Charles Chamberland in 1884 constructed a porcelain bacterial filter. Edward Jenner in 1798 used vaccination by taking out liquid material from cowpox lesions and introducing into people having small pox. But Pasteur and Chamberland developed of toxins produced by Corynebacterium diphtheria (causes disphtheria in human) its antitoxin was developed by Emil von Behring in 1890 and Shibasaburo Kitasato. He injected the inactivated toxin into rabbit that induced to produce antitoxin in the blood. The antitoxin inactivated the toxin and protected the disease. Similarly, tetanus antitoxin was developed by Behring. The traditional biotechnology is really the kitchen technology developed by our ancestors using the fermenting bacteria. Kitchen technology is old as human civilization. During Vedic period (5000–7000 BC), Aryans had been performing daily Agnihotra or Yan One of the materials used in Yan a is animal fat (ghee) which is a fermented product of milk Similarly, the divine soma a fermented microbial products used as beverage) had been offered to God. Summarians and Babylonians (6000 BC) were drinking the beer. Egyptians were baking leavened bread by 4000 BC. Preparation of curd, ghee, Wine, beer, vinegar etc., was the kitchen technology. In spite of all development, preparation of curd, ghee, vinegar alcoholic beverages jalabi, idli, dosa, have become an art of the kitchen of all Indians. The traditional biotechnology refers to the conventional

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technology which has been used for many centuries. Beer, wine, cheese and many foods have been produced using traditional biotechnology. Thus, the traditional biotechnology includes the process that is based on the natural capabilities of microorganisms. The traditional biotechnology has established a huge and expanding world market. In monetary term, it represents a major part of all biotechnology financial profits. Who can forget the story of Makhan Chori (butter stealing) by Lord Sri Krishna during Mahabharat period? Butter would have been produced following the same kitchen art. Besides breeding of strong productive animals, selection of desirable seeds for enhanced crop production has been the part of human activity since the time immemorial. The Egyptians (about 2000 BC) used to prepare vinegar from crushed dates by keeping for longer time. But the crushed dates produce intoxicants at first. In Egypt, Mesopotamia and Palestine (about 1500 BC) the art of production of wine from crushed grapes, and beer from germinated cereals (malt) using a bread leaven (a mass of yeast) was established. In Indian Ayurved, production of “Asava” and “Arista” using different substrate and flowers mahua (Madhuca Indica) or dhataki (Woordia fructicosa) has been well characterized till today since vedic period. In these methods various substrates are transformed into a number of products. Hence, the odor, color, and taste of final products are changed. Moreover, use of salt for preservation of various foods has been earlier developed even in Europe. Sill preservation methodology earlier developed even in Europe the mammies of Egypt noteworthy. Possibly, mummification would have been done through followed by use of mixture of salts largely sodium carbonate. Thus, the traditional biotechnology was an art rather than dehydration dead body science.

Role of microorganisms in fermentation The causes of fermentation could be discovered after observing microorganism using a microscope Antony van Leeuwenhoek (1673 1723) at Delf (Holland). During 18th century, a significant contribution was done by the chemists on the process and products of fermentation. In 1757, it was demonstrated that a milky precipitate could be obtained when the gas evolved from fermentation was passed through the lime water. It was called lime water test. A similar gas comes out from burning of charcoal. Henry Cavendish demonstrates that the gas evolved from brown sugar in water treated with yeast was absorbed by sodium hydroxide solution. Schele, J. Priestly (1772–1774) confirmed the identity of oxygen gas present in air. Using analytical technique for carbon estimation, Antoine Lavoisier gave the chemical basis of alcoholic fermentation. The study of microbiology was started since the first report of Louis Paster (1857) on lactic acid fermentation from sugar. He isolated the microorganisms

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(lactic yeast) that were associated with lactic acid and formed curd. The cells of lactic yeast were smaller than that of beer yeast Lactic acid production got increased when he added chalk powder to fermentation media. Pasteur showed the presence of lactic acid in curd by using a polarimeter. In 1860, Pasteur provided a detailed report on the use synthetic medium. He concluded that fermentation is carried out anaerobically by the living cells. During alcoholic fermentation in synthetic yeast increased the weight with increase in C and N contents of batch. The increase in yeast protein in synthetic medium was accompanied by a related decrease in the ammonium nitrogen in the medium. Fermentation of sugar was required for multiplication of yeast cells. Similar phenomenon occurred in fermentation of lactic acid, tartaric acid, batyric acid, etc. because of using pure culture, some of the fermentation processes were stopped growth and physiology of yeast differ when they are grown conditions was later on called as large amount of sugar was converted into alcohol, while under aerobic conditions large amount of sugar was converted into yeast cell mass. Pasteur suggested that high percentage of microbial population is killed by heating the juice at under aerobic and anaerobic (Pasteur effect) under anaerobic growth conditions a 62.8ºC (which now called Pasteurization). Robert Koch (I881) gave the method for establishing relationship of a pathogen with disease which is known as ‘Koch’s postulates’ or ‘pathogenicity test.’ Following this technique he proved that the anthrax disease caused Bacillius anthracts. Edward Buchner (1897) was the first to demonstrate enzymatically-mediated fermentation. He showed that cell-free yeast juice mixed with concentrated incubation the carbon dioxide and produced ethyl alcohol. Similar products were also produced aqueous solution of the other sugars such the dissolved substance in juice responsible for sugar fermentation the improved techniques of fermentation. Fermentation could also be demonstrated in test tubes with out a living organism. The work of chemistry had more role in understanding the fermentation than the microbiology. During 1890s, Hans Buchner and Martin Hahn developed more effective method of getting cell- free extracts after disrupting the microbial cells They ground the microbial cells with quartz and adding Kieselguhr (to get sufficient consistency in the resulting paste) Thus, sucrose fermentation using cell-free extracts from yeast cells (obtained by the above method) was demonstrated. Discovery viruses and their role in disease were possible when Charles Charmberland (1884) constructed porcelain bacterial filter. Edward Jenner (1798) used and introducing into people having smallpox. But Pateur and Chanberland developed an attenuated anthrax vaccine against anthrax disease.

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After the discovery of toxins produced Corynebacterium diphtheriae (causes diphtheria in human) its antitoxin was developed by and Shibasaburo Kitasato. He injected the inactivated toxin into rabbit that induced to produce antitoxin (antitoxin is an antibody with the ability to neutralize a specific toxin) in the blood. The antitoxin inactivated the toxin and protected against the disease. Similarly, tetanus antitoxin was developed by Behring.

1.6. MODERN BIOTECHNOLOGY •

There are the two major features which differentiates the modern biotechnology from classical biotechnology such as: • Capability of science to change the genetic material for getting new products for specific requirement through recombinant DNA technology. • The ownership of technology and its socio-political impact. Now the conventional technology industries, pharmaceutical industries, agro-industries, etc. are focusing their attention to produce biotechnologybased products.

Emergence of modern biotechnology The new or modern biotechnology embraces all methods of genetic modification by recombinant DNA and cell fusion technologies. It also includes the modern developments of traditional biotechnology processes. The new aspects of biotechnology founded in recent advancement of modern biology, genetic engineering and fermentation process technology are now increasingly finding wide industrial application. But the rate of application will depend on • • • • •

Adequate investment by the industries Improved system of biological patenting Marketing skill Economics of the new methods Public perception about the biotechnology products.

The traditional biotechnology associated with fermentation was gradually industrialized in the end of 19th century. This resulted in gradual growth of industries producing beer, whisky, wine, canned food and rum, etc. The first time in 1920 the Leeds City Council (U.K) established the Institute of Biotechnology. In the late 1960s, OECD was set up to promote policies

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for sound economic growth of the member countries. In 1978, The European Federation of Biotechnology was set up. The biotechnology brought industries and agriculture together in 20th century. The fermentation process were developed which produced the acetone from starch and paint solvent from automobile industries during the World War I. The antibiotic penicillin was discovered during World War II. Manufactured of penicillin shifted the biotechnological focus towards pharmaceuticals. Linking the fermentation with biochemistry, bioprocess, chemical engineering and instrument designing helped substantially in the progress of industries. The work on Microorganism dominated for the preparation of biological welfare, antibiotics and fermentation process. Suspected preparation of biological and chemical warfare led to US attack on Iraq in 2003 during Gulf War (1991). After the discovery of double helix DNA by Watson and Crick (1953), Werner Arber in 1971 discovered a special enzyme in bacteria which he called the restriction enzymes. These enzymes can cut the DNA strand and generate fragments. The cut ends of two fragments are single stranded sticky ends because the single stranded ends having identical base pairs can rejoin S. Chen and H. Boyer in 1973 removed a specific gene from a bacterium and inserted into another bacterium using restriction enzymes. This discovery marked the start of recombinant DNA technology or genetic engineering. Baltimore successfully transferred human growth hormone gene into a rabbit in 1976. The European Federation of Biotechnology was created in 1978. In 1978, a U.S. company “Genetech” used genetic engineering technique to produce human insulin in E. coli. In 1980, a trial of new hormone was conducted in the U.S.A., France, Japan, and the United Kingdom. The US Food and drug Administration gave marketing approval to “Humulin”, i.e., human insulin made by Eli Lilly (U.S.A) by the end of 1982. Another hormone somatotroping was produced on industrial scale. The first genetically engineered tomatoes in 1993, FlavrSavr, were sold in market. In 1996, the first clone lamb “Dolly” was borne successfully by the efforts of scientists of Scotland. Thereafter several cloned animals were produced. The sequence of the human genome was published in Nature and Science in 2001. The human genome project was completed by March in 2003 and in 2002, a designer baby was born to cure the genetic disease of her elder sister. A claim was made for the birth of a clone baby “Eve” on December 27, 2002 by the scientist of “Human Cloning Society,” the clonaid of France. In February, 2005 in the meeting of United Nations many countries supposed gene cloning

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in humans, while a few countries supported with request to allow for the sake of research only. In May 2005, scientists in South Korea have used a method called therapeutic cloning to produce stem cell lines. These are genetic matches to patients. Such stem cell lines could be used for disease research. The U.S.A. condemned this approach. In this method human embryos were produced through cloning and stems cells were obtained from blastocyst. The excised stem cells could be grown in vitro and used further. It is therefore interesting that the scientists are engaged in doing the most challenging task mass production of growth hormones, insulin, vaccines, immunogenic proteins and polypeptides, gene therapy, biofertilizers, biopesticides, producing disease and stress resistant plants, biomass, enzymes, antibiotics, acids, fuels, etc. Manu biochemical companies such as National Pituitary Agency (U.S.A), E.Lilly (U.S.A), Kabi Vitram A B (Swedan), Genetech Co. (U.S.A), Biogen (Switzerland), etc. are producing/trying to produce some of the above products by using genetic engineering techniques. Many Nobel Laureates including Dr. Har Govind Khorana are associated with these companies. Biotechnology is not a sudden discovery, rather a coming of age of a technology that was initiated several decades ago. By the middle of 20 century there had been a tremendous growth in the area of chemistry, physics and biology. Further knowledge of each branch has been advanced substantially. Multidisciplinary strategies were made for the solution of various problems. A novel spectrum of investigations occurred through the true interdisciplinary synthesis. This led to the evolution of biotechnology which is an outcome of integrated effort of biology with technology the root of which lies in biological science. The key difference between biology and biotechnology is their scale of operation. Usually the biologist works in the range of nanograms to milligrams. Biotechnologists working on the production of vaccine may be satisfied with milligram yields, but many other projects aims at kilograms or tonnes. Thus the main objective of biotechnologists consists of scaling up the biological processes (Smith, 1996). The main discipline of biology is microbiology, biochemistry, genetics, molecular biology, immunology, cell and tissue culture (Figure 1.6). However, on the engineering such as large scale cultivation of microbes and cells, their upstream and down stream process, etc. Theses processes can be separated into five major operations • • •

Strain selection and improvements Mass culture Optimization of cell responses

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Process operation Down stream processing

Figure 1.6: A tree of biotechnology. Many areas of biotechnology have arisen through the interaction between various parts of biology and engineering, biochemistry, biophysics, cell biology, colloid chemistry, embryology, ecology, genetics, immunology, molecular biology, medical chemistry, pharmacology, polymer chemistry, thermochemistry and virology. The modern biotechnology has developed several technologies extracting the basic knowledge from biology. Many areas of biotechnology have arisen through the interaction between various parts of biology and engineering, biochemistry, biophysics, cell biology, colloid chemistry, embryology, ecology, genetics. Immunology, molecular biology, medical chemistry, pharmacology, polymer chemistry, thermochemistry and virology. The modem biotechnology has developed several

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technologies extracting the basic knowledge from biology.

1.7. GLOBAL IMPACT AND CURRENT EXCITEMENT OF BIOTECHNOLOGY Each and every organism performs its function within its optimum limits. The excitement about the modern biotechnology is that the scientific methods (such as genetic engineering) have enhanced the natural capabilities of natural production of organisms. What a miracle is that a mouse of the size of rabbit can be produced. Bacteria like E. coli are producing mammalian, hormones such as insulin. somatostatin, somatotropin, etc. Yeast cells have been genetically manipulated to produce vaccine against hepatitis B virus (hepatitis disease). Myeloma cells (cancerous cells of bone-marrow) and B-cells of immunized mice were hybridized to produce hybrid cells that consist the characteristics of both the cells which were cell division and antibody production. Now the hybrid cells (hybridomas) are being used for production of monoclonal antibodies. In 1982, interferon (C, B and Y) were produced by genetically engineered E. coli cells. Techniques have been developed to produce rare and medicinally valuable molecules to change hereditary traits of plants and animals, to diagnose diseases, to produce useful chemicals and, to clean up and restore the environment. In this way biotechnology has great impact in the fields of health, food/agriculture and environmental protection. Due to rapid development the present situation is that there is no difference between pharmaceutical firms and biotechnology industry. However, approved products in the pipeline and renewed public confidence made it one of the most promising areas of economic growth in future. India offers a huge market for the products as well as cheap manufacturing base for export (Padh, 1996). Following are some of the areas where biotechnology has done the best.

1.8. BIOTECHNOLOGY IMPACT ON HEALTH CARE The maximum benefits of biotechnology have been utilized by health care. Biotechnology derived proteins and polypeptides form the new class of potential drugs. For example, insulin was primarily extracted from slaughter animals. Since 1982, human insulin (Humulin) has been produced by microorganisms in fermentor. Similarly, hepatitis B vaccines. Recombivax HB” (from Merk), Guni (from Shantha Biotechnics Ld, Hyderabad), Shanvac (Biological E. Laboratory),

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etc., are the genetically engineered vaccines produced biotechnologically. Since 1987, the number of biotechnology-derived new protein drugs has surpassed the new chemical drugs (Padh, 1996).

Some of the important products produced through genetically modified organisms. With the advancement of gene manipulation in organisms the science has led to a new revolution in biology which is called gene revolution. Obviously, it is a third revolution in the science after industrial revolution and computer revolution. Thus the roots of today’s biotechnology lie in chemistry, physics and biology. Currently there are about 35 biotechnology-derived therapeutics and vaccines approved by the USFDA alone for medical use, and more than 500 drugs and vaccines reach in market. Similarly, about 600 biotechnology diagnostics practices. About 130 gene therapy protocols have been approved by the US authorities imports of many immunodiagnostic are worldwide available in clinical. The major landmark in human history is the human genome sequence. The HGP is an international research programme. Almost the whole human genome has been sequenced and chrommosome map has been developed in various laboratories worldwide through coordinated efforts. Human chromosome mapping was completed by March 2003. There are about 3,000 functional genes in human. More than 97% genes are non-functional. They do not encode for any polypeptide chain. Objectives of human genome project are to: construct the detailed genetic and physical map of human genome, (i) determine the complete nucleotide sequence of human DNA, (ii) tore information in database, (iii) ocate the estimated 50,000–10,000 genes within the human genome, (iv) address the ethical legal and social issues LELSI) that may arise from the project, and (v) perform similar analysis on the genomes of several other organisms.

1.9. BIOTECHNOLOGY IMPACT ON ENVIRONMENT The natural biodegradability of pollutants present in environment has increased with the use of biotechnology. The bioremediation technologies have been found successful to combat the pollution problems. Bioremediation is the use of microorganisms to detoxify pollutants, present in the environment usually as soil or water sediments. The pollutants cause several health problems. Microorganisms which show potential to degradation of

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oil, pesticides and fertilizers belong to the genera of bacteria Pseudomonas, Micrococcus, Bacillus, and fungi Candida, Cladosporium, Torulopsis, Trichoderma, etc. Computer-based study and designing of genome is called genomics. Genomics deals with sequencing of the complete genome of a particular organism. Similarly, study of proteins present on genome using computer is called proteomics. The proleomics can be defined as the study of all the proteins present in genome of an organism. With the help of proteomics and genomics, the new molecules that can interact with the other partners could be identified. This gives us deep insight into biological pathway. Now the whole genome is available to biologists for scrutiny. There may be new small molecules as potential drug candidates. Therefore, interaction of some molecules can be studied in greater detail. The 33,000 genes of human beings are on a microchip, and have helped to design specific drugs for genetic diseases, for which there is no cure so far. For example, a specific gene expresses in breast cancer patients A designed drug (Herceptia) is good for treatment of breast cancer. Thus, the field of genomics has helped the growth of phamacologic, toxicologic, and protein studies on animals, therefore, and the new areas are called phamacogenomics, toxicogenomics, and proteogenomics, respectively. Bioinformatics: It is a new field of biotechnology linked with information technology. Bioinformatics may be defined as application of information sciences mathematics, statistics, and computer sciences) to increase the understanding of biology, biochemistry and biological data. The most remarkable success of bioinformatics to date has been is use in the ‘shotgun sequencing’ (breaking of a large piece of DNA into smaller fragments) of human genome.

1.10. BIOTECHNOLOGY IMPACT ON AGRICULTURE Biotechnology is making new ground in the food/agriculture area. Current public debate about BSTC bovine somatotropin (a hormone administered to cows to increase milk production) typifies an example of biotechnology product testing public acceptance. Similarly the FlavrSavr tomato (produced by transgenic plants engineered antisense technology to preserve flavor. texture and quality) is a new breed of value added foods. Food biotechnology offers valuable and alternative to food problems and a solution to nutritionally influenced diseases such as diabetes, hypertension, cancer, heart diseases, arthritis, etc. A transgenic golden rice has been produced by introducing three genes for the production

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of vitamin A in Taipei’ rice. Several insect (Bacillus thuringiensis) resistant transgenic Bt plants have been produced insecticidal toxin producing Bt gene of Bacillus thuringiensis into the desired plants. A transgenic cotton named ‘Ingaurd’ was released in Australia which contained Bt gene and provided resistance against insects. We can recall the Bt cotton prepared for Andhra Pradesh but sown in: (i)

Gujarat in 2001 which raised debate throughout the country. Biopesticides are coming to the market and their sales are increasing. Molecular Pharming is a new concept where therapeutic drugs are produced in farm animals, for example, therapeutic proteins secreted in goat milk. There are about a dozen companies that produce lactoferin. tPA, haemoglobin, melanin and interleukins in cows, goat and pigs (Padh, 1996). However, it is not surprising those vegetables producing vaccines, insulin, interferon and growth hormones would be available in market in 21st century, beside, human clones, and several other miracles. Pests are fast developing resistance to transgenic crops, according to a study carried out in Telangana and Andra Pradesh by entamologists from Prof. Jayashankar Telangana state agricultural university in Hyderabad and their counterparts in the US.

1.11. BIOTECHNOLOGY IN INDIA In most of the developing countries, the recombinant: DNA technology has become the major thrusts. In 1982, Government of India set up an official agency. The National Biotechnology Board (NBTB) which stared functioning under the Department of Science and Technology (DST). In 1986, NBTB was replaced with a full-ledged department, the Department of Biotechnology (DBT), under the Ministry of Science and Technology for planning, promotion and coordination of various biotechnological programmes. The DBT is making effort in promoting postgraduate education and research. Special MSc courses in Biotechnology in s DBT. The selection of students is d National Test, In addition, it also provides trained manpower for the rapidly growing biotech industry. It has also raised the level of biology education n certain areas of biotechnology in the country, Moreover, a considerable amount of basic biochemical and molecular biology is imported in these courses. Today, India has the DBT, DST, CSIR, ICAR, ICMR and IARI, and other working under the government. These agencies and the other National and International Industries are manufacturing Biotech products and marketing

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then after clinical trials. A Technology Development Board (IDB) has been set up by the government The TDB works with universities, industries and the National Institutes. The Technology Information Forecasting and Assessment Council (TIFAC) have prepared a vision 2020 document which consists of biotechnology also. There are over 30 companies in India which are producing the modern biotech products such a Wipro, Reliance, Life Science, Pennetia Biotech Ltd. Since 1980s, India has supported a lot to biotechnology industry and its products. Teaching and research of biotechnology have been included in University’s syllabi both at Under Graduate and Postgraduate levels DBTsupported departments are running in several Universities and institution. It is boned that India will play a key role in future as one of the largest market of the weed, and as a producer of biotech products. The United Nations Industrial Development Organization (UNIDO) recognized the potential of genetic engineering and biotechnology for promoting the economics progress of the developing countries. The initiation taken by UNIDO has led to the foundation of ICGEB. In 1981, in a meeting convened by UNIDO it was proposed to establish an international center of excellence to foster biotechnology in the developing world. In 1982, this concept was approved by a high level conference of developed and developing nations in Belgrade. The statutes of the center were signed by 26 countries with the entry into force of statutes on February 3 1994. The ICGEB has become a fully autonomous international organization composing of at present 33 member states The ICGEB has its two centers, one located in Trieste (Italy), and the other in Jawaharlal Nehru University, New Delhi (India). The Trieste component is currently occupying about 5,700 m2 area, whereas the New Delhi component is occupying about 10,000 m2 area. This center is functioning in a proper way since 1982. Many public and private institutions working under the Government departments and organization have advised the DBT to formulate the biotechnology programmes under the following areas: (i) Plant molecular biology and agricultural biotechnology, (ii) Biochemical engineering, process optimization and bioconversion, (iii) Aquaculture and marine biotechnology, (iv) Fuel fodder, biomass and green cover, (v) Medical biotechnology, (vi) Microbial and industrial biotechnology, (vii) Large scale use of biotechnology, (viii) Integrated systems in biotechnology, (ix) Veterinary biotechnology and (x) Infrastructural facilities. Pharmaceutical industry in India is very strong and vibrant with expertise for chemical drugs. It has little experience in biotech diagnostics and no experience in biotech therapeutics. Moreover, pharma industry is located between Mumbai and Ahmedabad (90% of drug production in India is in Gujarat and

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Maharashtra). There is no Government institution or university with expertise in this area to help pharma industry. However, for a variety of reasons, the Indian pharmaceutical industry will sooner or later enter in manufacturing of biotechnology based diagnostics and therapeutics (Padh, 1996).

1.12. BIOTECHNOLOGY IN WORLD Biotechnology products are increasing in world market day-by-day. The high value added biotechnology product to be used Countries which have boosted the UK, Japan, France, Australia, Russia, Poland, Germany, as well as India (among the developing Countries). The most effective me international networks developed to applied microbiology/biotechnology are the Regional Microbiology Network for South-East Microbiological Resource Centers medical field are now in domination for the last few years. The biotechnology R & D during the past two decades are of promotes enterprises through policy development and support international cooperation is through networks (MICREN). To faster biotechnology inventions, the U.S.A. promotes enterprises. Funding for basic scientific research at the National Institute of Health (NIH) has been supported. The US administration has boosted up the process for improving new medicines so that these may be quickly and safely small business development have also been encouraged through the incentives. The U.S. administration has geared up to intellectual property right protection. The international markets have been opened for biotechnology research and biotech products. One of the World’s best examples of partnership has been established by developing public databases. It enables the scientists to tie up with an enterprise. These have helped in developing partnership among University research-Government and private industries. Science education has been improved. Guidelines have been prepared so that science based regulatory programmes (i) can promote public biosafety, (ii) earn public confidence, and (iii) can guarantee fair and open international market. For promotion of technology products and bio-business, the other countries (such as the UK, Japan, Germany, France, Australia, etc.) have also prepared similar guidelines.

1.13. FUTURE DEVELOPMENT ACHIEVEMENTS OF BIOTECHNOLOGY A few developing countries like India have scientists technologists related

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to biotechnology where national strategies of development in biotechnology could be implemented. The scientific and technical manpower has to be properly shifted towards new biotechnology with the aim to produce expertise in biotechnology. In a keynote address, Bachhawat and Banerjee (1985) have described the impact of biotechnology on third world countries. They emphasized “Indian” bioscientist must be trained to utilize their knowledge and expertise for application and orientation, for example, a microbiologist must he trained in microbial genetics to be really useful in fermentation technology trained in cell culture, protoplast fusion or DNA recombination for practical utility and similarly people from traditional disciplines in life science may be trained to reorient their knowledge towards application and process of training read readjusted according to need. It is, therefore, necessary to encourage the biotechnological programmes industrial and educational levels. In higher education, teaching of biotechnology should be compulsory for undergraduate students to expand their understanding and knowledge of the scientific and engineering principles underlying biotechnology. Countries like USA, France, Germany and Japan have taken this issue seriously or a botanist must be In India, most of the universities have started teaching biotechnology at under-graduate level. However, at postgraduate level teaching and research have been initiated only by a few universities/ institutes on all India entrance test basis. Government of India has selected many thrust areas of national and international relevance as described earlier. Undoubtedly, the modern biotechnology is expected to solve many problems arising at the global level. In 21st century growth and economy of a country will certainly depend on operation of biotechnology. A revolution may occur in some of the areas like medical and health care, agriculture industry and environment. DNA fingerprinting has successfully helped the forensic science in the search of criminals making identity of individuals, solving parentage dispute, etc. Human diagnostics and therapeutic drugs have been discovered and commercialized. Gene therapy is hoped to solve the problems of genetic diseases, Biotechnology-based vaccines are the best the insulin. They have no side effects and pose no risk for the presence of live form of viruses in vaccines. Many transgenic plants and animals have been genetically improved. Now they are capable of producing new or improved products. The questions may be raised on complex, ethical, spiritual, and philosophical issues Hundreds of transgenic plants have been produced and many of them are being sown in field and products are available in market The plant biotechnology will reduce the dependence of farmers on pesticides and will help to utilized the new technologies In near future papers and chemicals with less energy and less pollution may be produced through biotechnology.

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Abatement of pollution using potential microorganisms (i.e., bioremediation) has been used in many countries. Thus the potential microorganisms are looked for better health, better products and better environment of future. Instead of chemical pesticides, biopesticides have been produced and commercialized by some industries in India and abroad. Similarly, biofertilizers (formulation of nitrogen fixing bacteria and blue green algae, i.e., cyanobacteria and phosphatesolubilizing bacteria and fungi) have also been made available to farmers in India. On the other hand, biotechnology has generated new jobs for the youth and stimulated the growth of small business. It has also encouraged the innovations in the industrial and agricultural sectors. The U.S.A. alone more than 15 lakh youth have been employed in industries

1.14. MISUSE OF BIOTECHNOLOGY The prevention off the misuse of biotechnology is stressed in many countries because it may cause a lot of mischief when given a free play in the hands of transnational companies engaged in ruthless persuit of profits. One of these concerns is the rapid pace of genetic erosion. This will lead to a situation where the base genetic material is available to only a few multinational companies in their gene banks. However, this genetic erosion has to be checked by saving the genetic diversity in its own environment, but not gene bank/germplasm bank, by the involvement of people proceeding from ‘green revolution’ to ‘gene revolution. Many new and transgenic plants and animals are being produced, and seeds, embryos, sperms are preserved. A day may come when farmers would have to depend on genetically engineered seeds. Still, there is doubt whether such seeds will suit for sustainable agricultural practice free from chemical poison. We are Moreover, the genetically modified organisms (GMOS) should be carefully researched and occur In addition, concerted monitored to ensure that the hazards to users and environment will not action should be undertaken to ensure that necessary consideration is given to the ethical and social effects of such studies. People should be known about the impacts of GMOS and genetically engineered products. Such efforts are already being made in some of the countries. The German Green party has called for a 5-year moratorium on commercial release of GMOS. The UK genetics forum is complaining for a partially mentally irresponsible application of biotechnology. In India, gene campaign against patenting of life forms and the misuse of biotechnology.

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1.15. BIODIVERSITY AND ITS CONSERVATIONS It is presumed that life on the planet-earth first originated around 4 billion years ago. This mega event marked a major step of transformation of the organic. Large and diverse communities of biota have, thus, occupied distinct climatic zones forming ecosystems. The concept of biodiversity first appeared in 1980. It is fact, the shortened form of two words: ‘biological’ and ‘diversity.’ It was coined by W.G. Rosen in 1985.

1.16. INTRODUCTION TO BIODIVERSITY Biodiversity or biological diversity can be defined as the vast array of species of microorganism, algae, fungi, plants and animals occurring on the earth either in the terrestrial or aquatic habitats and the ecological complexes of which they are a part.

1.17. DEFINITION AND EXPLANATION Biodiversity differs from place to place. It is so because environmental conditions of the area as well as the range of tolerance of the species determine whether or not a particular species can occur in that area.

1.18. ALPHA AND BETA BIODIVERSITY Alpha diversity: It refers to the diversity of organisms, i.e., number of species in the given community or habitat. Species diversity and species evenness, the two indices are used in combination to represent alpha diversity. Beta diversity: it appears in a range of communities due to replacement of species along a gradient of habitats or communities within a given geographical region.

1.19. LEVELS OF BIODIVERSITY In the biosphere, immense biodiversity exits at all levels of biological

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organization ranging from macromolecules within cells to biomes. The most important of these levels are genetic diversity, species diversity, community, and ecosystem diversity. •

•

•

Genetic diversity: it refers to the variation of genes within a species. The difference could be alleles, in entire genes or in chromosomal structures. Species diversity: species are distinct units of diversity, each playing a specific role in ecosystem. Species diversity refers to the variety of species within a region. The greater the species richness, THE greater is the species richness. Ecosystem diversity: Ecosystem diversity includes all the species, plus all the abiotic factors characteristic of a region.

1.20. RATE OF LOSS OF BIODIVERSITY Biodiversity loss has always existed as a natural process but threats biodiversity arise when the rate of extinction exceeds the rate of speciation.

1.21. CAUSES FOR THE LOSS OF BIODIVERSITY A. Hunting Man started hunting wild animals for food and safety, trade and fun ever since his appearance. Large scale hunting of wild animals causes destruction. B. Forest fires It is held that forest fires resulting from human activity or mistake have cause the elimination of many species of wildlife. C. Destruction of habitats and fragmentation Destruction of natural habitat causes the most serious threat to the biodiversity. 1. Deforestation 2. Pollution 3. Cleanliness D. Introduction of exotic species Intentional or chance introduction of exotic species into new island or countries by man adversely affect the native species E. Co-extinctions When a species become extinct, the plant and the animal species associated in an obligatory way with it also become extinct.

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1.22. USES OF BIODIVERSITY 1)

2)

3) 4) 5)

Source of food and improved varieties: Several thousands of species of edible plants and animals are known. Biodiversity play an very important role in creating novel varieties, as it provide the source material for breeding Fibers: Varieties fibers are available in India like cotton, jute, hemp, agave, flax, sun hemp, etc. are the major varieties. Plant species provide a variety of useful products such as gums, resins, dyes, perfumes, waxes lubricants, hydrocarbons, honey, latex, paper, tea, coffee, pearls ivory, fur, and skin. Drugs and medicines: Large numbers of substances with therapeutic properties are obtained from variety of plant species. Sport and recreation: Animals provide a good spot in their habitat to the hunters, and a good deal of fun and recreation to public in circus, zoos, parks, etc. Aesthetic value: Green forests, beautiful flowers, graceful beasts, songbirds and colorful fishes, birds, butterflies provide grandeur to biosphere.

1.23. EXTENT OF BIODIVERSITY IN PLANT Biodiversity refers to the variety and variability of life on earth. Biodiversity is typically a measure of variation the genetic, species, and ecosystem level.

1.24. EXPLORATION AND GERMPLASM COLLECTION It has attracted a great many adventurists, naturalists, travellers and plant hunters since distant past. The collection is based on ecogeographica/phyto-geographic surveys and more static/geographic approaches. It has floristics and flora of an area or region, etc. The regions of genetic diversity and utilization are areas and region wise as per utilization, such as cereals, pulses, fruits, vegetables, oilseeds, fibers, forages, etc. Crop genepool including its wild relatives in one basic unit involves population structure, incompatibility barriers, etc. crop germplasm includes landraces, primitive cultivators introgressed forms, obsolete and promising plant genetic resources on genetic approaches.

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1.25. INTRODUCTION AND EXCHANGE OF PGR (PLANT GENETIC RESOURCES) It has discovered related to genotypes of particular species, collected from different sources and geographical origins, for use in plant breeding to develop new cultivators. The sum total of hereditary material, i.e., all the alleles of various genes, present in a crop species and its wild relatives such as landraces, genetic stocks, framers verities, parental lines of hybrids, released varieties, primitive cultivators and wild and weedy relatives.

1.26. RED DATA BOOK AND ENDANGERED PLANT SPECIES The IUCN (International union of conservation of nature and natural resources) now called WCU (World conservation union) maintains a document called red list or red data book of taxa that are facing the risk of extinction. The IUCN has divided them into eight categories:

1. 2. 3. 4. 5. 6. 7. 8. • 1. 2. 3. 4. 5.

extinct; extinct of wild; critically endangered; endangered; vulnerable; lower risk; data deficient; and not evaluate. List of plant species Small whorled pogonia Sarracenia oreophila Plantanthera praeclara Showy lady’s slipprs Asclepias meadii

1.27. PLANT GENETIC RESOURCES It is the major biological resources on which humankind has relied for its very existence and its dependence on plant derived products will increase in the future due to greater demand form an increasing global population. New users

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will be found for plants and many new kinds of plants will be developed for their contribution to nutritional, medicinal, energy, shelter, fuel, amenity and cultural users. The consultative group on International Agricultural Research has taken the lead in these efforts as has the International plant genetic resources institute. On the technical side, new developments both in breeding technologies and in greater movement of germplasm ownership away from primary producers into industry-based ownership, offer great potential away from primary producers into industry-based ownership, offer great potential for plant breeding to create new kind of verities that will meet the future needs of humankind in quantity and in new kinds of food and other products.

1.28. PLANT QUARANTINE ASPECTS According to Ram Nath scientist insects, mites, nematodes, fungi, bacteria, viruses, MLOs, and other organisms are known to attack various crops of economic importance. These pests and pathogens not only reduced the quantity but also spoil the quality of the produce to a considerable extent. The potential food that has been aborted, spoiled or damaged would be enough to feed at least 75 million human beings. It may be defined as “Rule and Regulations promulgated by governments to regulate the introduction of plants, planting materials, plant products, soil, living organisms, etc. with a view to prevent inadvertent introduction of exotic pests, weeds and pathogens harmful to the agriculture or the envieonment of a country/region. It is designed as as safeguard against the harmful pathogens and pests exotic to a country of a region.”

1.29. SANITARY AND PHYTOSANITARY SYSTEMS (SPS) It is an agreement signed by WHO, at Uruguay round of the multi-trade negotiation which measures to protect humans, animals, and plants from diseases, pest, or contaminants. It applies to all sanitary (relating to animals) and phytosanitary (relating to plant) measures that may have a direct or indirect impact on international trade.

1.30. IN-SITU CONSERVATION The conservation of habitats and ecosystems where organisms naturally are

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occur. Sacred groves, biosphere, reserves, national parks, wildlife sanctuaries, etc.

1.31. EX-SITU CONSERVATION The conservation of elements of biodiversity out of the context of their natural habitats is referred to as ex-situ conservation, e.g., Zoos, botanical gardens, and gene banks.

1.32. CRYOPRESERVATION It is the use of very low temperatures to preserve structurally living cells and tissues.

1.33. GENE BANKS Gene banks are a type of bio repository which preserve genetic material. For plants, this could be by in vitro storage, freezing cuttings from the plants, or stocking the seeds.

1.34. CRYOBANKS Seattle sperm bank is a cryobank that currently focuses exclusively on providing donor sperm to women and families looking to conceive a child.

1.35. IPGRI (INTERNATIONAL PLANT GENETIC RESOURCES INSTITUTE) It is headquartered in Rome, Italy and has staff in eight countries worldwide. It is mission to strengthen the conservation and use of plant genetic resources, with special emphasis on the needs of developing countries. It is one of the 16 international agricultural research centers located around the world which are supported by the consultative group on International Agricultural Research (CGIAR) and consortium of 41 donor agencies.

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It is committed to advancing the conservation and use of plant genetic resources for the benefit of current and future generations. In its strategy diversity for development (1993), IPGRI recognizes that an important part of the genetic diversity of useful plants can be conserved in situ and the need to increase research and other activities on in situ and farm or community level conservation is note out. This strategy further recognizes that there are important socioeconomic and cultural dimensions to conservation and that these are particularly significant for in situ conservation.

1.36. FAO (FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS) It is help to raise levels of nutrition and standards of living, improve agricultural productivity, better the conditions of rural populations, and contribute to the expansion of the world economy. FAO highlights information’s as one of the priority areas in achieving agricultural development and food security.

1.37. NBPGR (NATIONAL BUREAU OF PLANT GENETIC RESOURCES) It was established by the Indian Council of Agricultural Research (ICAR) in 1976 with its headquarters at New Delhi. It has been given the mandate to act as a nodal institute at the national level for acquisition and management of indigenous and exotic plant genetic resources (PGR) for agriculture and carried out related research and human resources development for sustainable growth of agriculture. NBPGR is the second largest genebank in the world. It has the network of 10 regional stations covering different agro-climatic zones to carry out PGR activities including collection, characterization, maintenance, and evaluation of various crops.

1.38. CGIAR (CONSULTATIVE GROUP FOR INTERNATIONAL AGRICULTURAL RESEARCH) It is global partnership that unites organizations engaged in research for a foodsecured future. It is dedicated to reducing rural poverty, increasing food security, improving human health and nutrition and ensuring sustainable management of

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natural resources. It is carried out 15 centers that are members of the CGIAR consortium, in close collaboration with hundreds of partners including national and regional research institutes, civil society organization and development organizations and private sectors. It is generally run with other organization in partnership. There are number of members such as USA, UK, Germany, Switzerland, Japan, FAO, IFAD, World Bank, African Development Bank and Fund of the Organization of the Petroleum Exporting Countries.

1.39. PLANT BREEDERS RIGHTS It is generally protected in several countries through plant breeders rights (PBRs) plant variety rights (PVR). Through these rights further propagation of the variety is restricted. Under the earlier convention of 1978 due to International Union for the Protection of New Varieties (UPOV) the breeders rights did not prohibit the farmer from reuse plant back of farm saved seed of a variety form his own harvest for planting another crop freely used earlier as plant genetic resources for the purpose of breeding other varieties.

1.40. FARMERS RIGHTS It is a concept, which has been developed and adopted in FAO by all member countries. It recognizes the fact that farmers and rural communities have greatly contributed to the creation, conservation and exchange of plant genetic resources and also generated knowledge for the utilization of genetic diversity. Therefore it is the obligation of world community to help these farmers to carry out this task and also help them in utilizing the genetic diversity available with them. No, intellectual property system or any other mechanism exists for compensation or reward to the farmers for this work.

1.41. PROTECTION OF PLANT VARIETIES Various plan pests and pathogens inflict heavy crop loses both under field as well as under storage conditions. Plant quartine regulations promulgated by governments of different countries are designed to regulate the introduction and movement of plants, planting materials, plant products, etc. with a view to prevent the introduction of associated pests, pathogens and weeds exotic to a country or a region and which are harmful to its agriculture. Plant

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quarantine as a national service and its complementary role have been briefly discussed. Plant quarantine regulations with particular reference to India and the quarantine responsibilities of NBPGR in respect to the introduction of germplasm material for research use in the country have been discussed. There are number of organization that protected the crops from pest and pathogens such as National Institute of Plant Health Management, List of National Plant Protection Organization, Plant protection Advisor, Plant Protection (IPM), Plant Quarantine (PQ), Locus Control, and Pesticide Registration and Quality Control.

1.42. FARMERS RIGHTS ACTS After several amendments in the original draft, the latest draft of “Protection of Plant Varieties and Farmers Rights Act” 2001 was passed the Parliament (Lok Sabha) in August 2001. The president also gave his assent to the Bill November 5, 2001, so that the Bill was notified as Act No. 53 of 2001 in Gazette of India. It was to be enforced from a date that was to be notified by the Union Minister on Agriculture. One of the most important features of this bill is that it grants Framers rights by recognizing farmers as breeder’s cultivators and conservators of the seeds in their possession. It allows the farmers to use for re-sowing or sell their harvested seed, although they will not be allowed to sell the seed under a brand name. In other words the farmers will not be able to sell their harvested seed to another farmer for the purpose of raising a crop for commercial seed production.

2 Genes and Genomes and Genetic Engineering

2.1. NATURE OF DNA The DNA is found in all plants, animals, prokaryotes and some viruses. DNA is a double helical structure which consists of genetic material. It is joined by phosphodiester bond and it is acidic in nature. The entire genetic message that controls the chemistry of every cell of the body acting in a specific way is actually written in the language of four nitrogenous bases of DNA, i.e., purine and pyrimidines.

2.2. COMPOSITION OF DNA It is a polymeric compound containing four monomers known as deoxyribonucleotide monomers or deoxyribotids. Each DNA consists of pentose sugar, a phosphate group and a nitrogenous bases (Figure 2.1). Purines bases (adenine and guanine) are heterocyclic and two ringed bases and the pyrimidines (thymine and cytosine) are one-ringed bases.

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Figure 2.1: Nucleotides.

2.3. NUCLEOSIDES The nitrogenous bases combined with pentose sugar are called nucleosides.

2.4. NUCLEOTIDES A nucleoside linked with phosphate forms a nucleotide. Nucleoside = pentose sugar + nitrogenous base nucleoside + phosphate Nucleotide On the basis of different nitrogenous bases the deoxynucleotides are of following types: (i) Adenine (A) deoxyadenosine-3’/5’-monophosphate (3’75’-d AMP) deoxyguanosine-5’-monophosphate (5’-d GMP) deoxythymidine5-monophosphate (5’-d TMP); (ii) Guanine (G); (iii) Thymine (T); and (iv) Cytosine (C) deoxyeytidine -5-monophosphate (5’-d CMP). In addition to the presence of nucleosides in DNA helix, these are also present in nucleoplasm and cytoplasm in the form of deoxyribonucleotide phosphates, e.g., deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), deoxythymidine triphosphate (dTTP) (Figure 2.2). The advantage of these four deoxyribonucleotide in triphosphate form is that

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the DNA polymerase acts only on triphosphates of nucleotides during DNA replication. Similarly, the ribonucleotides contain ribose sugar, nitrogenous bases and phosphate. Except sugar, the other components are similar. However, uracil (U) is found in RNA instead of thymine. Generally, RNA molecule is single stranded besides some exceptions.

Figure 2.2: Nirogeneous base.

2.5. POLYNUCLEOTIDE The nucleotides undergo the process of polymerization to form a long chain of polynucleotide. The nucleotides are designated by prefixing ‘poly’ to each repeating unit such as poly A (polyadenylic acid), poly T (polythymidilic acid), poly G (polyguanidylic acid), poly C (polycytidilic acid) and poly U (poly uridylic acid). The polynucleotides that consist of the same are called homopolynucleotides such as poly A, poly T, poly G, poly C, and poly U.

2.6. CHARGAFF-EQUIVALENCE RULE By 1948, a chemist Erwin Chargaff started using paper chromatography repeating unit to analyze the base composition of DNA from a number of studies. In 1950, Chargaff discovered that in the DNA of different types of organisms the total amount of purines is equal to the total amount of pyrimidines, i.e., the

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total number of A is equal to the total number of T (A-T), and the total number of G is equal to the total number of C (G-C). It means that A/T = G/C, i.e., A+T/ G+C 1. In the DNA molecules isolated from several organisms regularity exists in the base composition.

2.7. PHYSICAL NATURE OF DNA 2.8. WATSON AND CRICK’S MODEL OF DNA Watson, J. D., and Crick, F. H. C. (1953) combined the physical and chemical data generated by earlier workers, and proposed a double helix model for DNA molecule. This model is widely accepted. According to this model, the DNA molecule consists of two strands which are connected together by hydrogen bonds and helically twisted. Each step on one strand consists of a nucleotide of purine base which alternate with that of pyrimidine base. Thus, a strand of DNA molecule is a polymer of four nucleotides, i.e., A. G. T, C. The two strands join together to form a double helix. Bases of two nucleotides form hydrogen bonds, i.e., A combines with T by two hydrogen bonds (A T) and G combines with C by three hydrogen bonds (G C). However the sequence of bonding is such that for every A.T.G.C. on one strand there would be T, A, C, G on the other strand. Therefore, the two chains are nucleotides on one chain are the photocopy of sequence of nucleotides on the other chain. The two strands of double helix run in antiparallel direction, i.e., they have opposite polarity. The left hand strand has 5–3’ polarity, whereas the right hand has 3’ 5’ polarity to the first one. The polarity is due to the direction of phosphodiester linkage complementary to each other, i.e., sequence of as compared to the first one. The DNA model also suggested a copying mechanism of the genetic material. DNA replication is the fundamental and unique event underlying growth and reproduction in all living organisms ranging from the smallest viruses to the most complex of all creatures including man. DNA replicates by semiconservative mechanism which was experimentally proved by Mathew, Meselson and Frank W. Stahlin in 1958. If changes occur in sequence or composition of base pairs of DNA, mutation takes place. Though the presence of adenine, guanine, thymine and cytosine is a universal phenomenon, yet unusual bases in DNA molecule is also occur. In some bacteriophages 5-hydroxymethylcytosine (HMC) replaces cytosine of the DNA molecule when methylation of adenine, guanine and cytosine occurs. This results in changes of these bases (Figure 2.3).

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Figure 2.3: Watson Crick’s model of DNA.

2.9. CIRCULAR AND SUPER HELICAL DNA Almost in all the prokaryotes and a few viruses, the DNA is organized in the form of closed circle. The two ends of the double helix get covalently sealed to form a closed circle. Thus, a closed circle contains two unbroken complementary strands. Some times one or more nicks or breaks may be present on one or both strands, for example, DNA of phage PM, Besides some exceptions, the covalently closed circles are twisted into super helix or super coils and is associated with basic proteins but not with histones found complexed with all eukaryotic DNA these histones like proteins appears to help the organization of bacterial chromatin structure with the result of nucleosome like structure, folding and super coiling of DNA and association of DNA polymerase with nucleoids. These nucleoid-associated proteins include H U proteins, IHF, proteins HI, Fir A, H-NS, and Fis. In archaeobacteria (e.g., Archaea) the chromosomal DNA exists in protein associated form. Histone like proteins have been isolated from nucleoprotein complexes in Thermoplasma acidophilum and Halobacterium salinarum. Thus, the protein associated DNA and nucleosome like structures are defected in a helix coils clockwise from the axis the coiling is termed as positive contrast, if the path of coiling is anticlockwise, the coil is called left handed or negative coil. The two ends of a linear DNA helix can be joined to form each strand continuous. However if one end rotates at 360°with respect to the other to produce; these histone like proteins appear to help the organization of bacterial DNA into a coiled variety of bacteria. In or some unwinding of the

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double joined resulting in formation of a twisted circle in opposite sense, i.e., opposite helix, the ends are to unwilling direction. Such twisted circle appears as 8, i.e., it has one node or crossing over point. If it is twisted at 720°before joining, the resulting super helix will contain two nodes. The enzyme topoisomerases alter the topological form, i.e., super coiling of a circular DNA molecule. Type I topoisomerases (e.g., E. coli top A) relax the negatively super coiled DNA by breaking the 5’-phosphoryl end, and then resealing the nicked phosphodiester backbone (Moat and Foster 1995). Type II Topoisomerases need energy to unwind the DNA molecules resulting in the introduction of super coils. One of type II isomerases, the DNA gyrase is apparently responsible for the negatively super coiled state of the bacterial chromosome. Super coiling is essential for efficient replication and transcription of prokaryotic DNA. The bacterial chromosomes is believed to contain about 50 negatively super coiled loops topological unit, the boundaries of which may be defined by the sites on DNA that limit its rotation (Wang, 1982; Mout and Foster, 1995). One of the phosphodiester bonds in dsDNA allowing the 3’-OH end to swivel around or domains.

2.10. ORGANIZATION OF DNA In addition to organization of DNA in prokaryotes and lower eukaryotes as discussed earlier in eukaryotes the DNA helix is highly organized into the welldefined DNA-protein complex termed as nucleosomes. Among the proteins the most prominent are the histones. The histones are small and basic proteins rich in amino acids such as cells there are five types of histones, e.g., H, H,A, H,B, H, and H. Eight histone molecules (two each of H, A, H, B, H, and H) form an octomer ellipsoidal structure of about I 1 nm 7 nm in diameter. DNA coils around the surface of ellipsoidal structure of histones 166 base pairs (about 7/4 turns) before proceeding onto the next and form. Thus a nucleosome is an octomer or four histone proteins complexed with DNA lyzine and/or arginine. Almost in all eukaryotic long and 6.5- complex structure, the nucleosome. The histones play important role in determining eukaryotic chromosomes by determining the conformation known as chromatin. The nucleosomes are the repeating units of DNA organization which are often termed as beads. The DNA isolated from chromatin looks like string 146 base pairs of DNA lie in the helical path and the histone DNA assembly is known as the nucleosome core. The stretch of DNA between the nucleosome is known as linker’ which varies in length from 14 to over 100 base pairs. The H1, is associated with the linker region and helps the folding of DNA into complex structure called chromatin or beads. As a result of

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m chromatin becomes visible as chromosomes during cell division.

2.11. STRUCTURE OF RIBONUCLEIC ACID (RNA) The RN TMV, yellow mosaic virus, influenza virus, foot and mouth disease is usually single stranded except one virus such as virus, etc. which have dsDNA. The virus, retrovirus, wound tumor entirely single strand of the RNA is folded either at certain regions or to form hairpin shaped structure. In the hairpin shaped structures the complementary bases are linked by hydrogen bonds, which give stability to the molecules. However, no complementary bases are found in the unfolded region. The RNA does not possess equal purine-pyrimidine ratio, as it is found in the DNA. Like DNA, the RNA is also the polymer of four nucleotides each one contains D-ribose, phosphoric acid and a nitrogenous base. The bases are two purines (A,G) and two is not found in RNA. Pairing between bases occurs as A-U and G-C pyrimidines (C, U). Thyamine is not found in RNA. Pairing between bases occurs as A-U and G-C. The nucleotides formed by the four bases are adenosine monophosphate (AMP), guanosine monophosphate (GMP), cytosine monophosphate (CMP), and uridine monophosphate (UMP). These are found freely in nucleoplasm but in the form of triphosphates, e.g., ATP, GTP, UTP and CTP. As a result of polymerization the ribonucleotides form a polynucleotide chain of RNA. If the RNA is involved in genetic mechanism, it is called genetic RNA as found in plant, animal and bacterial viruses. The DNA acts as genetic material and RNA follows the order of DNA. In such cells the RNA does not have genetic role. Therefore, it is called non-genetic RNA. The non-genetic RNA is of three types: (i) ribosomal RNA (rRNA), (ii) transfer RNA (1RNA) or soluble RNA (SRNA), and (iii) messenger RNA (MRNA) or template RNA. These three types of RNA differ from each other in structure, site of synthesis in eukaryotic cell and function.

2.12. GENE CONCEPT Although the role of hereditary units (factors) in transfer of genetic characters over several generations in organisms was advocated by Gregor John Mendel, yet the mystery of the ‘hereditary units was unraveled during early 1900s. In 1909, W. hereditary units; however, early work done by several workers proposes various hypotheses to explain the exact nature of genes. The gene has been defined as the unit of genetic information that controls a specific aspect of the phenotype. In 1906, W Bateson and R.C. Punnet reported the first case of linkage in sweet pea and proposed the presence or absence theory. According

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to them the dominant character has a determiner, and the recessive character lacks determiner. In 1926, T.H. Morgan discarded all the previous existing theories and put forth the particulate gene arranged in a linear order on string. In 1928, Belling granules would Johanson coined the term ‘gene’ that acts as theory. He thought that genes are the chromosome and look like beads on a proposed that the chromosome that appeared be the gene. This theory of gene was well accepted by the cytologists. In 1933, Morgan was awarded Nobel Prize for advoc the discovery of DNA as carrier of genetic informations, the A as re, it is necessary to understand both, the classical and modern concept of gene. According to the classical concepts a gene is a unit of: (i) physiological functions; (ii) transmission or segregation of characters; and (iii) mutation. In 1969, Shapiro and co-workers published the first picture of isolated genes. They purified the lac operon of DNA and took photographs through electron microscope. In 1908, the British physician Sir E.R. Garrod first proposed onegene-one product hypothesis. In 1941, G.W. Beadle and E.L. Tatum working at St Stanford University clearly demonstrated one-gene-one enzyme hypothesis, based on experiments on Neurospora crassa. They made it clear that genes are the functional units and transmitted to progenies over generations; also they undergo mutations. They treated N. crassa with X-rays and selected for X-ray induced mutations that would have been lethal. Their selection would have been possible when N. crassa was allowed to grow on nutrient medium containing vitamin B6. This explains that X-rays mutated vitamin B6 synthesizing genes. They concluded that a gene codes for the synthesis of one enzyme. In 1958, Beadle and Tatum with Lederberg received a Nobel Prize for their contribution to physiological genetics.

2.13. UNITS OF A GENE After much, extensive work done by the molecular biologists the nature of gene became clear. A gene can be defined as a polynucleotide chain that consists of segments each controlling particular trait. Now, genes are considered as a unit of function (cistron), a unit of recombination (recon) and a unit of mutation (mutan).

2.14. CISTRON One-gene-one enzyme hypothesis of Beadle and Tatum was redefined by several workers in coming years. A single MRNA is transcribed by a single gene. Therefore, one-gene-one MRNA hypothesis was put forth. Exceptionally,

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a single MRNA is also transcribed by and it is said to be polycistronic. Therefore, the concept has been given. As one-gene-one protein hypothesis. The proteins are the polypeptide chain of amino acids translated by MRNA. Therefore, it has been correctly used as one-gene-one polypeptide hypothesis more than one gene (Figure 2.4).

Figure 2.4: Cistron (A) Recon (B) Muton (C) Genes as a unit of function.

2.15. RECON Earlier, it was thought that crossing over occurs between two genes. In 1962, Benzzer demonstrated that the crossing over or recombination occurs within functional gene or cistron. In a cistron the recombinational units may be more than one. Thus, the smallest unit capable of undergoing recombination is known as recon. Benzer (1955) found that the cultures of T4 bacteriophage formed plaques on agar plates of Eshcherchia coli. Normal T4 formed small plaques of smooth edges, whereas the mutant T4 phage formed the larger plaques of rough edges.

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2.16. MUTON Benzer (1962) coined the term mutan to denote the smallest unit of chromosome that undergoes mutational changes. Hence, mutan may be defined as the smallest unit of DNA, which may be possible. The small’s largest unit in size followed by recon and at nucleotide level is changed in the nucleotide. Thus, changes unit of mutan is the nucleotide. Therefore, cistron is mutan. This can be explained that a gene consists of several cistron, a cistron contains many recon, and a recon a number of mutans. However, if the size of a recon is equal Lo mutan, there would be no possibility in recon for consisting of several mutans.

2.17. SPLIT GENES (OR INTRONS) During 1970, in some mammalian viruses (e.g., adenoviruses) it was found that the DNA sequences coding for a pieces. Therefore, these genes were variously named as interrupted genes or For the discovery of split genes in adenoviruses and higher organisms, Richards J. Roberts and Phillip Sharp polypeptide were not present continuously but were split into several split genes or introns (Gilbert, 1978 intervening sequences (Lewin, 1980), inserts (Weismann, 1978), Junk DNA were awarded Nobel Prize in 1993. DNA sequence codes for mRNA but the complete corresponding sequence of DNA is not found in MRNA. Certain sequences of DNA are missing in mRNA. The sequences present in DNA but missing in MRNA are called intervening sequences or introns, and the sequences of DNA found in RNA are known as exons. The exons code for m RNA.

2.18. RNA SPLICING In the initial stage, RNA transcript introns are synthesized which are removed later on by a process called RNA splicing. The junctions of intron-exon have a GU sequences at the intron’s 5-end, and an AG sequence at its 3’OH end. These two sequences are recognized by the special RNA molecules known as small nuclear RNA (snRNA) or snrnpS (Steitz, 1988) GUS These together with proteins form small nuclear ribonucleoprotein particles called snRNPs. Some of the snRNPs recognize the splice junctions and splice introns accurately. The UI-snRNP recognizes the 5’-splicing junction, and the U5 snRNP recognizes the 3 splicing junction. Consequently pre-mRNA is spliced in a large complex called a spliceosome (Guthrie, 1991). The spliceosome consists of pre-MRNA,

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five types of snRNPs and non-snRNP splicing factors (Rosbash and Seraphin, 1991). Robert and Sharp, the Nobel Prize winners in 1993, independently hybridized the MRNA of adenovirus with their progeny or DNA segments of virus. The MRNAS hybridized the ssDNA of virus where the complementary sequences were present. The mRNA-DNA complexes were observed under electron microscope to confirm which part of viral genome had produced the MRNA strand. It was found that MRNA did not hybridize DNA linearly but showed a discontinuous complexes pattern. Huge loops of unpaired DNA between the hybridized complexes clearly revealed the large chunk of DNA strand that carried no genetic information and did not take pa synthesis. The adenovirus MRNA contained four different regions of the DNA in protein The B-globin genes of mice and rabbits, and tRNA genes of yeast tyrosine-tRNA Consists of eight genes three of which have been studied in detail.

2.19. RIBOZYMES Thomas Cech (1986) discovered that pre- rRNA isolated from a ciliated protozoa, tetrahymena thermophila is self-splicing. Thereafter, S. Altman showed that ribonuclease cleaves a fragment of pre-tRNA from one end, and also contains a piece of RNA. This RNA fragment catalyzes the splicing reaction, i.e., acts as enzyme. Therefore, this RNA segment catalyzing the splicing reaction is called ribozyme. For this discovery Cech and Altman were awarded the Nobel Prize in 1989 in chemistry. The best-studied ribozyme activity is the self-splicing process is widespread and occurs in T thermophila pre-tRNA mitochondrial RRNA and MRNA of yeast and other fungi, chloroplast tRNA, IRNA and MRNA, and MRNA of bacteriophage of RNA. This The RRNA intron of T thermophila is 413 nucleotide long. The selfsplicing reaction needs guanosine and is accomplished in three steps: (i) the 3’-G attacks the 5’ group of introns and cleaves the phosphodiester bond; (ii) the new 3-OH group on the left exon attacks the 5-phosphate exons join and remove the intron; and (iii) the 3-OH of intron attacks the phosphate bond of nucleotide 15 residues from its end releasing the terminal fragment and cyclizing the intron exon.

2.20. OVERLAPPING GENES (GENES WITHIN GENES) In 1940s, Beadle and Tatum proposed one-gene-one protein hypothesis which

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explains that one gene encodes for one protein. However, if one gene consists of 1,500 base pairs of 500 amino acids in length would be synthesized. In addition, if the same sequence read in two a protein different ways, two different amino acids would be synthesized by the same sequence of base pairs. It means, the same DNA sequence can synthesize more than one protein at different time. It was realized for the first time when the total number of proteins synthesized by ØX174 exceeded from the coding potential of the phage genome. A similar phenomenon is found in the tumor virus SV40 where the total molecular weight of proteins (i.e., VP1, VP2, and VP3) synthesized by SV40 genes is much more than the size of the DNA molecule (5,200 base pairs, i.e., 1,733 codons). From this observation the concept of overlapping genes has emerged.

2.21. GENE ORGANIZATION The DNA molecules that make up the hereditary elements are called genome. The functional region of genome is called gene. In a complex genome only a small part is functional, in that it is coded into a protein with the amino acids sequence determined by the DNA sequence (Figure 2.5).

Figure 2.5: Presentation of Different regions in and around a gene in a genomic sequence.

2.22. GENE EXPRESSION The DNA has two important roles in the cells, first is replication and the second is expression. Gene expression accomplished by a series of events that contained in DNA is converted into molecule that take place in the call. The

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information contained in the DNA is converted into molecules that determine the metabolism of the cell (Figure 2.6).

Figure 2.6: Central Dogma.

2.23. GENE REGULATION The DNA of microbial cells consists of thousands of genes which do not express at the same time. At a particular time single or few genes expresses and synthesized the particular protein. The other genes remain silent and express when required. This shows that genes have property to switch on and switch off.

2.24. TRANSCRIPTION Synthesis of enzymes comes into the control of genetic material, i.e., DNA in living organisms. Enzymes synthesis occurs by two processes such as translation and transcription. Translation refers to the synthesis of mRNA. It is controlled by the promoter region of the gene.

2.25. TRANSLATION It is a process in which ribosomes in the cytoplasm or ER synthesize proteins after the process of translation.

Gene Machine Fully automated commercial instruments called automated polynucleotide synthesizer or gene machine is available in market which synthesizes predetermined polynucleotide sequence. Therefore, the gene can be synthesized rapidly and in high amount (Figure 2.7).

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Figure 2.7: Gene machine.

2.26. POLYMERASE CHAIN REACTION (PCR) Gene Amplification: Basic PCR and Its Modifications We discussed the techniques of recombinant DNA and gene cloning, which permit us to obtain an unlimited supply of identical copies of a gone sequence or DNA segment, which is cloned in a prokaryotic a eukaryotic cell with the help of a vector. This technique, discovered around 1975, proved extremely useful in experiments of molecular biology and genetic engineering and thus became an essential tool in all molecular biology laboratories. In 1985, yet another remarkable tool in molecular biology was discovered, which is known as polymerase chain reaction (PCR) nicknamed now as people’s choice reaction. The methodology is so important that the prestigious journal Science published from USA considered PR as the major scientific development of the year 1989, and had chosen Taq DNA polymerase, the enzyme used in PCR, as the mole of the year 1989. Kary Mullis who discovered this Scientific American, how he got the basic idea this reaction, while driving through the red wood mountains of California, USA. The polymerase chain reaction is such a powerful technique that may replace completely the gene cloning with vectors in due course of time.

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The Basic Polymerase Chain Reaction (PCR) In order to understand PCR, the readers should be familiar with the mechanism of DNA replication within the cells. The DNA replication involves polymerization of nucleotides using a template DNA strand with the help of an enzyme DNA polymerase, but this reaction invariably requires a primer strand to which further nucleotides can be added using the DNA polymerase enzyme. If the primer strand is not available, the reaction cannot proceed. In the living cells this primer strand is not a DNA strand but is a small single stranded RNA molecule synthesized with the help of RNA polymerase enzyme. In PCR, a similar reaction takes place in an ‘eppendrof tube,’ where the primer strand is added from outside in the form of a deoxyoligonucleotide, and DNA polymerase enzyme is added to help in polymerization. Unlimited supply of amplified NA is obtained by repeating the reaction, which is made possible by regular denaturation of freshly synthesized double stranded DNA molecules by heating it to 90–98°C. At this high temperature the two strands separate. Once the double stranded DNA is made single stranded by heating up to 90–98°C, the mixture with two primers, recognizing the two strands and bordering the sequence to be amplified is cooled to 40–60°C. This allows the primers (which are in excess) to bind to their complementary strands through renaturation. The presence of Taq DNA polymerase enzyme and all the four essential nucleoside triphosphates allow synthesis of complementary strands in the usual manner. In a thermal cycler this process is automatically repeated 20 and 30 times, so that in a single afternoon, a billion copies of the sequence flanked by the left and right primers can be produced. In order to continue the synthesis, the temperature of the mixture is alternately increased (for denaturation) and decreased (for renaturation) once every 1–3 minutes (as fixed by the computer device). Therefore during temperature rise the enzymatic activity of DNA polymerase should not be destroyed, otherwise one may have to add a fresh aliquot of enzyme in each cycle of amplification. This became possible only by the discovery of thermostable enzyme Taq DNA polymerase, isolated from Thermus aquaticus growing in hot springs. This enzyme acts best at 72°C and the need denaturation temperature of 90°C does not destroy its enzymatic activity. Later, other thermostable enzymes like Pflu DNA polymerase isolated from Pyrococcus fiuriosus and Vent polymerase isolated from Thermococcus litoralis, were discovered and were found more efficient. These enzymes allowed automation of the entire process and automatic PCR thermal cyclers are now available (each for a price of around Rs. 2.00 lakhs), which can amplify DNA sequences at a fast speed unattended (Figure 2.8).

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As pointed out earlier, PCR may eventually replace gene cloning technique. This technology is much easier and requires much smaller quantity nanogram or ng 10g of DNA. For cloning, DNA is needed, as the starting material, in microgram (ug) quantities.

Different Schemes of PCR In the previous section, we discussed the basic outline of PCR technique, outlining how billions of copies of a DNA sequence can be obtained within a few hours, without the use of a vector and a host as done in gene cloning. However, there are several variations to this basic PCR technique, which allow varied applications of this technique.

Inverse PCR In this technique, the amplification of those DNA sequences takes place, which are away from the primers and not those flanked by the primers. For instance, if the border sequences of a DNA segment are not known and those of a vector arc known, there the sequence to be amplified may be cloned in the vector an sequences of vector may be used as polymerization sequence flanked by the primers and towards the DNA inserted segment. Similarly, if the gene sequence is known, then it can use for sequences as primers, for an inverse, to amplify the sequences, e.g., the regulatory sequences.

Figure 2.8: Polymerase chain reaction.

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Anchored PCR In the basic PCR technique and the inverse PCR, one has to use two primers representing the sequences lying at the ends of sequences to be amplified. But sometimes, we may have knowledge about sequence at only one of the two ends of the DNA sequence to be amplified. In such cases, anchored PCR may be used, which will utilize only one primer instead of two primers. In this technique, due to the use of one primer, only one strand will be copied first, after which a poly G tail will be attached at the end of the newly synthesized strand. This newly synthesized strand with poly G tail at its 3’ end will then become template for the daughter strand synthesis utilizing an anchor primer with which a poly C sequence is linked to complement with poly G of the template. In the next cycle, both the original primer and anchored primer will be used for gene amplification.

PCR for Site Directed Mutagenesis This technique is used for introducing mutations at the desired place in a DNA sequence by sequences of primers. Since mutation through prime A variation of this technique allows mutation to be introduced mutations are limited to the ends of the gene sequence to be introduced at any place of interest in the gene the method is described as overlap extension which works as follows: In two separate PCR reactions, a particular gene is amplified into two separate segments. In each of these two reactions there is one primer at the end of the gene and the other (with desired alteration for mutation) internal to the sequence. The internal modified primers in two reactions are complementary to one another, so that the amplified products will have their ends internal to the original sequence actions will overlap. The internal ends of the two PCR products, when mixed, function as primers. Extension of these primers by taq polymerase results in the formation of a complete gene, with mutations incorporated at internal sites.

Cloning and Expression of the PCR Product The amplified PCR product can be cloned, if restriction sites for specific enzymes are introduced at the 5’ end of each primer. These restriction sites do not interfere with amplification and can be used for cloning with a variety of vectors in the usual manner. Similarly, incorporation of a T7 promoter at the 5’ end of one primer will allow copies of PCR product own the use of RNA polymcrase enzyme to get RNA copies of PCR product

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Asymmetric PCR for DNA Sequencing PCR products can also be used as templates for direct DNA sequencing (ligation mediated PCR – LMPCR). Single stranded DNA can be produced for this purpose using asymmetric PCR, in which the two primers are used in 100:1 ratio, so that after 20–25 cycles of amplification, one primer is exhausted so that single stranded DNA is produced in the next 15–10 cycles. For more details about PCR assisted DNA sequencing, the readers are advised to consult the next chapter.

2.27. POLYMERASE CHAIN REACTION APPLICATION During the last few years, with the improvement of PCR protocols and due to the availability of automatic thermal cyclers commercially the applications of PCR have increased manifold. However, for application of PCR, we need a pair of primers which should be based on the knowledge of nucleotide sequence of DNA to be amplified. Therefore, non-availability of this information about the DNA segment or gene to be amplified, becomes a limitation in the applications of PCR, although this difficulty has been overcome for the study of DNA polymorphism, through the primers. Some of the applications of PCR technology of random DNA primer are described in this section.

Study of DNA Polymorphism Using PCR Using PCR, DNA polymorphism can be studied sequence at loci with (using primers based on known sequence) or at random site primers having unknown but random DNA sequences these are called random primers). In either case, DNA may be amplified from one or more genomic DNAs and subjected to digestion with one or more to electrophoresis and the restriction patterns may be photographed t reveal polymorphism. Sequences of prolamin gene and phytochrome gene have been used for the study of polymorphism for these genes in different species of Oryza (wild and cultivated rice). Similarly, storage protein genes in wheat and many genes in other animal and plant species have been specific restriction endonucleases. The digested DNA may be subjected used, for the study of DNA polymorphism. The non-availability of sequences of specific genes for designing primers for DNA amplification has been overcome by a variety of approaches including the following: (i) In case of multigene families like those for RNA, tRNA and heat shock proteins, there are conserved consensus sequence known,

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which are used for designing universal primers. Similarly for amplification of antibody genes also, universal primers have been used in PCR. (ii) Gene bank Sequence Data have also been used for designing primers for amplification of microsatellites like poly (A), poly (G), pol (TC) and poly (TG), etc. This is used for a study of length polymorphism in these microsatellites, also described as ‘short tandem repeats or simple sequence repeats SSR. (iii) The consensus sequences for intron exon junctions or intron splice junctions (ISJ) have also been users which have been used for amplification of intron or exon sequences to study DNA polymorphism. (iv) Conserved sequences at the ends of SINEs (short interspersed elements) and LINEs (long interspersed elements) have also been used for a study of polymorphism in the distribution and lengths of these repeat sequences. (v) In certain viruses, DNA polymorphism could be studied by designing degenerate primers on the basis of amino acid sequence of a protein using Ti for designing prime genetic code.

PCR and RAPD Markers The most important use of PCR for e study of DNA polymorphism, however, involves the use of random prime giving Random Amplified Polymorphic DNA. This technique is considered simpler and offers several advantages over more commonly used technique known as RFLP and RAPDs have actually been used for a study of polymorphism chromosome mapping in maize, soybean, mouse, humans, etc.

PCR and VNTR Loci Alec Jeffreys and his colleagues in U.K suggested the use of PCR for study of DNA polymorphism at VNTR (variable number tandem repeats, also known as ‘minisatellites loci in humans. Primers were developed for the conserved flanking regions of VNTR loci so that all available VNTR loci could be amplified. The PCR products differ according to the number of repeat units present in the different VNTR loci and this variation is visualized in the positions occupied by different bands after electrophoresis.

PCR and SSR Loci The VNTR loci discussed above consist of repeat electrophoresis units in the range of 11–60 base pairs in length. A much simpler type repeat units exist in the form of dinucleotides, trinucleotides or tetra nucleotides, e.g., (CA)n, (GT) n (AAT)n or (AGAT)n. These repeat units were described as microsatellites, but later also called short tandem repeats or simple sequence repeats (SSR). Some of these SSR loci, such as (CA)n or (GT) n occur in the human with

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n varying from 10 to 60. The DNA sequences flanking SSRs are conserved, allowing the selection of PCR primers that will amplify the intervening SSR in all genotypes of the target species PCR reaction includes a small amount of 3P labeled nucleotide (only one of the four nucleotides is labeled) or it may include primers, one or both of them being end labeled. This allows visualization of PCR products using autoradiography after electrophoresis on a standard sequencing gel. Variation in the length of products is a function of the number of SSR units. The principle underlying is detection polymorphism using two parents and their F1 hybrid. Markers resulting from SSR length polymorphisms are placed on genetic maps relation RFLP’s, RAPD’s, VNTR’s, other SSR’s and even the morphological markers. A comparison of very limited sequence data from plants (including algae) those of vertebrates (including humans) indicated that SSR’s plants are found with the same frequency as in vertebrates. Recently DNA sequence data for five tree species and Zea mays have been screened for the presence of (AC)n and (AG)n and the frequency in these six species was found to be close to one microsatellite (SSR) per 5 x 3 x 10 bp. Other plant and animals species have also been examined for the presence of a number of SSR’s and a wide occurrence of these markers in plants and animals preparing saturated was suggesting their utility in preparing saturated molecular maps. Molecular mapping using PCR Genetic and physical chromosome maps have also been prepared, both in plants and animals, using PCR. Once RAPD’s are detected, recombinant inbreeds (RI’s) or F2/backcross segregating populations, or doubled haploids (DH) derived from haploids (in plants) can be used to detect linkage and recombination frequencies. The molecular marker which cosegregate RI’s or F,’s or DH’s suggest linkage and the frequency of cosegregation among RIs or Fa/backcross plants will be a measure of the strength of linkage. The molecular markers identified through PCR, can also be assigned to specific chromosomes using aneuploids or alien addition lines, e.g., wheat-barley additions -21 pairs of wheat chromosomes +1 pair of a specific barley chromosome). A number of molecular markers have been mapped using PCR in maize, soybean, barley, mouse, humans, etc.

Sequence Tagged Microsatellite Sites (STMS) or SSR Loci It has also been examined and mapped using PCR. The gene sequence databases are screened for a microsatellite and the flanking sequences are used for designing primers for PCR. Several STMS or SSR loci using this technique have been mapped in mouse, where as many as 200 microsatellites have been detected in the genome database. Besides SSR’s or STMS’s, other markers including sequence tagged sites or STS’s (a short DNA sequence in a molecular genomic DNA marker) and expression sequence tags or ESTs (a short DNA

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sequence from a cDNA clone representing an expressed gene) have also been for genetic mapping. In both these cases, PCR can be profitably utilized for screening RI’s (mouse), somatic hybrid cell lines (humans) or F2 individuals (in plants). This will allow detection of linkage and calculation of recombination frequencies leading to the preparation of genetic maps. Complete molecular maps of several human chromosomes could be prepared using STS’s and PCR.

Gene Tagging Using PCR PCR has also been utilized for developing molecular m to specific genes of economic importance. For instance, in tomato 144 random primers were developed to produce 625 PCR products from a set at markers closely link instance in tomato, near isogenic lines (NIL’s, which differ only for the presence and absence of a specific gene) for Pseudomonas resistance. At least three of PCR products were present in one and not in the other of a pair NIR’s showing linkage with Pseudomonas resistance gene (Pio). The PCR products could be mapped close to Pro. Such tagging of gen through PCR will be used in future for plant breeding (selection desirable plants in segregating populations) and also for isolation of genes.

PCR for Confirming the Presence of Transferred Gene When a gene is transferred with a vector to cultured cells or primers can be designed to conduct PCR for amplification of the sequence so transferred. This technique allows the confirmation of transfer and maintenance of the gene of interest. In gene therapy experiments, the transfer and presence of a marker gene for neomycin resistance (NeoR) could be detected in the blood of patients, even after 60 days, although the frequency of marked lymphocytes gradually decreased necessitating a second transfusion of such marked cells. Similarly, ADA (adenosine deaminize deficiency) gene was transferred for therapeutic purposes and was used to detect the presence of this gene up to 6–5 months, s that the patient (4 year old girl) was then placed on a programme of maintenance infusions at 3–5 months intervals. Thus PCR proves to be immerse help in monitoring a gene in genetic engineering or gene therapy experiments.

Human Genetics Using PCR PCR has also found extensive use in human genetics. Some of these uses include the following:

Prenatal Diagnosis Using PCR Prenatal diagnosis of sickle cell anemia with enhanced sensitivity was perhaps the

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first application of PCR. Using PCR, this is done in less than one day in contrast to several weeks needed, when Southern blots are used for hybridization with a probe. The test has also been used for diagnosis of phenylkeptonuria (PKU), β thalassemia, hemophilia, etc. The PCR product in all these cases is examined using a labeled probe, to suggest whether or not mutant sequence causing the disease times, RFLP pattern of PCR products in healthy and defective feti singular-fetus) differ, thus enabling prenatal diagnosis. In still other cases PCR product may be sequenced to reveal the difference (for sequencing is found or n with PCR, consult next chapter).

Recombination Data Using PCR PCR can be used for DNA amplification utilizing individual sperms, which are the products of meiosis and recombination. Therefore, by studying PCR products from a large number of individual sperms one can calculate the proportion of sperms that are recommbinant for linked markers. This allows construction of genetic maps a resolution which is not possible with pedigree analysis. Microdissected chromosomes bands are also used for PCR to allow physical location of recombination at genetic lesions.

Sexing of Embryos Using PCR Since DNA sequences single cells can studied using PCR, sex of human or livestock embryos, fertilized in-vitro, can be determined before implantation. For this purpose, PCR primers and probes specific for sex chromosomes (e.g., ZFY or ZFX sequences) can be used. Y-specific primers and probes humans are available for this purpose. This technique can also be used to detect sex-linked disorders in the fertilized embryos.

2.28. DNA FINGERPRINTING DNA fingerprinting is more successfully used in forensic science to search out criminals, rapists, solving disputed parentage and uniting the lost children to their parents or relatives by confirming their identity. This is done through making link between the DNA recovered from samples of blood, semen, hairs, etc. at the spot of crime and the DNA od the suspected individuals or between child and his/her parents/relatives. We will discuss briefly the use of DNA fingerprinting in forensic medicine for identification of criminals and disputed parentage. Some well characterized sequences of microsatellites being used for designing primers, so that DNA fingerprinting may be achieved through PCR. PCR allows amplification of DNA from individual hairs, stains of blood or

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seminal fluid having partially degraded DNA, which could not be used earlier for characterization of individuals.

3 Recombinant DNA Technology and Tools of Genetic Engineering

3.1. CHIMERIC DNA In this chapter, we will describe about different type of restriction enzyme used regularly as a routine in all DNA cloning and recombinant DNA enzymes.

3.2. RESTRICTION ENZYMES FOR CLONING For cloning of DNA, often we need to cut DNA at specific sites, recognized and cleaved by specific enzymes. Also by locating the positions of cleavage site of a number of restriction enzymes, restriction maps can be prepared. These restriction enzymes recognize short sequences of double stranded DNA as targets for cleavage. Different enzymes recognize different but specific sequences, each ranging in length from 4 to 8 base pairs. The enzymes are named by a three letter than which are the identifies their origin. Roman numerals are added to distinguish several enzymes with same origin. For instance, EcoRI is derived from E. coli and HpaI is derived from Haemophilus parainfluenzae. Besides cleavage, modification in the form of methylation is also brought about by some enzymes called modification enzymes. This methylation distinguishes genes in different states of functioning. There are also some enzymes which performs the functions of both restriction and modification. Based on the attributes, restriction enzymes have been grouped into two classes: (i) Type II restriction enzymes systems which have separate enzymes for modification and restriction; and (ii) type I and type III enzymes systems, in which same enzyme possesses both activites, although the restriction and modification sites differ in position. The above two classes of restriction enzymes, type II enzymes are most important for cloning purposes. The target

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sites for these enzymes are 4–8 bp with symmetry. Most of type II restriction enzymes cleave DNA at unmethylated target site. Some enzymes introduce staggered cuts, others generate blunt ends. Enzymes with 4 bp target sites are used when frequent cuts are desired and those with 8 bp are used when rare cuts are desired to get long DNA segments. Otherwise majority of enzymes used have 6 bp target sites. Some of them can cleave both methylated as well as unmethylated targets, but majority of them cleave only unmethylated targets.

3.3. TECHNIQUE OF RESTRICTION MAPPING The recombination frequencies, which are the function of distances between genes, are not completely independent of (i) the nature of mutants used; (ii) the position of these mutants on chromosomes; (iii) the genetic background; and (iv) the environmental conditions and a variety of other factors. Consequently, the distances between genes on a genetic map may not correspond to the distances between them on the DNA molecule of which they are a part at the molecular level. Gaps may also be present on a genetic map due to non-availability of mutants in that region. At the molecular level, the fine structure of a gene can be studied through determination of nucleotide sequence of the concerned DNA segment Sometimes this is done through isolation of DNA segment corresponding to a gene followed by sequencing of the DNA segment, which needs time and energy. Instead we can prepare a map of the DNA by its cleavage at specific sites with the help of restriction endonucleases, which recognize very short specific DNA sequences and cleave the DNA at these specific sites. These sites of cleavage can be identified and mapped to give rise to a restriction map. In a restriction map, the nucleotide sequence of DNA between sites close to one another (300 bp or less) can be determined. These regions can then be connected into a sequence of the entire gone, whose nucleotide sequence, then can compared with amino acid sequence of protein coded by it. This can be further extended to adjoining genes to saturate the physical map, an objective which is difficult, but is being attempted for human genom. On a restriction map, is found a linear sequence of sites, each for a specific enzyme and the distances between them are measured in terms of number of base pairs of DNA. This technique can be used both in prokaryotes and eukaryotes, although, all regions of chromosomes cannot be easily mapped in eukaryotes. Different steps involved in restriction mapping include the following.

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3.4. RESTRICTION CLEAVAGE AND GEL ELECTROPHORESIS If we take a particular DNA molecule or DNA sample, digest it with a specific restriction enzyme and than subject to gel electrophoresis we will notice a series of bands on the gel slab or cylinder. The bands can be observed under ultravoilate light after staining with ethidium bromide. The position of different bands will depend on DNA fragment size, such that smaller the fragment, more rapidly it will move, and longer the segment more slowly it will move. It will mean that the fragment away from the loading site will be smaller and that close to the loading site will be longer DNA fragments. The gel can be calibrated by using a mixter of DNA fragments of known length so that the position of the bands on this standard gel can be compared with the bands in the experimental DNA digest and the fragment length in each band of DNA digest can thus be determined sometimes using a computer device. The results of digestion of 5000 bp long hypothetical DNA molecule digested separately by two enzymes A and B. As shown in the figure, the enzyme A cleaves the DNA into four fragments of lengths 2100, 1400, 1000 and 500 bp, while the enzyme B cleaves it into three fragments of length 2500, 1300, 1200 bp. These data with some additional experiments can be used to generate a restriction map.

3.5. CONSTRUCTION OF A RESTRICTION MAP The data on DNA digestion by more than one endonucleases as discussed above can be utilized to arrange the sites of cleavage in a defined order. This can be done by several methods. This method involves successive digests with two individual enzymes, where we digests with cither enzyme A or enzyme B and then cleave it with the other enzyme. The original DNA sample is also digested by a mixture of both the enzymes to confirm the results of individual successive digests. When the fragment A-2100 is obtained and digested with enzyme B, it is cut into two fragments of 1900 bp and 200 bp. Fragment B-2500 (obtained from individual digest with enzyme B) is similarly cut by enzyme A into two fragments of 1900 bp and 600 bp, suggesting overlap of A-2100 and B-2500 in the region of fragment of 1900 bp which is obtained by one cut with enzyme A and the other by enzyme B. The DNA fragment of 1900 bp is also available, when DNA is digested with a mixture of both enzymes A and B (double digest). Using this information, we the overlapping regions in A and B digests and find out the sites of cleavage by A and B. This will then allow us to prepare the restriction map.

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3.6. USE PARTIAL DIGESTS, END LABELING HYBRIDIZATION IN RESTRICTION MAPPING The above technique of individual and double digests can be supplemented into with other techniques for actual construction of maps. For instance, by permitting incomplete digestion, fragments longer than those obtained by complete digestion may be obtained. These will be called partial digests. In the example used in the last section, in partial digests with enzyme A, instead of getting A-1000, A-2100, A-1400, A-500, we may get A-3100 A-1400 and A-500, which will suggest that A-1000 and A-2100 lie in the adjacent regions. In another partial digest if we get A-1000, and A-500, this will suggest that A-2100 and A-1400 are adjoining, which will mean that A-1000 and A-1400 are found on the two sides of A-2100s. This technique will thus allow the arrangement of fragments in a linear order. Another useful technique involves labeling of the ends (end labeling) of DNA ends can be identified due to labeling, even alter digestion. In the a example, using end labeling, if A-1000 and A-500 are found to be radio-actively labeled, these will be present at the two ends of the restriction map. Some of the features of a restriction map can also be confirmed by nucleic acid hybridization. A molecule before digestion, so that the fragments containing these 2100 and B-2500 have an overlapping region of 1900bp, they should hybridize with each other, which will confirm overlapping. The above technique will help in accurate completion of a restriction map, but will require that we have a complete set of restriction fragments which make the entire DNA region being mapped. Once the restriction map (which is a physical map) is ready, it can be compared with the genetic map. Although large changes, already located on the genetic map can be easily located on the restriction map, but point mutations cannot be always and easily located on the restriction map, since the restriction sites often do not change due to mutation. In such a situation, one may like to determine the nucleotide sequences on individual fragments and compare them in normal and mutant individuals. Since sequencing of nucleotides is more laborious and cannot be easily undertaken for the whole genome, another technique of molecular genetic markers in the form of restriction fragment length polymorphisms (RFLPs) has been utilized during the 1980’s to generate genetic linkage maps in human, mouse, rat, pig, sheep, fruitfly, (Drosophila), nematode (Coenorhabditis elegans), yeast, maize, wheat, rice, barley, tomato, potato, lettuce, rice, pepper, pea, etc.

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3.7. CONSTRUCTION OF CHIMERIC DNA DNA molecules are produced by inserting a foreign DNA segment into the DNA molecule of a vector. These chimeric DNA molecules are the means of cloning genes or other small DNA sequences. The construction of chimeric DNA, as we know now, involves joining of the broken ends of the vector with the two ends of the sequence to be cloned. There at least three methods available for the construction of this chimeric DNA by palindromes and staggered cleavage. This is the most common meth palindromic sequence, a restriction enzyme causes staggered cuts producing short complementary single stranded stickily ends (a palindromic sequence is one, which has complementary sequences at the two ends of a single strand, e.g., AT.GAT). This can be illustrated using the example of enzyme EcoRI, which cuts the sequences at specific positions. When another DNA molecule is similarly cut by the same order enzyme, similar sticky ends having same sequences in the single stranded ends will be produced, so that when it is mixed with the previous molecule similarly treated, the two will anneal producing a chimeric DNA or chi plasmid if plasmid DNA is involved. Enzyme DNA ligase helps in joining the bonds at the cut ends of two molecule The above technique has the advantage of regenerating two restriction sites (e.g., EcoRI site) in the chimeric DNA, so that the foreign DNA segment can be retrieved rather easily from the cloned copies of chimeric DNA by cleavage again with the same enzyme. There are also following disadvantages with this technique. •

•

The two cleaved ends of a vector or of a foreign DNA may join end to end before getting inserted. Therefore while isolating chimeric DNA, one will have to select chimeric molecules each having only a single insert. This can be achieved by separating molecules of different sizes by gel electrophoresis. The recognition site, particularly in the sequence to be cloned may not lie at a convenient position, so that sometimes only a part of the desired segment will be inserted.

3.8. ADDING POLY DA AT THE 3’ ENDS OF THE VECTOR AND POLY DT AT THE 3’ ENDS OF THE DNA CLONE In this method, DNA can be cut at the desired position both in the vector and in the clone, without staggered cleavage. Using precursor dATP, poly dA is added at both the 3’ cut ends of the vector with the help of enzyme terminal

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transferase. Similarly, using precursor dTTP, poly dT is added at both the 3’ cut ends of the DNA sequence to bc cloncd. The vector and clone can then be joined by annealing the poly dA with poly dT tails and then ligating them using DNA ligase enzyme. In this technique, there is no chance of reannealing between the two cut ends of the same DNA molecule or of the two similar DNA molecules so that one of the disadvantages of the first method has been overcome in this case. However, in this technique it will not be easy to retrieve the cloned DNA, because the recognition site of the enzyme duc to insertion of poly dA and poly dT has been lost in this case. But if poly dG and poly dC are used instead (which serve the same purpose), cloned sequence can be retrieved, since poly dG: poly dC regenerate the recognition for the enzyme Pstl.

3.9. BLUNT END LIGATION BY T4 DNA LIGASE In this technique, a restriction enzyme is used to cut a duplex DNA at the same place in both the DNA strands. The broken ends are then used to joining with the two ends of another DNA molecule irrespective the sequences present at the broken ends of the two DNA molecules. The T4 DNA ligase is used for this joining reaction. The disadvantage of this technique is that any two broken ends may join including those belonging to the same DNA molecule. This leads to the production of a variety of products and one will have to select the desired product from a mixture of products. This blunt end ligation is used for developing a method in which the DNA to be cloned can be easily retrieved whenever required. This method makes use of short DNA duplexes (linkers) that contain EcoRI palindrome or some equivalent palindrome, which being small in size, can be synthesized chemically. These linkers can be linked to the blunt ends of vector DNA of an insert by blunt end ligation. This will allow the creation an EcoRI site in the linker region of the vector, so that when a DNA segment is now cloned, the inserted DNA segment can be retrieved by with EcoRI. However, in this method there is no restriction on the original choice of sites to generate the cut ends. Therefore, with this method it is now possible to insert a foreign DNA segment at a particular site in the linker region of the vector and then retrieve this foreign segment whenever necessary.

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3.10. DNA CLONING IN BACTERIA AND EUKARYOTES 3.11. CLONING IN BACTERIA Bacteria are often used as hosts for multiplication of chimeric vectors or recombinant DNA molecules. Therefore, a bacterial strain should be available, which should be the host for the vector and should be modified or selected for high transformation efficiency. Bacteria can be classified into. • •

gram-negative bacteria, e.g., Escherichia coli. Pseudomonas, Rhizobium and gram-positive bacteria, e.g., Bacillus subtilis and Streptomycetes sp. Ordinarily same plasmid cannot maintain itself in stable form in both classes of bacteria and, therefore, plasmid vectors for two classes usually differ. While gram-negative bacteria can be used for plasmids, phages and cosmids vectors, gram-positive bacteria are used as hosts for only plasmids. Chimeric vector is inserted bacterial cells and transformed bacterial colonies are selected and for multiplication in suspension cultures. The chimeric DNA is retrieved whenever required

3.12. CLONING IN EUKARYOTES Since chromosomes found in the nucleus of eukaryotes are separated from the rest of cell through nuclear membrane, and since many of the gene are split genes with exons and introns, genetic engineering with eukaryotes requires new methods and tools. When eukaryotic genes are cloned prokaryotes, the split genes cannot be correctly expressed, because prokaryotes do not have the equipment necessary for splicing out the RNA transcribed from the introns of a gene. In view of this, eukaryotic cells may sometimes be needed for cloning and particularly for expression of cloned eukaryotic genes. Among eukaryotes, DNA cloning has been done in yeast, mouse and to some extent even in some higher plant species. In yeast, a plasmid called 2u DNA (63 bp) is found, which is an appropriate cloning vehicle. An efficient transformation method is also available, which involves protoplast production followed by PEG directed introduction of DNA into protoplast. In animal cells like mouse cells, special animal viruses were used as cloning vehicles. Simian

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virus 40 (SV40) is one such virus, in which globin gene could be integrated. This gene integrated in SV40 could be transcribed and translated in mouse kidney cells. A variety of human cell lines in culture are also used for cloning of foreign DNA, using some animal viruses as vectors.

3.13. MOLECULAR PROBES Molecular probes are small DNA or RNA segments that complementary sequences in DNA or RNA molecules and thus allow identification and isolation of these specific DNA sequences from an organism. Antibodies are also occasionally used as probes to recognize specific protein sequences. Although, initially, these probes were developed and used for genetic engineering research but arc now frequently used for a variety of purposes including diagnosis of infectious diseases, identification of food contaminants, variety of microbiological tests, forensic (e.g., fingerprinting of murderers or rapists, etc. Probes can also be used to identify strains of an organism, e.g., varieties of a crop species (plant breeder likes to have a quick test to identify his variety to maintain his patent or breeder’s right, so that a competitor may not use it in another name leading to infringement of rights). For basic studies in molecular biology laboratories, the molecular probes are frequently used for identification and isolation of genes or related sequences. In theory, any nucleic acid (or rarely protein, e.g., antibody) can be used as probe, provided it can be labeled to permit identification and quantitation of the hybrid molecules formed between the probe and the sequence to be identified. In practice, double and single stranded DNAs, mRNA and other RNA’s synthesized in vitro are all used s probes. DNA/RNA probe assays arc faster and sensitive, so that many conventional diagnostic tests for viruses and bacteria involving culturing of the organism, are being fast replaced by antibody and DNA probe assays. While culture tests can take days or even months, molecular probe assays can be performed within few hours or minutes. Therefore, the use of DNA probes has become today’s most sophisticated and sensitive technology for a variety of uses involving biological systems both in basic and applied studies including their commercial use.

3.14. PREPARATION OF PROBES 3.15. GENOMIC DNA PROBES Simple, reproducible methods have been devised for the preparation of molecular probes. A random or specific DNA sequence (probe) can be isolated

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from the genomic DNA (DNA extracted from cell nuclei), using following steps extract DNA from plant or animal tissue.

Digest extracted DNA with a restriction enzyme (e.g., EcoRI or HindIII), which cuts DNA on specific sites or positions where a specific sequence recognized by the enzyme is found; (ii) Run the digested DNA on an agarose or polyacrylamide gel for electrophoresis to separate fragments of different sizes; (iii) Isolates DNA of specific fragment size from a particular band identified through Southern blots by hybridization with specific labeled mRNA or c DNA molecules, if available. If random probes are needed, no hybridization of Southern blots is required (Southern blots are DNA bands transferred from gel to nitrocellulose membrane with the help of a buffer rising through capillary action; s isolate DNA of specific. (iv) Clone this DNA in a vector allow chimeric vector to infect bacteria for multiplication, where it can make billions of copies. The steps (ii) to (vi) above are also used for preparing cDNA probes. From the transformed bacteria, the chimeric vector can be obtained and used in one of the following ways: (i) It may directly as probe (the presence of vector DNA will not interfere, so that it is not really necessary to remove it). (ii) The segment may be separated (or retrieved), by using the same enzyme which was used for cloning. In the latter case, cleaved chimeric vector DNA will be again electrophoresed for separating the inserted segment on the gel. This inserted segment thus retrieved can now be used a probe. (iii) The chimeric DNA may be used for PCR, using flanking sequences as primers; the PCR probe) product can be separated and used as a probe. (i)

3.16 CDNA PROBES A DNA sequence corresponding to a part of as gene(s) (an oligonucleotide) can be obtained by reverse transcription of mRNA. cDNA thus obtained can be cloned and used as a probe.

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3.17. SYNTHETIC OLIGONUCLEOTIDES AS PROBES DNA probes with nucleotide sequence can also be synthesized chemically using automated DNA synthesizers now available commercially. These synthetic probes will be efficient only when they are not more than 20 to 40 nucleotides in length.

3.18. RNA PROBES OR RIBOPROBES High specific activity RNA probes or expression vector. This is achieved through RNA synthesized in vitro (on DNA inserted in vector) and labeled simultaneously employing specific RNA polymerase and labeled nucleotides which are supplied b commercial firms in a kit for riboprobe preparation. RNA probes off several advantages over DNA probes, since these are single stranded, and provide improved signal or hybridization blots. However, widespread presence of ribonucleases creates some problems in their preparation and riboprobes may also be synthesized from DNA templates cloned in a use, because these ribonucleases will cause degradation of RNA which is de synthesized. Therefore, extreme care is needed in the preparation o of riboprobe, keeping all glassware ribonuclease free (no glassware should be touched with bare hands, because ribonucleases may be available on or skin).

3.19. LABELING OF PROBES The detection of homologous sequences after hybridization with the probe is like finding a needle in the haystack. Therefore, for the success of DNA probe assay, it is necessary to develop simple, safe and sensitive techniques for their use. As probes transmit no signal of their own, they have to (b) either labeled with radioactive isotopes or (i) methods are used t couple nonradioactive signal molecules to the probes Without impairing the hybridization ability of these probes. These signal molecules may include fluorescent antibodies and enzymes that produce color changes in yes off and chemiluminescent catalysts.

3.20. RADIOLABELED PROBES Traditionally radioactively labeled probes are used for a variety of experiments.

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p is the most commonly used radioisotope, although other labels such as H, 35S and 1251 have also been used. Conventional labeling replaces a proportion of the nucleotides in a nucleic acid sequences, with 32P derivatives (e.g., 32 PUCTP) or adds 32P to be end of the molecule. At least three methods are available for labeling of the probe (i) nick translation involves creation of nicks in the probe DNA followed by extension of the broken ends using a labeled deoxyribonucleotide with help of DNA polymerase I (or know fragment of this enzyme); (ii) oligonucleotide labeling involves the use of short random oligonucleotides, which are used as primers for copying the probe in presence of labeled deoxyribonucleotides; (iii) riboprobe preparation this involves synthesis of labeled RNA, using DNA probe as template, in presence of a labeled ribonucleotide. After hybridization with labeled probe, hybrids are detected by autoradiography. P32 has the advantage over other radioisotopes, since it has high specific activity. However, in general, radioisotopes, have some disadvantages. They are difficult to handle and expensive to dispose off. Detection by autoradiography, while sensitive, may take a long time if there arc few counts in the hybrid. Furthermore, radioisotopes have a short half life (halflife of 14.3 days) and therefore experiments should be completed preferably within one hall life.

Non-Radioactiveprobes (a) Biotin labeled probes Recent advances in nucleic acid technology now offer alternatives to radioactively labeled probes. One procedure that is becoming increasingly popular is biotin labeling of nucleic acids. This system exploits the affinity, which the glycoprotein ‘avidin’ has for biotin (vitamin H). Avidin is commonly found in egg white. Biotinylated probes are prepared through a nick translation derivatives. Alter hybridization and washing, detection of hybrids is done by a series of cytochemical reactions which finally give a blue color whose intensity is proportional to the amount of biotin in the hybrid. There are several advantages of using biotinylated probes. For example, these assays employ in advance in bulk and stored at –20ºC for repeated uses. Detection technology is that very small probes non-toxic materials, whose half-life is longer. These probes can be prepared hybrids is much faster than by radioactive probes. A limitation of this technology is that very small probes contain only a small number of biotinylated sites, limiting the intensity of signal obtained has been solved by adding long tails’ of biotinylated nucleotides to the probes through enzymatic methods. Sometime the probe does not need to be labeled with biotin, but only coupled with a tail. Another disadvantage f biotin labeled probes is that the

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cytochemical visualization reactions lead to precipitation of insoluble material which cannot be removed and therefore, the filter cannot be reused, while in radiolabeled probes, the filters can be used repeatedly for hybridization with a number of probe one at a time.

(b) Digaxigeain Labeled Probes Digoxigenin is another chemical derived from plants and used for nonradioactive labeling of probes. An antibody associated with an enzyme (antidigoxigenin-alkaline phosphatase conjugate) is used for the detection of the presence of digoxigenin. The probe may be labeled with digoxigenin 11 dUTP supplied with a digoxigenin kit (these kits are available fram any commercial firm, e.g., Boehringer Mannheim). The labeled and denatured probe may be used for hybridization with denatured DSA on Southern blots or for in-situ hybridization. After hybridization the membrane or the slide may be washed and the membrane or the slide is transferred into detection buffer containing 20 rg/ml of antidigoxigenin fluorescein and 5 % (w/v) BSA (bovine serum albumin). The system is incubated for 1h at 37°C, and then the membrane/slide is washed in detection buffer three times (8 min each at 37ºC). Alkaline phosphatase activity can be detected using 0–17 mgml BCIP (5-bromo-4 loro-3 indolyl phosphate) and 0.33 mg/ml NBL (nitroblue tetrazolium) as dye substrate.

(c) Alternatives to biotin and digoxigenin labeling The techniques of non-radioisotopic label have been further expanded and new methods Am. In have DN that have heen devised for attaching other ligands (e.g., hapten determinant 2, 4-dinitrophenol, arsenate derivatives, etc.) to nucleotides without hampering their ability to be incorporated into DNA. These alternatives require binding of attached ligands to specific proteins that can b to monitor many probes simultaneously by using several different ligands since each ligand would yield a different. A chemiluminescent (light emitting) probe system has also been developed in which two different m R probes complementary to a continuous segment of DNA hybridize adjacent segments of a gene. The first label is a chemiluminescent complex that emits light at a specific wavelength; this emission excites the label Molecule on the second probe to emit light at a different wavelength which can be detected using a photomultiplier device. This process called nonradioactive energy transfer, can occur only if the two probes hybridize correctly and the two labels are close to each other. This system has great fidelity and provides basic technology for a homogeneous assay. In addition, DNA does not need to be immobilized and no washing steps are necessary which may be an additional advantage for large scale testing.

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3.21. AMPLIFICATION OF DNA PROBE SIGNALS In recent years, DNA probe assays and immunoassays (using antibodies) have been competing with each other for a variety of purposes. While DNA probes have an advantage of detecting the presence of gene rather than its product, the immunoassay has the advantage of getting amplified target (the gene product) firstly due to transcription (several mRNA molecules can be synthesized on single copy of gene) and secondly due to translation (several protein molecules can be produced from same mRNA molecule). The amplified target gives an amplified signal. Therefore, if DNA probes have to compete with monoclonal antibodies for immunoassay, DNA probe signals need to be amplified. Following devices have been used for the signal amplification: •

•

•

•

Probes have been developed for TRNA, since rRNA genes are present in thousands thus amplifying the target (as done by companies like Gene-Probe and Gene-Trak systems in USA). Polymerase chain reaction (PCR) technology (which utilizes a thermostable polymerase) is used to generate millions of copies of target DNA sequence, which can then be identified by standard methods. Signal generating capacity of the probe can also be increased by any one of the following methods: (i) concentrating more label at the site of target molecule by attaching multiple enzyme molecules to each of its DNA probe or (i) by using multiple probes, or (ii) by attaching details consult next Chapter) Amplification probe technology uses multiple secondary probes (each having one or more enzyme molecules) that hybridize to multiple target specific primary probes. The secondary probe is independent of target but helps in amplifying the signal. These approaches have been multiple enzymes to each of the multiple probes. Described as Christmas Tree or Christmas Forest approaches. The above amplification systems will play a significant role in the u of DNA probes in future for a variety of purposes.

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3.22. TECHNIQUES USED IN MOLECULAR PROBING 3.23. SEPARATION OF DNA FRAGMENTS USING AGAROSE OR POLYACRYLAMIDE GEL ELECTROPHORESIS When genomic DNA, extracted from any tissue of a plant or animal species, is digested with a restriction enzyme, it is cleaved into segments. The segments of different sizes can be separated through gel electrophoresis before a molecular probe is used to detect the segments which have sequences similar to those in the probe. Gel electrophoresis involves movement of fragments or molecules under a high voltage electric current. The mixture of DNA fragments is loaded in a well created on one edge of the gel. The gel may be a cylinder or a slab (usually a slab for cloning expt.), about 10 cm long and 0–5 cm thick. The rate of movement of fragments is inversely correlated with the size of fragments molecules, so that heavier fragments will remain closer to the site of loading and the lighter fragments will move away. Fragments of different sizes will appear as bands on the gel and can be examined or isolated for further study. More often agarose gels are used, but for separation of fragments differing by few base pairs, polyacrylamide gels are used. Polyacrylamide gels are more commonly used for DNA sequencing experiments.

3.24. SEPARATION OF LARGE DNA MOLECULE MOLECULES USING (PFGE) The technique of gel electrophoresis is used for separation of DNA molecules of different sizes. However, DNA molecules of large size not be handled in this technique. In recent years, a new technique called Separation of large DNA molecules (whole chromosomes) using PFGE The technique of gel electrophoresis is used for separation of DNA pulsed field gel electrophoresis (PFGE) has been used, for separation large sized DNA molecules, sometimes representing whole chromosomes. Using this technique, separation of DNA molecules belonging to each of individual chromosomes of yeast has become possible technique, short pulses of electricity are used in two different directions and DNA is embedded and used

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in the form of agarose plugs to avoid fragmentation of large DNA molecules. Using the technique: PFGE and a more refined technique CHEFE (contour clamped homogeneous electric field electrophoresis), genome of several fungi could be resolved into chromosomal bands and used for mapping of DNA In this sequences on specific chromosomes.

3.25. SOUTHERN, NORTHERN, AND WESTERN BLOTTING A mixture of DNA, RNA or protein fragments can be separated by gel electrophoresis and the separated bands can be stained and visualized directly in the gel. “However, to confirm the identity of these bands or to find similarity of one or more of these bands with a known and available molecular probe, it is possible to hybridize these bands with a labeled probe. To facilitate this hybridization, the bands are often transferred to a nitrocellulose technique described as blotting. When called Southern blotting (after the name of E.M. Southern); when RNA bands are thus transferred it is described by the jargon term Northern blotting and similarly when protein bands are transferred, the technique is described as Western blotting.”

(a) Southern blotting For Southern blotting, DNA sample is first digested with a restriction enzyme and digested sample is gel electrophoresed. The DNA bands in the gel are denatured into single strands with the help of an alkali solution. “Subsequently, the gel is laid on top of a buffer saturated filter paper, placed on a solid support (e.g., glass plate), with its two edges immersed in the buffer. A sheet of and a stack of many A weight of about 0.5 nitrocellulose membrane is placed on top of the gel papers (paper towels) on top of this membrane is placed on top of paper towels. The buffer solution is drawn up by filter paper wick, and passes through the gel to the nitrocellulose membrane and finally to the paper towels. While passing through the gel, the buffer carries with it single stranded DNA, which binds on to the nitrocellulose when the buffer passes through it to the paper towels. After leaving this arrangement for a few hours or overnight, paper towels are and discarded. The nitrocellulose membrane with single stranded eA bands blotted on to it, is baked at 80°C for 2.3 hours to fix the DNA removed a his hybridization, brane through a us blotted, it is m); when RNA term Northern the technique DNA permanently on the membrane. This membrane now has t DNA bands from agarose gel, and can be used for hybridization with radioactively labeled DNA or RNA probe. The membrane may then be washed to remove any

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unbound DNA and X-ray film is exposed to the hybridized membrane to get autoradiographs.” The above steps involved in a replica. Sample is first ample is gel into single the gel is laid support (e.g., r. A sheet of stack of many of about 0.5 s drawn up by membrane gel, the buffer

(b) Northern blotting Initially the technique of Southern blotting used for DNA transfer from gel to the membrane could not be used for blot-transfer of RNA. Instead mRNA bands from the gel were blot-transferred into a chemically reactive paper, prepared by diazotization of aminobenzyloxymethyl paper. The technique being related to Southern blotting was called Northern blotting (not after name of any scientist as in Southern blotting). Later, it was shown that mRNA bands can be blotted firstly onto nitrocellulose membrane, a technique which has been widely adopted. The mRNA bands blótted onto nitrocellulose membrane can be hybridized with a labeled DNA or RNA probe The single stranded regions of the probe are removed by nuclease (mungbean nuclease or $–1 nuclease), so that quantitative estimation of hybridized mRNA can also be made.

(c) Western blotting This technique is used to detect proteins of a particular specificity. Particularly when a transferred gene expresses in transformed cells, the translated product in the form of protein can be identified by this technique. The extracted proteins are subjected to polyacrylamide gel electrophoresis (PGE) and are then transferred onto nitrocellulose, to which they bind. Nitrocellulose membrane is then used for probing with a specific labeled antibody (antibody will not hybridize 125 with protein, but bind with it). The antibody may be labeled with and the signal is detected again with autoradiography. If radioactive label is not used, bound antibody may be detected by a second antibody tagged with an enzyme.

3.26. DOTS AND SLOT BLOTS Another variety of Southern or Northern is dot blots or slot blots. In dot blots, cloned or pure extracted DNAs to be tested are spotted adjacent to each other on a nitrocellulose membrane. DNA blots thus produced arc immobilized and denatured so that they are bound on the membrane as single stranded DNA blots. “The membrane is then hybridized with radioactively labeled probe (DNA, or RNA). The dot representing sequence related to probe will light up autoradiography. The intensity of the dot will indicate the relative concentration of a sequences related to the probe in the DNA sample used for a particular dot.

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No steps involving digestion with enzyme concentration of a sequence related to the probe in the DNA electrophoresis or transfer from gel to membrane are needed in this technique.” Therefore, it is considered much more convenient, when o restriction sites are needed to be studied. Sometimes, an apparatus is used for placing spots on the membrane through slots made in this equipment. The spots made thus are in form of oblong slots rather than round blots. These slots are used just like dot blots and are described as slot blots.

3.27. APPLICATIONS OF MOLECULAR PROBES Molecular probes can be used for both basic and applied studies in field of molecular biology and biotechnology. These uses will be discussed in detail in the different subsequent chapters of this book at appropriate but will be described briefly in this section. Use of molecular probes in restriction fragment length polymorphisms RFLPs) and related analyzes.

(a) What are RFLPs When genomic DNAs from each of several individuals belonging to one or more species are digested separately with restriction enzymes, electrophoresed, blotted a membrane, and probed with a labeled DNA clone, polymorphism the hybridization pattern is sometimes revealed and attributed to sequence differences between the individuals. Such variation has been termed as restriction fragment length polymorphism” (RFLP). Variation m one DNA fragment obtained with a specific enzyme is treated as RFLP.

(b) Detection of RFLPs A RFLP can be demonstrated using following steps: (i) Extract and purify DNA from several individuals which may differ in some respects among themselves. (ii) Digest with a restriction endonuclease. (iii) Subject each “DNA digest to gel electrophoresis separately, but on the same gel slab. (iv) The DNA fragments of different lengths resulting from digestion can be separated by gel electrophoresis, but the differences in the distribution patterns of fragments in several DNA samples due to digestion by several endonucleases cannot be detected directly. This is because the number of fragments is large and the range in size is rather continuous, such that they form a continuous smear on the gel. Electrophoresis is, therefore, followed b the following steps. (v) Fragments are transferred from

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gel to nitrocellulose filters using the technique of Southern blotting. (vi) The filters with small fragments, fully or partially homologous to the probe, can be detected b autoradiography after hybridization. Since repetitive DNA may hybridize with many fragments, they will give a smear on autoradiography. Therefore, unique DNA sequences are generally used as probes for detecting RFLPs. The DNA fragments hybridizing with the probe, can be visualized by autoradiography, and will be called RFLPs” at the phenotypic level. In results of a hypothetical experiment are shown where RFLPs are detected by using four plants (which differ by point mutation as well as by insertions) and three enzymes individually and in combination.

(c) RFLPs for evolutionary studies The restriction fragment length polymorphisms (RFLPs) can be studied in a set of related species, using a random or a specific DNA probe. The similarities and differences can be used to infer phylogenetic relationships. This has actually been done in a number of cases, both in plants and in animals.

d) RFLP maps and linkage of RFLPs with specific genes RFLPs have been used to prepare chromosome maps in humans, mice, fruitful and in Mendelian markers for genetic mapping is sometimes limited due to nonavailability of mutants. The list of markers can be extended by using molecular markers, which are examined in the form of RFLPs. plants including maize, tomato, lettuce and rice. The use of Mendelian marker for genetic mapping is sometimes limited due to non-availability of mutants. The list of markers can be extended by using molecular markers, which may be examined in the form of RFLP. “Once a large number of RFLPs are available in a species, the parents, F, and F2’s can be used to study their inheritance and linkage relationship and genetic linkage maps can be prepared. They have been assigned to specific chromosomes using monosomics in maize and efforts are being made to relate them with morphological and economic traits so that they can be used for practical plant breeding. A difference in restriction maps between two individuals or a RFLP can be used as a genetic marker in much the same way as any other phenotypic marker. The polymorphism at the molecular level may be due to the presence or absence of a restriction site. Shows how the presence of an additional site will produce two fragments and its absence will produce only one fragment. Although, there may be linkage between a molecular marker and a phenotypic trait, the change in molecular map (restriction map) may not affect the phenotype. In order to

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study linkage relation between a molecular marker and present in the DNA shown parents that differ morphologically as well as for molecular markers By we may examine the restriction fragment patterns and the phenotypes of making crosses between two such parents, we can estimate the recombination frequencies between molecular markers and phenotypic markers using” a test cross. In, we used a hypothetical example showing linkage of red and white eyes with different molecular markers in Drosophila. “It can be seen that as a result of recombination, in test cross, 15 % progeny shows association of red eye with a molecular marker associated the white eye in the parent, and vice versa. Therefore, it is concluded that restriction marker is 15 map units away from the marker eye color although it has no causal relation with eye color. Such a linkage between molecular marker and phenotypic marker allows identification of genetic loci at the molecular level. A tight linkage of such a molecular n with disease resistance may also be recommended as a tool for identification of disease resistant cultivars in many of our crop plants and will be increasingly utilized in future Molecular markers are also being used for selection of plants at the seedling stage for plant breeding purposes.”

Use of molecular probes in molecular cytogenetics (a) Isolation of gene or related sequence This can be facilitated through the use of a probe carrying a known sequence. For this purpose sequences may be hybridized following any one of the following techniques: (i)

(ii)

(iii)

Southern blots can be prepared (from genomic DNA or cDNA) to isolate DNA corresponding to a band that hybridizes with the probe. This DNA can then be used for cloning experiment to get a partial genomic library or cDNA library (see later in this chapter for details); The genomic or cDNA library can be screened through colony hybridization or plaque hybridization to identify and isolate the clone carrying the desired sequence;, sometimes the step (ii) is not used for complete genomic/cDNA libraries can be prepared and used in step The identified clone should carry the gene or sequence of interest, that can be multiplied and the sequence or gene retrieved according to standard procedure.

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(b) In situ hybridization (ISH) for location of sequences chromosomes A molecular probe can also be used for in situ hybridization “where chromosomes in miotic or meiotic preparation are used for hybridization with the probe (without extracting DNA). In a number of cases, rye chromosomes in wheat background have been identified using this technique. Sat-chromosomes with NORs (nucleolar organizing regions) are recognized using probes for ribosomal DNA as done in many In wheat and can be identified using a D genome can be identified using a D genome specific probe. A large (wheat, barley, etc.). Telomeres have been identified in human eukaryotes using eukaryotes using a telomeric sequence as a molecular probe, showing of all chromosomes carry the same sequence. In D genome telom related species, chromosomes of genome specific probe (pASI separated from Aegilops be cited where the use of molecular probe u hybridization served a very useful purpose.

Use of molecular probes for human health care DNA probe are being extensively utilized for diagnosis of diseases caused by parasitic Protozoa and Helminths. They are also used for antenatal diagnosis of congcnital diseases to allow advice on abortion of fetus, if desired. Similarly probes have been designed for the diagnosis of a number of sexually transmitted diseases.

Use of molecular probes in DNA fingerprinting With the help of probes prepared from human minisatellite DNA dispersed throughout human genome, it has become possible to identify individual humans, sin the possibility of any two humans (except identical twins) showing same pattern of fingerprinting is remote.” This aspect is already playing an important role in identifying criminals like murderers and rapists. This subject will also receive a detailed treatment.

3.28. CONSTRUCTION AND SCREENING OF GENOMIC AND CDNA LIBRARIES In order to isolate one or more rclated genes from a genome, we like to prepare a mixture of clones each carrying DNA derived either from the genomic DNA or from cDNA (derived from the mRNA isolated from a specific metabolically active tissue of an organism). This mixture may contain thousands of clones, which when derived directly from the genomic DNA are collectively called a

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genomic library. Similarly when these clones are derived from cDNA, they are collectively called a cDNA library construction and use of these libraries will be discussed in this section.

Genomic library by shotgun experiment Cloning an entire genome in the form of a library of random genomic clones (without identifying them) is often called a shotgun experiment. “This experiment, genomic DNA is extracted, broken into fragments reasonable size by a restriction endonuclease and then inserted into a cloning vector to generate a population of chimeric vector molecules. A set of fragments cloned in this manner is called a genome library. Once such a library is available, then clones can be perpetuated indefinitely in a plasmid vector and retrieved whenever needed for a variety of poses, including identification and isolation of a gene, when a specific probe is available. Genomic libraries can be prepared by using a number of restriction endonucleases, one at a time, so that fragments of varying sizes having different places of the genome will be available. However this may to cuts at inconvenient places, including sites within a gene, so that is having complete genes will be difficult to obtain. In order to come this difficulty, we use the following strategy in the shotgun experiment: (i) We use restriction endonucleases, which have short (4 bp) recognition sequences, so that such a sequence may be frequently distributed. (ii) Conditions are used which give only partial digests, so that a particular restriction site is only occasionally cleaved, and long fragments without having any breaks on recognition sites available within a gene can be easily obtained. This technique of shotgun experiment leads to the construction of a random genomic library, in which all fragments have same fragment ends thus helping retrieval of a fragment from the vector with the help cuts of the same enzyme.” The number of fragments representing every sequence of the genome increases with genome size. For instance, for a probability level of 99 that all sequences are present in our library of a species, we may need 1,500 cloned fragments for E. coli, 4,600 for yeast, 48,000 for Drosophila melanogaster and 8,00,000 for a mammal like human being, Libraries reaching these desired limits have been prepared in all these cases.

3.29 CDNA LIBRARY FROM MRNAS Complementary DNA (cDNA) libraries can also be prepared by isolating.

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“As from tissues which are actively synthesizing proteins, like roots in plants, ovaries or reticulocytes in mammals, etc. The mRNA for copying it into cDNA through the use of reverse transcriptase. The mRNA and leaves is used for copying it into cDNA through the use of re cDNA molecule can be made double stranded, and cloned. However cDNA clones will differ from genomic clones in lacking the introns present in split genes, but the advantage of being capable to be expressed bacteria, which do not have the machinery to process HnRNA (obtained cDNA clones from split genes) mRNA.”

3.30. COLONY (PLAQUE) HYBRIDIZATION FOR SCREENING OF LIBRARIES Once a genomic library or cDNA library is available, we may like to it for isolation of a gene sequence. This can be achieved by colony hybridization technique. In this technique, bacterial transformmed chimeric vectors are grown into colonies, which are lysed on nitrocellulose filters. Their DNA is denatured in situ and fixed on the filter, which is hybridized with a radioactively labeled probe carrying sequence related to the gene to be isolated (usually a cloned cDNA for screening of a genomic library). “Colonies carrying this sequence will be identified by dark spots after autoradiography, so that the original chimeric vector carrying the desired gene sequence can be recovered from one or more colonics in the original master plate and used for further experiments. This technique is described as colony hybridization. It is possible that probe may identify more than one clones or that a gene is fragmented the library. In such a case, one needs to reconstruct the desired sequence using several overlapping sequences available in the library. This is a very routine exercise whenever we like to isolate specific DNA sequences from the genome of a species, or from cDNA derived from mRNA of specific tissue of species. Sometimes the library may be available not in the form of bacteria is formed with chimeric DNA molecules, but in the form of chimeric particles carrying the cloned segments. In such a situation, a bacterial is infected with a mixture of chimeric phage particles (i.e., the library) and a large number of plaques develop overnight.” These plaques can be treated just like the colonies in colony hybridization to identify and isolate the chimeric phage particle carrying the gene of interest. This technique is then described as plaque hybridization.

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3.31. CHROMOSOME WALKING AND CHARACTERIZATION OF CHROMOSOME SEGMENTS When a probe is used for the identification of a gene sequence in a genomic library, the probe may hybridize with a number of clones, each carrying a part of a large gene fragmented during preparation of genome library. “If we obtained partial digests (by digesting the DNA only partially) from the genome, different genomes (from large number of cells) may give fragments which have overlapping sequences, because sites cleaved different genomes of the same organism, will differ being random. Since hone of these fragments may have entire sequence represented in the probe, overlapping, sequences may be used to construct the original genome scquence. Identification of fragments with overlapping sequences may be a key to the reconstruction or characterization of large chromosome regions. This is achieved by the technique popularly described as chromosome walking. The technique of chromosome walking involves the following steps: (i) from the genomic library select a clone of interest (identified by a probe) and subclone a small fragment from one end of the clone (there is a technique available to subclone a fragment form the end); (i) the subeloned fragment of the selected clone may be hybridized with other clones in the library and a second clone hybridizing with the subclone of the first clone is identified due to presence of overlapping region; (ii) the end of the second clone is then subcloned and used for hybridization with other clones to identify, a third clone having overlapping region with the subcloned end of the second clone; (iv) the third clone identified as above is also sub cloned and hybridized with clones in the same manner and the procedure may he continued; (v) restriction map of each selected clone may be prepared and compared to know the region of overlapping, so that identification of few overlapping restriction sites will amount to walking along the chromosome, or along a long in chromosome segment.” Regions of chromosomes approaching 1000 kb have been mapped following the above technique. Restriction maps of entire chromosomes can be prepared in this manner following the technique of chromosome walking.

3.32. REVERSE GENETICS AND CHROMOSOME JUMPING (OR HOPPING LIBRARIES) In most cases, we identify the genes by their protein products, which help in the isolation and cloning of a specific gene. However, for many human disorders

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which are each controlled by a single gene (e.g., Huntington disease, cystic fibrosis, Duchene muscular dystrophy, etc.), the gene product is not known despite intensive investigations. Such genes are cloned process often called identified primarily by its map position. Once the gene is cloned, it can be utilized for getting information about the encoded protein, the gene mutation and the metabolic defect that may be associated with the disease. Since the information about the is coming in the reverse order, this is popularly described his reverse genetics approach involves the following two steps: (i) locate the gene to a particular chromosome through RFLP linkage analysis; (ii) map the position of the gene with respect to molecular marker, both through recombinational analysis (in terms of recombination aluc in cM=centiMorgan units) and by physical mapping (in terms of kilobase pairs) with pulsed field gel electrophoresis (PFGE) and preparation of restriction map. Even if the distance is 1 cM, which is the limit of resolution, this will be equivalent to 1,000 kb. Therefore, if the segment carrying the gene is to be cloned using the linked molecular marker such a segment will be too large to be cloned. In order to bring the molecular marker close to the gene of interest, ‘chromosome jumping approach has been utilized. The technique of ‘chromosome jumping’ is based on the following steps depending upon the distance between the gene and the marker, decide about the distance of jumps’ or ‘hopsize’ (e.g., 100 kb or 200 kb): (i) genomic DNA molecules in the range of selected size (say 80 kb-130 kb in case of 100 kb ‘hopsize are 16)-240 kb for ‘hopsize’ of 200 kb) are selected through pulsed-field gel elcctrophoresis (ii) for circularization of DNA segments, ligation been two ends of each long linear DNA molecule was allowed using T4 ligase in the presence of supF+ (iv) DNA circles obtained; (ii) above are digested with EcoRI; (v) the vector iCh3A A lac, an amber mutated phage vector (supF” is also cut with EcoRI and used for cloning mal DNA fragments representing the junctions of the circularized genomic molecules and carrying sup F; (vi) the cloned DNA fragments and in(v) above repreśent the jumping library, which can be plated on the host and screened through the technique of plaque hybridization described earlier. The above technique of chromosome jumping will help narrowing the gene and available molecular markers. After several cycles the jumping followed by cloning the regions that are close to e to approach very close to the desired gene and clone it. It is thus obvious that in the field of ‘reverse genetics’ using technique of chromosome jumping, it will characterize genes, whose gene products are unknown. The gene for cystic fibrosis has recently been isolated using this technique. It could also be shown that ‘cystic fibrosis’ disease is caused by a single base substitution in this gene. This technique of ‘reverse genetics’ will become a very active area of research particularly in human genetics in the coming years. The term ‘reverse genetics’ has however been redefined recently.

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The classical genetics, sometimes described as forward genetics’ involves a study which starts from the phenotype, and follows through the identification of the gene and finally concludes with the isolation and sequencing of the gene. In view of this, if ‘reverse genetics’ has to use steps in the reverse order for a genetic study, then it should start with DNA molecules or clones with unknown effect and should conclude with the determination of the phenotype which it controls. This can be achieved by preparing random genomic DNA clones, which can be subsequently utilized for a genetic study in the reverse order. This will then be described as ‘reverse genetics.’ This definition of ‘reverse genetics’ differs from the earlier conventional meaning of this term, where ‘reverse genetics’ was used to describe cases of genes with unworn products, which are isolated (with the help of phenotype and its linkage with molecular markers) and then studied in some detail.

3.33. ISOLATION, SEQUENCING, AND SYNTHESIS OF GENES Molecular manipulation of specific genes is of research in biotechnology. These genes, isolated or artificially synthesized before they are manipulated and used or transformation heading to the production of transgenic animals and plants that are discussed respectively, genes are also sometimes sequenced for a better understanding of its structure, that needs to be manipulated. We therefore, discuss the gene one of the very popular areas should be cither technology available for isolation, sequencing and synthesis of specific genes in this chapter.

Isolation of Genes During the last two decades significant progress has been made in the techniques for isolation of a variety of genes, including those for (ribosomal RNA; (ii) specific protein products; (iii) phenotypic traits with unknown product and those for (iv) regulatory functions, e.g., promoter genes, etc. Different techniques have been used for the isolation of the se different types of genes and will be briefly described in this section. Early attempts for isolation of ribosomal RNA and protiens As we know, ribosomes consist of ribosomal RNA (rRNA) and proteins Ribosomal RNA (IRNA) can be of four sizes namely, 28S, 16S, 58S and 5S (S stands for Svedberg units, a measure of speed with which a molecule sediments on centrifugation). This ribosomal RNA makes 80 per cent d cellular RNA and is synthesized on ribosomal genes, which could be isolated. Isolation of ribosomal genes was considered convenient due B their following three characteristic features (i) availability of homogenies rRNA; (ii) differences

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between ribosomal RNA genes and other gene due to relatively high G + C content in rRNA (rRNA has 45–60 % G + C while the remaining RNA has only 40 % G + C); (iii) ribosomal genes are resent in multiple copies, their number within several thousands. A cell sometimes approaching. In view of the above, ribosomal RNA genes were isolated for the first time (in 1965) in an amphibian named Xenopus, by Hugh Wallace and Max L. Birnstiel, working at the University of Edinburgh (UK) Following steps were involved in this isolation: (i) RNA was isolated from ribosomes of Xenopus and made radioactively labeled, due to its replication in a medium containing tritiated uridine; (ii) ribosomal DNA was isolated by density gradient centrifugation followed by its denaturation (G +C content of rDNA differs from that of bulk DNA and helps in separation by centrifugation); (ii) single stranded DNA was fixed on filter paper; (iv) labeled rRNA was added on filter paper carrying single stranded DNA; (v) DNA-RNA hybridization was allowed to take place (vi) excess labeled RNA was washed (vi) radioactivity was measured and duplex hybrids isolated, which on denaturation, gave single stranded DNA, which could be made double stranded. Isolation of ribosomal DNA allowed the first characterization of ribosomal genes, which was followed by the isolation of 5S genes by Donald Brown. For a detailed account of isolation of ribosomal genes in Xenopus as originally done, readers are referred to an articlè Isolation of Genes in Scientific American (August, 1973)

Isolation of genes coding for specific proteins Isolation of genes for specific proteins became possible only after the discovery of reverse transcriptase enzyme in 1970. This enzyme can be easily used for the synthesis of copy DNA or complementary DNA (cDNA) from mRNA. This complementary DNA can then be used for the isolation of the corresponding gene from genomic DNA. It is, therefore, obvious that for the isolation of a specific gene, techniques should first be available for the isolation of specific mRNA. For this purpose, antibodies are produced against a specific protein for which the gene is to be isolated. Therefore, isolation of a gene coding for a specific protein involves the following basic steps. G purification of the protein product of gene; (ii) production of antibodies against this protein product by immunizing animals like rabbit or mouse; (ii) precipitation of polysomes engaged in synthesizing specific protein with the help of the antibody raised in step (i): (iv) isolation and purification of mRNA from the polysome fraction; (v) using mRNA for synthesizing cDNA with the help reverse transcriptase; (vi) cloning of cDNA thus synthesized for preparation of a cDNA library, (vi) immunological and electrophoretic analysis translation products of cDNA clones to identify specific cDNA meant for the specific protein in mind; (vii) use of

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the specific DNA probes selected in step (vii) the identification and isolation of the gene from genomic DNA through screening a complete or partial genomic library (for preparation of cDNA and genomic DNA libraries, consult Chapter 3). In step (vi) above for preparation of cDNA library, cDNA may be cloned in expression vectors (vectors in which gene can be expressed due to the presence of promoter sequences for RNA polymerase enzyme) like Ågtll, which can accept gene insertions into B galactosidasegene. The chimeric vector will then produce hybrid protein if correctly expressed. The hybrid protein can be identified using antibody as above. Plaques belonging to the chimeric vector carrying the desired gene can also be identified by the reaction of antibody attached to a radioactive protein This procedure was used to clone a number of genes including a gene regulating zein synthesis in maize. The cDNA clones may also be used directly for gene manipulation I transformation experiments. However, when cDNA clone is used, it be described as synthesis of gene rather than as isolation of gene, cDNA has been artificially synthesized, and not isolated from the organism. The above technique for isolation of gene has now been successfully utilized both in plants and animals. However, the first genes isolated were those which existed in multiple copies and were expressed at high levels in specific tissues, e.g., ovalalbumin gene in chicks, globin and immunoglobulin genes in mouse, genes for storage proteins in cereals and legumes, amylase genes in barley, actin genes in some legumes, etc.

Isolation of genes which are tissue specific in expression It is much easier to isolate genes which are expressed in specific tissues. For instance, genes for storage proteins are expressed only in developing seeds, oval albumin gene is expressed in oviduct or globin gene is expressed. Such genes can be easily isolated because mRNA extracted in crythrocyt from these specific tissues will cither exclusively belong to the gene of interest or it will be rich in this species of mRNA. Other mRNA molecules in minor quantities can be eliminated, since these can be identified through their isolation from tissues where this gene is silent. This strategy which was actually followed isolation and cloning carrot genes expressed during the development of the somatic embryos, an also for the isolation of several genes for storage proteins in crop is outlined.

Isolation of genes using DNA or RNA probes Specific molecular probes (whether DNA or RNA probes), if available can be

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used for isolation of specific genes. These probes may be available either from another species for the same gene or may be artificially synthesized using a part of the amino acid sequence of the protein product of the gene.

Use of heterologous probes The probes obtained from one if the species of hererologous probes. These effective in identifying gene heterologous probes have been found to be effective in identifying blots. For instance, the gene for chalchernthese or on Southern clones during colony hybridization or plaque hybridization or on Southern blots synthase has been isolated from Anuirhinum majus and Petunia hybrida heterologous cDNA probe from parsley. Similarly, heterologous probes from maize were used for isolation of barley genes barley be used with cDNA library and not with the genomic library since vector (e.g., pGEM-4 blue) can also be used for getting 89 waxy gene) and Al (aleurone gene). Heterologous probes should be the latter case, unrelated genes or pseudogenes (which do not isolated and cloned). These heterologous clones if available robes, which have been found to be more sensitive and efficiernt.

Use of cDNA or synthetic probes We have already discussed the cDNA probes above. However if protein, purified using the technique of two dimensional gel electrophoresis, is used for microse of 5–15 consecutive amino acids, this information can be used the synthesis of oligonucleotides (using automated DNA synthesizers) These oligonucleotides may then be directly utilized for screening of cDNA genomic libraries for isolation of specific genes. These can also be used for designing primers for the amplification of a gene in DNA extracted from a tissue. A 32 kilodalton (KD) glycoprotein (isolated from style) associated with the S2 incompatibility allele of Nicotiana alaia, was used for synthesis of an oligonucleotide leading to the isolation of a related gene.

Isolation of genes coding for unknown products In some cases, we are interested in isolation of a gene whose phenotypic effect is known, but the gene product has not been identified or cannot be isolated. Such genes include those for morphological traits like dormancy photoperiodicity, disease resistance, etc. This area of research, in which genetics is studied by isolating the gene first without knowing the gene product is often described as reverse genetics. The method used for the isolation of these genes is different from those used for genes coding for known proteins.

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Use of transposable elements (transposons tagging) The transposable elements (TEs), in some cases, have been effectively utilized for isolation of s, when the gene product is unknown. In this case a transposon works mutagen and therefore as a gene tag. Following steps are involved in this procedure: (i) Clone a known h a scorable phenotypic effect. (ii) TE is transposed to this gene get an unstable alicle. (ii) This unstable allele is cloned and TE is isolated s unstable allele (this is to select a TE which can produce unstable as gene wit om allclc effect, to prbdt; (iv) This TE is transposed to a gene of interest with known phenotlo produce unstable allele. (v) The DNA is extracted from this ant. (vi) TE sequence is used as a probe to isolate and clone the mutant (Carrying inserted TE), so that we can then isolate the gene of interest. In maize, TE like Ac Ds, En/Spm and Mu1 have been isolated using Adh1. Similarly, TEs like Tam3 and Tam7 have been isolated from snapdragon (Antirhinum mojius). These TEs have been used experiments leading to isolation of genes. In maize, several P, Al, CI and C2 have been isolated successfully using gene for gene tagging method. For transposon tagging, often transposable elements endogenous to like maize and snapdragon have been used However, rarely transposons available from one plant species can be moved into the genome of another plant species, whose gene is to be isolated. For instance, Ac element of maize has heen transferred to tobacco, where it can integrate into any locus permitting transpose tagging and gene isolation.

Mutant complementation In this technique, DNA clones from the selected which should be able to complement the wild type strain are mutants which are thus transformed into wild type. Once these DNA clones are available, the protoplasts derived from the mutant plant may be transformed using wild type clones and the transgenic plants are produced. Once this is done, the gene of interest can be isolated from DNA extracted from the transformed plants using wild type complementary clone as a probe.

Use of RFLP maps or chromosome walking for gene isolation In recent years, RFLP (restriction fragment length polymorphism) maps have been prepared in maize, tomato, rice, etc. Linkage of RFLP loci with genes of interest arc also being worked out as an aid to plant. If RFLPs are known which are very close to the target gene (as may be done through RFLP maps or chromosome walking), there by locating the RFLPs on either side of the target

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gene, a long intervening DNA segment may be isolated. This fragment may be used for subcloning leading to the isolation of the desired gene. If RFLPs arc examined in two near isogenic lines (NTLs) differing for a single gene, the difference in RFLP maps will help in locating the position of gene on molecular map which can then be utilized for isolation of the gene

Use of chromosome jumping for gene isolation. We described the technique of chromosome jumping for walking long chromosome distances thus bringing the available known sequence (related to the available probe) closer to the gene of interest. Once this has been eyed, the techniques described in the previous section may be conveniently used for isolating this gene for further study.

Isolation of novel genes by Asis Datta at JNU, New Delhi Recently (in 1992), Prof Asis Datta of JNU, New Delhi was selected for he Birla Award for Science and Technology for cloning and characterization he following two novel genes, relevant to human health.

Gene for oxalate decarboxylase In 1991, Prof Asis Datta laboraton reported for the first time the cloning and characterization of the oxalate decarboxylase gene, which degrades and will thus reduce the content of oxalate or oxalic acid, which is harmful in many ways including the following. (a) Several green leafy vegetables (e.g., Amaranthus, spinach, etc.) being rich in oxalate, if ingested in large quantity are toxic, since oxalate chelates calcium and also destroys renal tissues in kideny. (b) The attack and spread of a fungus (Whetzelinidsclerot iorium), which causes damage to crops like sunflower involves accumulation of oxalic acid the infested tissue. (c) Oxalic acid is an essential substrate for the synthesis of the neurotoxin called 3-N-oxalyl 1-La, B diaminopropionic acid (ODAP) found in different parts of the plants of Lathyrus sativus (khesari dal Consumption of this legume causes neurolathyrism, a well known disease causing damage to leg muscles, etc. ODAP interferes with metobolism of glutamic acid, which is involved in transmission of nerve impulses in the brain, so that despite its rich protein content L. sativus cannot be used as a foodsource. In all the above cases (leafy vegetables, sunflower & L Sarivus), transfer of single gene for oxalate decarboxylase to produce transgenic plants will improve these plant species by reducing the oxalate content. This new gene will also allow the development of a diagnostickit to measure oxalate in blood and urine,’ since prevalent methods for clinical tests are quite expensive.

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Gene for a seed specific protein with nutritionally balanced com position of amino acids Prof Datta’s laboratory recently also reported for the first time (in 1992) isolation of a gene from Amaranthus that encodes a seed specific protcin rich in all essential amino acids including lyzine and S-amino acids. Interestingly, the amino acid composition of this protein corresponds to that of WHO recommended protein standard for the optimum human nutrition. It is known that seed proteins in ceric as well as in legumes (peas, etc.) are deficient, the cereal proteins being deficient in lyzine and pulses being deficient in sulfur containing amine acids. In view of this, the gene isolated from Amarenthus will be utilized in future for compensating the amino acid deficiencies proteins, once it is genetically engineered into the target crop plants.

Sequencing of Gene or a DNA Segment Once a gene or a DNA fragment has been cloned, its further study involves DNA sequencing.(The technique of DNA sequencing was very laborious till 1975, when a breakthrough was made in DNA sequencing methods Two different methods are now routinely used for determination of DNA sequences.

3.34. MAXAM AND GILBERT’S CHEMICAL DEGRADATION METHOD In this method, which is illustrated, following steps are involved: (i) Label the 3’ends of DNA with 32P. (ii) Separate two strands, both labeled at 3’ends. (iii) Divide the mixture in four samples, each treated with a different reagent having the property of destroying either only G, or only C, or ‘A and. G’ or T and C. The concentration of reagent is so adjusted – that 50 % of target base is destroyed so that fragments of different sizes having 32p are produced. (iv) electrophoresis each of the four samples in four different lanes of the gel. (v)Autoradiograph the gel and determine the sequence from positions of bands in four lanes.

3.35. SANGER’S DIDEOXYNUCLEOTIDE SYNTHETIC METHOD Fred Sanger (who won Nobel Prize twice) had initially developed a method for DNA sequencing, which utilized DNA polymerase to extend DNA chain

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length. This was termed plus-minus method. Subsequently, he developed a more powerful method, utilizing single stranded DNA as template for DNA synthesis, in which 2,’3’ dideoxynucleotides were incorporated leading to termination of DNA synthesis. These dideoxynucleotides are used as triphosphates (dd! TP) and can be incorporated in a growing chain, but they terminate synthesis, since they cannot form a phosphodiester bond with next incoming deoxynucleotide triphosphate (dNTP), Following steps are involved in Sanger’s dideoxy method for DNA containing single stranded DNA sample (cloned in M13 phage) to be sequined, all four dNTPs (radioactively labeled) and an enzyme for 9 sequencing.(i) Four reaction tubes are set up, each containing single stranded DNA sample to be sequined, all four dNTPs and an enzyme for DNA synthesis. Each tube also contains a small amount (much smaller amount relative to four dNTPs) of one of the four dNTP, so that four tubes have each a different ddNTP, bringing about nation at a specific bascadcnine (A), cytosine (C), guanine (G) and thymine (T). (ii) The fragments, generated by random incorporation of diNTP leading to termination of reaction, are then separated by electrophoresis on a high resolution polyacrylamide gel. This is done for all the four reaction mixtures on adjoining lanes in the gel. (iii) The gel is used for autoradiography so that the position of different bands in each lane can be visualized. (iv) The bands on autoradiogram can be used for getting the DNA sequence. A variant of the above dioxy method was later developed, which has allowed the production of automatic sequencers. In this new approach, a different fluorescent dye is tagged to the oligonucleotide primer in each of the four reaction tubes (blue for A, red for C, etc.). The four reaction mixtures are pooled and electrophoresed together in a single polyacrylamide gel tube. A high sensitivity fluorescence detector, placed near the bottom of the tube, measures the amount of each fluorophore as a function of time. The sequence is determined from the temporal order of peaks corresponding to four different dyes.

3.36. DIRECT DNA SEQUENCING USING PCR (ALSO CALLED LIGATION MEDIATED PCR LMPCR) Polymerase chain reaction (PCR) has also been used for sequencing the amplified DNA produçt. This method of DNA sequencing is faster and more reliable and can utilize either the whole genomic DNA or cloned fragments for sequencing a particular DNA segment. The DNA sequencing using PCR involves two steps: (i) generation of sequencing template (double stranded or single stranded)

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using PCR; and (ii) with the PCR products either with the thermolabile DNA polymerase or with the method or thermostable Tag DNA polymerase. Thus the DNA sequencing using PCR eliminates the need of cloning the DNA in single stranded DNA phage vector, i.e., M13. Although double stranded DNA product difficulty due to can be utilized for sequencing, this may cause difficulty due to reassociation after denaturation thus preventing the sequencing from annealing to its complementary sequence to allow extension. To reduce this problem, either a variant of the standard method for sequencing double stranded DNA is employed or single stranded DNA templates are produced following asymmetric PCR earlier. A number of thermolabile DNA polymerases have been used for sequencing of in vitro amplified DNA Alternatively, Tag polymerase (thermostable enzyme) used for PCR, can also be used for sequencing reaction. In either case, Sanger’s synthetic method involving incorporation of dideoxynucleotide (ddNTP) for chain termination, is used for sequencing However, Maxam and Gilbert’s chemical degradation method can also be used. In Sanger’s method, as usual, four mixtures are prepared, each using one of the four ddNTP. The sequencing primer is labeled with 32P and the mixtures with amplified DNA, Taq polymerase and appropriate buffer are incubated at 70°C for 5 min. The reaction is stopped by addition of formamide stop solution in each tube and mixtures on polyacrylamide sequencing gel to obtain the ladders, which can be read by computer or manually. This method using PCR helped in automation of DNA sequencing.

3.37. SYNTHESIS OF GENES There are two approaches available for the synthesis of genes: (i) when the detailed structure of a gene is available, this gene can be synthesized by a purely chemical method as done by HG Khorana for the synthesis of gene for a tRNA (reported for the first time in 1970). (ii) If the detailed nucleotide sequence of the gene is not available, one may utilized the ‘RNA directed DNA polymerase’ enzyme for the synthesis of the gene in question in the form of complementary DNA (cDNA) from the mRNA of the gene isolated in its pure form. As discussed above, cDNA can also be used for isolation of a gene either (i) from DNA extracted from living cells utilizing the technique of Southern blot hybridization, or (ii) from a genomic library.

Chemical synthesis of tRNA genes As outlined above, before one can start the chemical synthesis of a gene, the

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structure of gene should be known. The structure of a gene earlier could not be worked out by direct chemical analysis, since there were no means for isolating a gene (techniques for isolation of genes were developed later and were described earlier in this chapter). The structure of gene could, therefore, be inferred only from its product. For instance, if a gene is responsible for giving rise to a polypeptide chain and the structure of this chain is known, then from the genetic code dictionary, structure of the gene could be easily inferred. Such genes, were initially considered to be too long to be synthesized because an average gene contains about 1,500 base pairs. On the other hand, since tRNA molecules are fairly small in size (about 80 nucleotides), a gene responsible for giving rise to a tRNA molecule was within the reach for synthesis.

Synthesis of gene for yeast alanyl tRNA As readers may know, structures of large number of tRNA molecules are now known. The first RNA whose structure could be known was yeast alanyl tRNA. R.W. Holley who died in early 1993 and his coworkers (in 1965) gave the detailed structure of yeast alanyl tRNA. This information was immediately used by Khorana and his coworkers to deduce the structure of the gene for yeast alanyl tRNA. The structure of this gene would obviously be such that one of the two strands of DNA (gene) would be complementary to base sequence of yeast alanyl tRNA. The other strand would then automatically have the same sequence as in tRNA except that in place of cil of tRNA there would be thymine in DNA. Synthesis of such a long chain (77 base pairs) of double stranded DNA was rather difficult in 1965 (not in 1990’s) but Khorana and coworkers had extensive experience of synthesizing DNA of known bate sequences. It was decided that such a long chain could not be synthesized by adding a single base each time. Therefore, it was decided that small oligodeoxyribonucleotides ranging in length from 5 to 20 nucleotides should first be synthesized. These segments would be single stranded and would cover the whole length of both the strands of DNA. These would then be joined to form double stranded DNA, 77 nucleotide pairs long In actual synthesis of yeast alanyl tRNA gene, the following steps were involved bis (a) Synthesis of oligonucleotides. Fifteen oligonuclcotides ranging from pentanucleotide (5 bases) to an icosanucleotide (20 bases) were synthesized Synthesis was conducted through condensation between hydroxyl group the 3’ position of one nucleotide and phosphate group at 5’ position d the second nucleotide. In order to bring about condensation, all other functional groups, not taking part in condensation were protected using specific protective groups. After protecting the groups, reaction between a nucleotide with protected 5’ end

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and another nucleotide with protected end proceed. Subsequently condensation consisting also brought about between groups of two, three or four nucleotides. (b) Synthesis of three duplex fragments: With the help of 15 tides (single stranded), three duplex fragments, each with a li stranded end, were prepared. These three fragments consisted of the following: (i) the first 20 nucleotides, with the nucleotides17–20 single stranded, this fragment was called ‘A’; (ii) the nucleotide residue 17–50 with single stranded regions 17–20 and 46–50 segment called B’; and (iii) the nuclcotide residues 45–77 with single stranded 17–20 as region 46–50;this segment was called ‘C.’ (c) Synthesis of gene from three duplex fragments: The three fragments ‘A,’ ‘B’, and ‘C’ were joined to give complete gene in each of the two following ways; (i) In one scheme, ‘A’ was joined to ‘B’ taking advantage of the overlap in residues 17–20; C was then added, with the overlap in the region 46–50 residues. The complete double stranded DNA with 77 base pairs representing the gene was thus ready. (ii) In the other scheme, B’ was first added to ‘C and to this A’ was added in the end to obtain the complete gene. This gene, which was synthesized in 1970, was hopefully used for subsequent work, since this gene could replicate and make its own copies. (d) Synthesis of gene for a true precursor tRNA: Before Khorana could complete the synthesis of gene for yeast alanyl IRNA in 1970, as outlined above, it became obvious that tRNA was not the direct product of transcription. Instead a precursor molecule is first synthesized which subsequently, after losing segments of RNA by cleavage, gives risc to tRNA. Obviously, therefore, the actual gene for yeast alanyl tRNA will be longer than the DNA duplex synthesized by Khorana. In view of this Khorana subsequently initiated synthesis of a gene for E coli tyrosine suppressor tRNA precursor. DNA duplex which will give rise to this tRNA precursor, was synthesized in the form of 26 small oligonuclcotide segments. These were then arranged into six DNA duplex fragments having sin stranded ends. These six fragments gave rise to presumed gene for E. coli tyrosine suppressor tRNA precursor. This gene, however, still la promoter region and other sequences essential for processing.

Later, in 1979, Khorana reported completion of the total synthesis of a biologically functional tyrosine suppressor transfer RNA gene carry all regulatory sequences. This gene was 207 base pairs long and include the following: (i) a 51 base pairs long DNA promoter region; (ii) a 126 base pairs long DNA corresponding to the precursor tRNA; and (iii) a 25 base pairs long duplex DNA corresponding to 16 base pairs adjoining CCA end of tRNA and the remainder, a modified sequence including EcoRI endonuclease specific sequence. The complete synthetic gene was cloned in the vcctor bactcriophage (lambda virus) by gene cloning method discussed.

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On transformation of E. coli, the phage could multiply with the cloned gene.

Total synthesis of a human leukocyte interferon gene Interferon is a protein, which has a property of inhibiting viral infection and cell proliferation and thus can be used for treatment of viral infection and malignancies (cancer). These properties have generated great interest in human interferons (IFNs). At least three kinds of interferon genes are known in human genome: (i) leukocyte interferon genes (IrNα- genes, (ii) fibroblast interferon gene (IFN-3 genes), and (iii) immune interferon gene (IFN-y genes). Genes for different kinds of interferons differ and details of base sequences are not known for all of them. In 1980, Weismann and coworkers published the nucleotide sequence gene derived from cloned IFN-a cDNA this information, total synthesis of this human leukocyte interferon gene 514 base pairs long, was achieved and published in 1981 (Edge et al., 1981). Since there are 514 base pairs in the gene, it involved synthesis of DNA strands with 1028 nucleotides in predetermined way. This was the longs gene synthesized till 1982. The synthesis involved coupling of initial nucleotide onto a polyacrylamide resin to which further nucleotides could be added in pairs. Sixty six (66) oligonucleotide segments varying in size be added from 14 to 21 were first synthesized, which were than arranged in a predetermined way and joined. This gene has been incorporated into a plasmid and in capable of synthesizing a interferon. Further details about the synthesis of this IFN- gene are available in the original article published in Nature (Vol. 292, pp. 756–761, 1981). The 514 base pairs code for 100 amino acids 498 base pairs, and include the initiation and termination signals. There are no introns sequences present in this gene (introns are noncoding sequences, common, in eukaryotic genes, that are known as split genes). The plasmids with this newly synthesized gene have been transferred into bacteria (E. coli), which are being utilized now for synthesis of interferon in industry. This has greatly reduced the cost of production of this drug (interferon), which was earlier sold at the rate of Rs.16 million per 50 mg.

3.38. GENE SYNTHESIS MACHINES Methods for synthesis of DNA molecules of known base sequences have improved dramatically during 1980s and 1990s. Therefore, genes can now be synthesized rapidly and in high yields. For instance, a gene for a IRNA tock 20 man years effort during 1965–70 (described earlier in this chapter) but the same gene can now be synthesized within a matter of few days with the help of Gene

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Synthesis Machines developed in 1980’s. The key innovations which made the availability of gene synthesis machines possible, due to the, the following: (i) development of silica based supports, which are e and provide support for solid phase synthesis of DNA chains and (ii) development of stable protonated deoxyribonucleoside phoramidites as synthase, which are remarkably stable and hydrolysis and thus are ideal for DNA synthesis. These two procedures can be used to prepare DNA molecules more than 100 nucleotides long within a few days. Different steps involved in solid phase synthesis of a DNA chain. Several versions of gene machines are now available. These machines, under the control of a microprocessor, synthesize specific short sequences of single stranded DNA automatically. The desired sequence is entered on a keyboard and the microprocessor automatically opens the valves of the containers of successive nucleotides, reagents and solvents needed at each step, into a synthesizer column, which is packed with tiny silica beads. These beads provide support on which DNA molecules are assembled.

3.39. SYNTHESIS OF GENES FROM MRNA H. Temin and D. Baltimore, in 1970 discovered the presence of RNA directed DNA polymerase enzyme which has the ability of synthesizing DNA on RNA template. This enabled molecular biologists to synthesize complementary DNA (cDNA) using mRNA as a template. If mRNA transcribed from a specific gene is made available in purified form, the elementary DNA (cDNA) synthesized with its help will represent the synthesized gene. To what extent this synthesized gene is a faithful copy of native gene will depend on the fidelity of copying mRNA and also on the stability of DNA thus synthesized. Moreover, since mRNA of a gene does not have the complete transcript of the gene in vivo, the synthesized gene will be smaller than the gene in vivo. By copying eukaryotic purified mRNA, several genes have been artificially synthesized. Most important of these genes are the genes for sea urchin histone proteins, ovalbumin gene in chicken and globin genes in mammals. The gene synthesized as cDNA from a B globin mRNA, has been inserted into a plasmid in order to study its behavior. As earlier pointed out, this gene will lack any additional regulatory sequences absent in mRNA but present in a globin gene in vivo. This type of gene will also lack intron sequences found in eukaryotic split genes. Another factor causing a difference between synthesized gene and the gene in vivo will depend on the sincerity with which the enzyme will synthesize cDNA, because in many cases cDNA was found to be smaller than the mRNA itself. These genes (cDNA) have already become a very important tool in molecular biology experiments.

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3.40. GEL PERMEATION It is a technique in which polymeric organic compound is used to prepare a porous medium. The pore size is determined by degree of cross-linking of polymeric chains. Solutes present in the mixture are separated on the basis of their size and shape when they pass through a column consisting of packed gel particles (Figure 3.1).

Figure 3.1: Gel permeation chromatography (a) spatial accessibility of molecules during gel filtration (b) movement of molecules of different sizes through microporus gel forming stationary phase.

3.41. CHROMATOGRAPHY It is a technique of separation in which small sized particle or liquid substances are separated in a buffer solution.

3.42. ION EXCHANGED CHROMATOGRAPHY When molecules are dissolved in solvent, they dissociate into charged ions. Therefore, they develops polarity. On the basis of polarity, i.e., presence of charges they can be separated. Ion–exchange chromatography is a powerful technique used for separation of two protein which are very similar to each to each other (Figure 3.2).

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Figure 3.2: Ion exchange chromatography: (a) cation exchange; (b) anion exchanger.

3.43. ELECTROPHORESIS It is a method of separation of charged molecules applying an electric field. When the charged molecules are placed in an electric field they migrate depending on their net charge, size, shape and applied current.

3.44. AGAROSE GEL ELECTROPHORESIS Agrarose is a linear polymer of D-galactose and 3,6–anhydro–L-galactose which is extracted from seaweed. At neutral pH the negatively charged DNA migrates towards the anode after application of electric field across the gel. It is a standard technique of nucleic acids.

3.45. PULSED FIELD GEL ELECTROPHORESIS (PFEG) It is a process to separate the several megabases long DNA molecules. PFEG varies a little from agrose gel electrophoresis and can resolve DNA molecules of 100–1000kb. It changes the direction of the electric field in such a way that larger DNA fragments are align more slowly with the direction of the new field than do the smaller DNA molecules. The pulsed field electric fields applied to a gel force the DNA molecule to reorient before continuing to move like snake through the gels (Figure 3.3).

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Figure 3.3: Pulse filed gel electrophoresis. Polyacrylamide gel electrophoresis (PAUL). It is most widely used electrophoresis for separation and characterization of proteins and nucleic acids. Polyacrylamide gel offers several advantages such as inertness to chemicals, superior resolution stability over wide range of pH, temperature and ionic strength. Polyacrylamide gel is prepared by using the following (i) monomers (e.g., acrylamide, N,N-methylene bisacrylamide, (ii) initiator NN.N.N-tetramethylethylene diamine (TEMED), (iii) propagators (ammonium persulfate riboflavin, and (iv) terminator or inhibitor (oxygen/air). Pore size of polyacrylamide gel can be changed by varying the concentration of acrylamide and bi-acrylamide monomers in a fixed volume of gelatin solution. Free radicals formed by ammonium sulfate activate the acrylamide gel. Long polymer chain is thus produced after reacting the activated acrylamide with successive acrylamide molecules. Further, bisacrylamide brings about gelation and cross-linking through polymerization. This results in formation of complex network of acrylamide 4. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDA PAGE) Separation of some of the proteins does not occur due to similar charge such proteins are treated first with an ionic detergent called sodium dodecylsulfate (SDS) before start and during the course of electrophoresis (PAGE). Therefore, such electrophoresis is called SDSPAGE electrophoresis. Identical proteins are denatured by SDS resulting in their sub-units. The polypeptide chains get opened and extended. On the basis of their mass but not the charges, the molecules are separated. SDS-PAGE is a high-resolution method used universally for analyzing the mixture of proteins according to their respective size. SDS solubilized in soluble proteins makes possible the analysis of the other insoluble mixtures. Separation of the proteins does not occur due to similar charge: mass ratio (z/m). Therefore, such proteins are treated first with an ionic detergent called SDS before start and during the course of electrophoresis. Therefore it is called SDA-PAGE.

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3.46. TWO-DIMENSIONAL ELECTROPHORESIS A mixture of protein is separated using two- dimensional electrophoresis. The first dimension uses the iso-electric focusing, and the second dimension is sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) Isoelectric Focusing (IEF): The biomolecules such as proteins, DNA and RNA have electric charges which depend on molecule to molecule and the conditions of the medium (pH of buffer in which dissolved). Charged molecules can be separated by N electrophoresis in gels. Due to the differences in amino acid composition proteins have a unique mass and charge. Hence the proteins have net negative charge and net positive charge or iso-electric point (no charge) at a given pH of buffer. The amphoteric substances such as proteins which differ in their isoelectric points can be separated by IEF. Isoelectric point is a pH value at which the net charges on molecules are zero Ampholytes (i.e., complex mixture of synthetic polyamino-polycarboxylic acids) are introduced (Figure 3.4).

Figure 3.4: Isoelectric focusing.

3.47. SPECTROMETERY In 1900, for the first time J.I. Thomson introduced mass spectrometer which

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employed fixed magnetic and electric fields to separate ions of different mass and energy. He recognized that charged particles differing in momentum behaved differently in an electric field, and used this property for separation of ions with different mass. Its extensive use for research in various field of biological science was started in 1980s. DuFing 1990–2000, mass spectrometry became an important technique for genomics and proteomics research leading to the 2002 Nobel Prize in Chemistry to J.B. Fenn and K. Tanaka. Twodimensional electrophoresis is more powerful when coupled with MS. The unknown protein spot is cut from the gel and cleaved by trypsin digestion into mass spectrometer and the mass of fragment is fragments which are then analyzed by finger print can be used to estimate plotted. This mass the probable amino acid composition of each fragment and tentatively identify the protein. The proteome and its changes can be studied very effectively by employing the two techniques together The MS can also provide valuable information about covalent modification of proteins which can affect their activity. Mass spectrometry is very useful technique. It is used in (i) identification of unknown compounds, quantification of known compounds and determination of structural and chemical ties of compounds when present in small the production of ions of the materials in sample, (ii) their separation on the basis of their mass: charge (m:e) ratio, and (iii) determination of relative abundance of each ion. Therefore spectrophotometer consists of three components: the source. It does not directly measure the molecular mass but detects m: e ratio. Mass is measured in terms of Dalton (Da). One Dalton = 1/12th of mass of a single atom of isotonic carbon (3C) unit (106–10 g). This technique involves: a mass of ion, an analyzer, and a detector.

3.48. MATRIX-ASSISTED LAYER DESORPTION-IONIZATION (MALDI) MALDI was first developed by Karas and Hillenkamp in 1988. It co-precipitates the large excess of a matrix material with the analyte molecule. In this method a sub-microliter of the mixture (matrix +analyte) is pipetted onto a metal substrate and allowed to dry. The dried mixture is irradiated by laser pulse (337 nm) which specifically absorbs the selected solid of matrix. The irradiation causes energy transfer and desorption resulting. With the help of matrix ions the nonabsorbing analyte molecules also get desorbed into the gas phase and ionized. The charged molecular ions of analyte are detected by MALDI-TOF mass spectrometer. Generally, MALDI is used for the study of molecules having mass above 500 Daltons (i.e., oligonucleotides and peptides). Proteome analysis will be crucial for understanding different biological processes in the post-genomic

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era. To achieve this objective mass spectrometry is extensively used for: peptide sequencing, identification of protein, protein expression in different tissues and conditions, identification and post-transcriptional modification (e.g., phosphorylation, glycosylation, etc.) of proteins in response to different stimuli, and characterization of protein interactions that include protein-ligand, proteinprotein and protein-DNA interaction n gas phase matrix ions t improvement in MS has significantly improved its application in the study of protein structure and function. For the study of macromolecules, the Indian Society for Mass Spectrometry (ISMAS) was established in 1978 with it’s headquarter at BARC (Bhabha Atomic Rescarch Center), Mumbai. This society conducts a workshop each year on mass spectrometry. In India, facilities for MALDI- TOF MS have been created at several centers including the Center for Cellular and Molecular Biology (CCMB), Hyderabad, Indian Institute of Sciences, Bangalore, BARC, Rajiv Gandhi Center for Biotechnology (RGCB), Thiruvanantapuram, etc.

3.49. SURFACE ENHANCED LASER DESORPTIONIONIZATION (SELDI) It is a unique combination of affinity chromatography and time of flight (TOF) mass spectrometry on a single platform, a combination designated ionization time of flight mass spectrometry). This system enables protein capture, purification, ionization and analysis of complex biological mixtures directly on protein chips array surface and detection of the purified proteins by laser desorption-ionization time of flight (LDI-TOF), MS analysis. SELDI system consists of three parts: (i) protein chip arrays (eight spots of 2 mm diameter); (ii) protein chip reader (LDI-TOF MS); and (iii) specialized software.

3.50. ELECTROSPRAY IONIZATION (ESI) In the late 1980s, J.B. Fenn developed ESI mass spectrometry and awarded Nobel Prize in 2002 in chemistry. In this method the sample is dissolved in liquid with mobile phase (i.e., water: acetonitrile: methanol) and pumped through a hypodermic needle at a high voltage (about 4,000 V which causes strong electric field at the top of nozzle of the metal needle). This disperses electrostatically electrosprays the highly charged small droplets of about one micrometer in size (Figure 3.5).

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Figure 3.5: Electrospray ionization source. These droplets are electrostatically attracted to the inlet of MS. Dry gas or heat is applied. The solvent around these droplets soon evaporates and impart charge increases with decrease in their size. Hence equal charges repel each other. This ionization is very gentle which causes no breakdown of analyte ions in the gas phase or onto the analyte molecules.

3.51. FLUORESCENCE SPECTROSCOPY Fluorescence refers to emission of light from a molecule after absorption of radiant energy When a molecule absorbs radiant energy, its electrons are elevated to excited state. If there is no chemical reaction, dissociation, rearrangement or energy transfer by collision, the molecule return to ground state and excessive energy is lost as heat or light, i.e., photons of energy (v). Emission of light is called fluorescence. A compound may absorb energy in UV region and fluoresce in visible region. In fluorescence the absorbed excited light is of a smaller wavelength (more energy) and the wavelength of the emitted light is longer (less energy). More increase in wavelength of emitted light than the excited light is called stoke shift. The fluorescence shown by the natural compounds is known as intrinsic fluorescence. There are certain chemical compounds which do not fluoresce. However, fluorescence can be detected after coupling them with a fluorescence probe. This phenomenon is called extrinsic fluorescence.

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3.52. POLYMERASE CHAIN REACTION In polymerase chain reaction (PCR) provides a simple and ingenuous method for exponentially amplification of specific DNA sequences by in vitro DNA synthesis. This technique was developed by Kary Mullis at Cetus Corporation in Emery Ville, California in 1985. Kary Mullis shared the Nobel Prize for chemistry in 1993. This technique has made it possible to synthesize large quantities of a DNA fragment without its cloning. It is ideally suited where the quantity of biological specimen available is very low such as a single fragment of hair or a tiny bloodstain left at the site of a crime. The details of PCR techniques and its mechanism are described by Erlich (1989) in his edited book ‘PCR Technology.’ The PCR technique has now been automated and is carried out by a specially designed machine. The PCR requires the following: (i) (ii)

(iii)

DNA Template: Any source that contains one or more target DNA molecules to be amplified can be taken as template. Primers: A pair of oligonucleotides of about 180–30 nucleotides with similar G+C contents act as primers. They direct DNA synthesis towards one another. The primers are designed to anneal on opposite strands of target sequence so that they will be extended towards each other by addition of nucleotides to their 3’ ends. Enzyme: The most common enzyme used in PCR is a thermostable enzyme called Taq polymerase. It is isolated from a thermostable bacterium called Thermus aquaticus. It survives at 95°C for 1–2 minutes and has a half-life for more than 2 hours at this temperature. The other thermostable polymerases can also be used in PCR.

3.53. DNA AMPLIFICATION FINGERPRINTING (DAF) The DAF has been described by Caetano-Annoles and Gresshoff (1994) where a single arbitrary primer (about 5 bases long) is used to amplify DNA by using PCR. In this technique the parameters are carefully optimized. But for early and easy determination of amplified products, automation and fluorescent tagging of primers are extremely amenable. DAF differs from RAPD as (i) it requires higher concentration of shorter primers (5–8 nucleotides), (ii) two temperature cycles contrast to three cycles in RAPD, (iii) it produces a very complex banding pattern, and (iv) DAF products are used in Sed on acrylamide gels and detected by using silver staining.

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3.54. APPLICATION OF PCR After the discovery of PCR, the modern biology has been revolutionized in each and every aspect. “Some of the areas of application of PCR have briefly been discussed herewith. (a) Diagnosis of Pathogens: There are several pathogens that grow slowly. Therefore the cells are found less in number in the infected cells/tissues. It is difficult to culture them on artificial medium. Hence, for their diagnosis, PCR-based assays have been developed. These detect the presence of certain specific sequences of the pathogens present in the infected cells/tissues. Besides, it is useful in detection of viral infection before they cause symptoms or serious diseases. (b) Diagnosis of Specific Mutation: in humans there are thousands of genetic diseases. Mutations are also related to genetic diseases. Presence of a faulty DNA sequence can be detected before establishment of disease. By using PCR sickle cell anemia, phenylketonuria and muscular distrophy can also be detected. The other diseases can also be diagnosed by using PCR. For example PCR based diagnostic for AIDS. Chlamydia, tuberculosis, hepatitis, human pappiloma virus, and other infectious agents and diseases are being developed. The tests are rapid, sensitive and specific. (c) In Prenatal Diagnosis: It is useful in prenatal diagnosis of several genetic diseases. If the genetic diseases are not curable, it is recommended to go for abortion. (d) DNA Fingerprinting: In recent years, DNA fingerprinting is more successfully used in forensic science to search out criminals, rapists solving disputed parentage and uniting the lost children to their parents or relatives by confirming their identity. This is done through making lines between the DNA recovered from samples of blood, semen, hairs, etc. at the spot of crime and the DNA of suspected individuals or between child and his/her parents/relatives. (e) In Research: In addition, DNA fingerprinting of new microorganisms isolated from various extreme environment (soil. water, sediments, air, extreme habitats, etc.) is also carried out to confirm their identity by comparing with the DNA sequences of known microorganisms. Their DNA and RNA can be amplified. Besides, it is also useful to determine the orientation and location or restriction fragments relative to one another. (f) In Molecular Archacology (Palaentology): PCR has been used to clone the DNA fragments from the mummified remains of humans and extinct animals such as the woolly mammoth and linosaurs from the remains of ancient animals as recently epitomized in Michel Crighton’s Jurassi Park. DNA from buried humans has been amplified and used to trace the human migration that occurred in ancient time. (g) Diagnosis of Plant Pathogens: It is also applied in diagnosis of plant diseases. A large number of plant pathogens in various hosts or environmental samples are detected by using PCR, example, viroids (associated with flower mosaic virus, bean yellow mosaic-virus, plum pox virus, potyviruses), mycoplasmas bacte (Agrobacterium umifaciens,

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Pseudomonas solanacearum, Rhizobium leguminosarum, Xanthomon Compestris, etc.), fungi (e.g., collectotrichum gloeosporioides, Glomus spp., Laccaria spp., Phytophthora spp. Verticillium spp),” and nematodes (e.g., Meloidogyne incoginta M. javanic. apple, pear, grape, citrus, etc.), viruses (such as TMV (Henson and French, 1993; Chawla, 1998).

4 Tools of Recombinant DNA Technology (Cutting and Joining of DNA)

4.1. EXONUCLEASE These enzymes act upon genome and digest the base pairs on 5’ or 3’ ends of a single stranded DNA or at single strand nicks or gaps in double stranded DNA (Figure 4.1).

Figure 4.1: Action of exonuclease (a) and endonuclease (b).

4.2. ENDONUCLEASE They act upon genetic material and cleave the double stranded DNA at any point except the ends, but their action involves only one strand of the duplex (Figure 4.2).

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Figure 4.2: Host controlled restriction and modification of phage I in E. coli strain K and strain B.

4.3. RESTRICTION ENDONUCLEASES The restriction enzymes are called as ‘molecular scissors.’ These acts as foundation of recombinant DNA technology. These enzymes are present in bacteria and provide a type of defense mechanism called the ‘restrictionmodification system. Molecular basis of these systems was elucidated first by Werner Arber in 1965. However, there was no understanding about cutting and joining of DNA molecules using DNA enzymes, before 1970. During the early 1950s, it was discovered when phage A infects E. coli, phage DNA is delivered inside the cell. Host-control restriction modification system provides defense to E. coli. Bacterium protects itself through: (i) restriction mechanism (i.e., identifies the introduced foreign DNA and cuts into pieces called restriction endonucleases, and (ii) modification system (i.e., methylation of certain bases of its own DNA by methylase so that phage A-expressed restriction endonuclease could not cleave its DNA)

4.4. NOMENCLATURE The restriction enzymes are named based on some of the following principles:

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•

Name of the organism is identified by the first letter of genus name and the first type letters of the species name to form a three letter abbreviation in the italic, for example E. coli = Eco and H. influenzae = Hin, etc. • A strain or type identified is written as subscript, e.g., EcoK for E. coli strainK. Hind for H. influenzae strain Rd. • In such cases where the restriction and modification systems are genetically specified by plasmid, the extra chromosomal element is identified by a subscript, e.g., Ece RI, EcoPI, etc. • When a strain has several restriction and modification systems, these are identified by Roman numerals, for example HindI, HindII, HindIII for H. influenzae strain Rd., etc. These Roman numerals should not be confused with those in the classification of restriction enzymes into Type I. a) Type III Restriction Enzymes: The Type III enzyme is made up of two subunits, one specifies for site recognition and modification and the other for cleavage. In a reaction, it moves along the DNA and requires ATP as source of energy and Mg as co-factor. ATPase activity is lacking in these enzymes. Some examples of Type III enzyme are Hpal, MbolI, Fokl, etc. They have symmetrical recognition sites and cleave DNA at double stranded DNA two sites in opposite orientation must be present. One strand of double stranded DNA is cleaved about 25–27 bp away from the recognition site which is located in its immediate specific non-palindromic sequences. Since the cleavage products of these enzymes are homogeneous population of DNA fragments. Since, the cleavage products of these enzymes are they cannot be used for genetic engineering experiments.

4.5. EXAMPLES OF SOME ENZYMES There are some other restriction enzymes, which do not fall under the Type I-III. Hence there is need for reclassifying restriction enzymes under the other categories from Type I to Type IV. Examples of the other restriction enzymes are Eco 571, MCR (modified cytosine reaction), etc. Eco571 is made up of single polypeptide. It shows both nuclease modification and nuclease activity. The S-adenosyl methionine stimulates nuclease activity of this enzyme. It bears some other properties that resemble with Type II enzymes. Therefore, Eco 571 should be put under Type II to Type IV.

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4.6. NUCLEASE It degrades the single stranded DNA or single strand protrusion of double stranded DNA Cohesive ends. As a result of action of nuclease cohesive ends are converted into blunt ends Therefore, if nuclease is used to remove the incompatible ends so that overlapping ends may be developed for annealing of two fragments of DNA molecules, mode of action of S1 nuclease.

4.7. DNA LIGASES Mertz Davis (1972) for the first time demonstrated that DNA cleaved DNA molecules could be covalently sealed with E. coli DNA ligase and were able to produce recombinant DNA molecules (Figure 4.3), which has 5–33 ‘-OH (hydroxyl) termini. There are two enzymes which are extensively used for covalently joining restriction fragments the ligase from that encoded by T4 phage. The main one of DNA phage, hence, the enzyme is known as T4 DNA ligase.

4.8. ALKALINE PHOSPHATASE When plasmid vector, for joining a foreign DNA fragment, is treated with restriction enzyme, the greatest difficulty arises at the same time. Because the cohesive ends of broken plasmids, instead of joining with foreign DNA join the cohesive end of the same DNA molecules and get recircularized. To overcome this problem, the restricted plasmid (i.e., plasmid treated with restriction enzymes) is treated with an enzyme, alkaline phosphatase, that digests the terminal 5‘phosphoryl group. The restriction fragments of the foreign DNA are not treated with alkaline phosphatase. Therefore, the 5 ‘end of foreign DNA fragment of the plasmid. The hybrid ligase will only join 3 ‘and 5’ ends of recombinant DNA together if the 5 ‘end is phosphorylated. Thus, alkaline phosphatase and ligase prevent recircularization of the vector and increase the frequency of production of recombinant DNA molecules. The nicks between two 3 ‘ends of DNA fragment and vector DNA are repaired inside the bacterial host cells during the transformation can covalently join to 3 ‘end or recombinant DNA obtained has a nick with 3’ and 5’ hydroxy ends (Figure 4.3).

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Figure 4.3: Alkaline phosphates.

4.9. REVERSE TRANSCRIPTASE In addition to these enzymes, reverse transcriptase is used to synthesize the copy DNA complementary DNA (CDNA) by using MRNA template. Reverse transcriptase is very useful in the synthesis of CDNA and construction of or as a cDNA clone bank. Until recently, it was known that the genetic information’s of DNA pass to protein through MRNA. During 1960s, Temin and co-workers postulated that in certain cancer causing animal viruses which contain RNA as genetic material, transcription of cancerous genes (on RNA into DNA) takes places probably by DNA polymerase directed by virus RNA. Then DNA is template for synthesis of many copies of viral RNA in a cell. In 1970, S. Mizutani, H.M. most used as Temin and D. Baltimore discovered that information’s can also pass back from RNA to DNA. They found that retroviruses (possessing RNA) contain RNA dependent DNA polymerase which is also called as reverse transcriptase. This produces single stranded DNA, which in turn functions as template for complementary long chain of DNA.

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4.10. DNA POLYMERASE This enzyme polymerizes the DNA synthesis on DNA template (or cDNA template) and also catalyzes a 5’–3’ and 3’-5’exonucleolytic degradation of DNA (Kornberg, 1974).

4.11. T4 POLYNUCLEOTIDE KINASE The T4 polynucleotide kinase catalyzes the transfer of y PO, of ATP to 5’ terminus of a depho phorylated DNA (i.e., DNA lacking -PO, residue) or RNA as below: 5’GGOH 3 3’-CCTTAAp 5’-GGOH Polynucleotide kinase -3’ 3’-CCTTAA5’.S- 7P-ATP. This enzyme shows both phosphorylation as well as phosphatase activities; therefore, two types of reaction can be used accordingly. In phosphorylation activity the y-PO is transferred to the 5’end of dephosphorylated DNA. When ADP is in excess, it causes the T4 polynucleotide kinase to transfer the terminal phosphate (attached to a nucleotide) from phosphorylated DNA to ADP. By using radio- labeled gamma-phosphate (PO) from P-ATP. This shows that the enzyme plays a dual role of phosphorylation activity and phosphatase activity. it was found that DNA is rephosphorylated by transfer of any phosphate solution yp. Due to the presence of both properties, this enzyme is used in radiolabeling of the 5’ end of duplex during DNA sequencing (see DNA sequencing method), constructing the terminally labeled DNA, phosphorylating the synthetic linkers/oligonucleotide linkers/other DNA fragments lacking terminal 5’phosphates, etc.

4.12. TERMINAL TRANSFERASE This enzyme is also known as from calf thymus. It has a property to add ‘oligodeoxynucleotide tail’ to 3’-OH end of double stranded DNA fragments. Thus it extends homopolymer tails and this phenomenon is called homoplymer tailing, deoxynucleotidyl transferase. It has been isolated and purified

4.13. USE OF LINKERS AND ADAPTORS As discussed earlier that treatment of DNA with Type II restriction enzymes results in formation of fragments having sticky ends or blunt ends. Such DNA fragments cannot be ligated with cloning vectors. Therefore, artificially

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cleavage sites at blunt end can be added as linker or ecules. Chemically synthesized double stranded oligonucleotides are called linkers. They are added double stranded DNA molecules that act as adaptor mole are added recognition site for restriction enzyme. For example the following oiligonucleotide possess recognition sequence for EcoRI (asterisk nucleotides). It can be ligated to blunt end of any double stranded DNA and cut by EcoRl to generate sticky ends to DNA9 (Figure 4.4).

Figure 4.4: Linker and ADAPTOR molecules.

5 Tools of Genetic Engineering (Cloning Vectors)

5.1. CLONING AND EXPRESSION VECTORS In recent years, techniques for manipulating prokaryotic as well as eukaryotic DNA have witnessed a remarkable development. This has allowed breakage of a DNA molecule at two desired places to isolate a specific DNA segment and then insert it in another DNA molecule at a desired position. The product thus obtained is called recombinant DNA and the techniques often called genetic engineering. Using, this technique we can isolate and clone single copy of a gene or a DNA segment in to an indefinite number of copies, all identical. This became possible because vectors like plasmids and phages reproduce in a host in their usual manner ever after insertion of foreign DNA, so that the insertion DNA will also replicate faithfully with the parent DNA. This technique is called gene cloning. With this technique, genes can be isolated, cloned and characterized, so that the technique has led to significant progress in all areas of molecular biology. A variety of vectors have been developed which not only allow multiplication, but may also be manipulation in such a way that the inserted gene may express in the host. Due to the importance of a variety of these cloning and expression vectors in genetic engineering experiments, they are discussed in some detail in this chapter. The techniques used for inserting foreign DNA molecules will be discussed in next chapter. The commercial use of these techniques will be discussed in subsequent chapters.

5.2. CLONING VECTOR FOR RECOMBINANT DNA One of the most important use of recombinant DNA technology in the cloning of (i) random DNA or cDNA segments, often use as probe or (ii) specific genes,

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which may be either isolated from the genome or synthesized organochemically or in the form c DNA from m RNA. This other DNA molecule is often used in the form of a vector, which could be a plasmid, a bacteriophage, a derived cosmid or phagemid, a transposon or even a virus. Techniques should also be available, which will allow selection of chimeric genomes obtained after insertion of foreign DNA from a mixture of chimeric and the original vector. Another critical desired feature of any cloning vector is that it should posses a site at which foreign DNA can be inserted without disrupting any essential function. Therefore, in each case an enzyme will also have to be selected which will cause a single break. Sometimes vectors are modified by inserting a DNA segment to create unique site for one or more enzymes to facilitate its use in gene cloning. This inserted DNA with restriction sites for several enzymes is sometimes called a polylinker.

5.3. PLASMIDS AS VECTORS Plasmids are defined as autonomous elements, whose genomes exist in the cell as extrachromosomal units. They are self-replicating circular duplex DNA molecules, which are maintained in a characteristic number of copies in a bacterial cell, yeast cell or even in organelles found in eukaryotic cells. These plasmids can be single copy plasmids that are maintained as one plasmid DNA per cell or multicopy plasmids, which are maintained as 10–20 genomes per cell. These are also plasmids, which are under relaxed replication control, thus permitting their accumulation in very large numbers. These are the plasmids which are used as cloning vectors, due to their increased yield potential. Circular plasmid DNA which is used as a vector, can be cleaved at one site with the help of a restriction enzyme to give a linear DNA molecule. A foreign DNA segment can now be inserted, by joining the ends of broken circular DNA to the ends of foreign DNA, thus regenerating a bigger circular DNA molecule that can now be separated by electrophoresis on the basis of its size. Selection of chimeric DNA is also facilitated by the resistance genes, which the plasmid may carry against one or more antibiotics. If a plasmid has two such genes conferring resistance against two antibiotics and if the foreign DNA insertion site lies within one of these two genes, then the chimeric vector loses resistance against one antibiotic, the gene for which has foreign DNA inserted within its structure. In such a situation, the parent vector in bacterial cells can be selected by resistance against two antibiotics and the chimeric DNA can be selected by retention of resistance against only one of the two antibiotics.

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5.4. PBR322 AND PBR327 VECTORS The naturally occurring plasmids may not possess all the above and other essential properties of a suitable cloning vector. Therefore, one may have to restructure them by inserting genes of relaxed replication and genes for antibiotic resistance. This has actually been done and suitable plasmid vectors have been obtained. One is pBR322, which is 4,362 bp DNA and was derived by several alterations in earlier cloning vectors. It has genes for resistance against two antibiotics an origin of replication and a variety of restriction sites for cloning of restriction fragments obtained through cleavage with a specific restriction enzyme. Another vector pBR327 was derived from pBR322, by deletion of nucleotides between 1427 to 2516. These nucleotides are deleted to reduce the size of the vector and to eliminate sequences that were known to interfere with expression of the cloned DNA in eukaryotic cells. pBR327 still contains genes for resistance against two antibiotics. Both pBR322 and pBR327 are very common plasmid vectors (Figure 5.1).

Figure 5.1: pBR322 vector.

5.5. PUC VECTORS Another series of plasmids that are used as cloning vectors belong to pUC series. These plasmids are 2700 bp long and possess: (i) ampicillin resistance gene, (ii) the origin of replication derived from pBR322, and (iii) the lac Z gene derived from E. coli. Within the lac region is also found a polylinker sequence having unique restriction sites. When DNA fragments are cloned in the region of pUC, the lac gene is inactivated. These plasmids when transformed into an appropriate E. coli

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strain, which is lac, and grown in the presence of IPTG and X-gal, will give rise to white or clear colonies. On the other hand, pUC having no inserts and transformed into bacterial will have an active lac Z gene and therefore will produce blue colonies, thus permitting identification of colonies having pUC vector with cloned DNA segments. The cloning vectors belonging to pUC vector with cloned DNA segments. The cloning vectors belonging to pUC family are available in pairs with reversed orders of restriction sites relative to lac Z promoter. Puc8 and Puc9 make one such pair. Other similar pairs include pUc12 and puc13 or puc18 and puc19 (Figure 5.2).

Figure 5.2: puc19 cloning vector. As discussed above, in pBR322and Pbr327, the DNA is inserted at a site located in one of the two genes for resistance against antibiotics, so that it will inactive one of the two resistance genes. The insert bearing plasmid can be selected by their ability to grow in a medium containing only one of the two antibiotics and by their failure to grow in a medium containing both the antibiotics. The plasmids caring no insert on the other hand, will be able to grown in media containing one or both the antibiotics. In this manner, the presence of lac Z gene in pUC and resistance genes against ampicillin and tetracycline in pBR322 and pBR327 allow selection of E. coli colonies transformed with plasmids carrying the desired foreign cloned DNA segment.

5.6. YEAST PLASMID VECTORS Special vectors are also known for introducing DNA segments in yeast cells,

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a eukaryotic system that has been used extensively for developing genetically engineered yeast cells. Although E. coli plasmids or phages can be are used for transfer of genes to yeast cells, the frequency of transformation is rather low. For instance, yeast cells that are LEU could be transformed using chimeric plasmid carrying LEU + gene yeast. This involves integrative transformation having low frequency. To overcome this difficulty of low frequency transformation, effort were made to prepare a variety of vectors for yeast. The possible examples of yeast plasmid vector are: (i) Yip or yeast integrative plasmids, which allow transformation by crossing over and have no replication origin; (ii) Yep or yeast episomal plasmids, which carry 2 micron DNA sequence including the origin of replication and rep genes; (iii) YRp or yeast replication plasmids, which carry autonomously replicating sequence; this sequence is very common with many yeast genes so that stable transformation can be achieved by crossing over; (iv) YCp or yeast centromere (CEN) containing plasmids, which function as true chromosomes and segregate during mitosis and meiosis; (v) pYAC or yeast artificial yeast chromosome vector which carries both centromere and telomere sequences and, therefore, can be used to obtain artificial chromosomes. The above vectors have been extensively utilized for a study of yeast genome and are also used as shuttle vector, which allow genic sequences to be routinely transferred back and forth between yeast and E. coli cells, provided they contain origins of replication active in both yeast and E. coli. These vectors also have markers enabling the selection of E. coli cells or colonies transformed with these vectors. The vectors can therefore be amplified in E. coli and then used to transform yeast cells.

5.7. RETRIEVER VECTORS Retriever vectors are another class vectors, which are used to retrieve specific genes from the normal chromosome of an organism like yeast through recombination. They will multiply both in E. coli and yeast. In such a vector the gene in question is removed by enzymes to produce gapped vector having flanking sequences. Through recombination they acquire lost sequences from the host. Retriever vectors are very useful in isolation of genes from yeast for further molecular studies including sequencing.

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5.8. TI AND RI PLASMIDS AS VECTOR FOR HIGHER PLANTS Among higher plants, Ti plasmid of Agrobacterium tumefaciens or Ri plasmid of A. rhizogenes is the best-known vector. T-DNA, from Ti or Ri plasmid of Agrobacterium, is considered to be a very potential vector for cloning experiments with higher plants. This will involve the following steps: (i) foreign DNA has to be first cloned into T-DNA of Ti or Ri plasmids; (ii) modified hybrid T-DNA can be transferred to the genome of plant cells by Agrobacterium infection. Recombinant Ti plasmid can also be obtained by cloning of a large fragment of Ti plasmid in Pbr322. These recombinant Ti plasmids can then be used for transformation of higher plants. This system can be widely used, since Agrobacterium infects nearly all dicotyledonous plants. Such cloning in plants has proved to be of immense use to modify agricultural plants to increase their productivity. Monocotyledonous plants to which our cereal crops belong are however, difficult materials to be used for improvement through such techniques; some success, however, has been achieved in cereals also in recent years (Figure 5.3).

Figure 5.3: Octopine plasmid (a) Nopaline plasmid (b).

5.9. BACTERIOPHAGES AS VECTORS Bacteriophages provide another source of cloning vectors. Since usually a phage has a linear DNA molecule, a single break will generate two fragments, which are later joined together with generate two fragments, which are later joined together with foreign DNA to generate a chimeric phage particle.

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The Chimeric phage can be isolated after allowing it to infect bacteria and collecting progeny particles after a lytic cycle. The use of phage particles as vector imposes a limitation on the size of foreign DNA is too long, size of phage DNA may not be accommodated in phage head. In order to overcome this problem, those segments of DNA, which do not contain essential genes, may be removed. Such a technique has been followed in phage lambda to create smaller vector genome having single restriction site for the enzyme Eco RI. Since the reduced size also fails to be adequately packed in phage head, this automatically provides a selection method, in which only the chimeric particles will be obtained in the phage progeny, and the vector particles lacking cloned segment will be eliminated due to its reduced size.

5.10. LAMBDA PHAGE VECTORS Plasmid vectors described in the previous section are often used for cloning DNA segments of small size. However, while preparing a genomic library in a eukaryote, the cloned fragments should be large enough to contain a whole gene. This will also therefore can be screened without serious difficulty. The above properties and other requirements of cloning whole genome in eukaryotes are fulfilled by the phage lambda and cosmid vectors, the former permitting cloning of segments up to 20–25 kb long and latter accommodating segments up to 45kb long. Phage lambda however, is easier and more efficient for making genomic and cDNA libraries.

(a) lambda gt 10 and lambda 11 Lambda 10 and lambda gt 11 are modified lambda phages designed to clone cDNA fragments. The major difference between those two vectors is that gt11 is an expression vector, where inserted DNA is expressed as B- galactosidase fusion protein. Lambda gt 10 is a 43 kb double stranded DNA for cloning fragments that are only 7 kb in length. The insertion of DNA Inactivation cl+ gene generating a cl- bacteriophage. Non-recombinant lambda gt 10 an forms cloudy plaques an appropriate E. coli host, while recombinant plaques. Further, in an E. coli strain carrying hf La150 mutation only cl- phage will form plaques, become cl+ will forms lysogenes and will not undergo lysis to form any plaques. Recombinant lamba gt 10 plaques can thus be easily selected. Lambda gt 11 is a 43.7kb double stranded lambda phage for cloning DNA segments, which are less than 6 kb in length. Foreign DNA can be expressed as

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beta galactosidase fusion proteins. Recombinant lambda gt 11can be screened using either nucleic acid or antibody probes. The recombinant lambda gt 11becomes gal-, while non –recombinant lambda gt 11 remains gal+ so that an appropriate E. coli host, and with recombinant phage will form white or clear colonies and that with recombinant phage (gal+) will form blue colonies permiting screening in the presence of IPTG (inducer) and X gal (substrate).

(b) EMBL3 and EMBL4 (Replacement Vectors) EMBL3 and EMBL4 are the two vector that are designed so that a central nonessential part of 44 long phage can be replaced by a foreign DNA. Cleavage of the phage with an appropriate enzyme generates three fragments. The central fragment representing 40% of the phage genome is nonessential for propagation of the phage and can be replaced by foreign DNA that may be as long as 20– 30kb. Non –recombinant DNA resulting from the fusion of left arm and right arm will give a DNA that is too small for packaging, so that there is automatic selection against non-recombinant phages. EMBL3 and EMBL4 are two such replacement vectors used for preparing genomic libraries in eukaryotes with cloned fragments, 15–25 kb in size. The two vectors have polylinkers with reverse orders of restriction sites with respect to each other.

(c) Charon 34 and Charon 35 Charon 34 and Charon 35 differ from each other only in their central fragments and will accept fragments 9–20 kb long. They have more extensive range of restriction targets within their polylinkers than do EMBL3 and EMBL4. In these vectors, physical separation of lambda arms from the central fragments is required before ligation to donor fragments. In EMBL3 and EMBL4, removal of central fragments is not required, because there is a mechanism which ensures that 98% of packagable DNA are recombinants.

M13 phage as cloning vector for DNA sequencing M13 is a filamentous bacteriophage of E. coli and contains a 7.2 kb long single stranded circular series of cloning vectors which can be used for cloning of a wide variety of DNA. M13 phage has been variously modified to give rise to a M13 mp series of cloning vectors which can be used for cloning of a wide variety of DNA fragments particularly for the purpose of sequencing through sanger’s method od dideoxy chain termination. Cloning vectors of M13 mp series have a lac Z gene that complements gal host giving blue colonies. But if a foreign DNA segments is inserted at one of the polylinker sites associated with

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lac Z gene, it Inactivates lac Z gene and no complementation is possible. Thus on transformation, only white or clear plaques are obtained thus permitting selection of recombinant M13mp plaques. Once the foregin DNA is cloned in M13 vector, commercially available oligonucleotide primers are used for copying the insert in presence of dideoxynucleotides, so that fragments of different sizes with known termini are produced, permitting the determination of the sequence. Reversed order of the restriction sites in polylinker is present in a pair of vectors like M13mp8 and M13mp9 permitting sequencing from both the ends of double stranded DNA molecule. The extension of the commercial primer or cloned fragments in M13 vector in the presence of radioactively labeled nucleotides will also allow generation of radioactivity single stranded probes.

5.11. COSMIDS AS VECTORS Cosmids are plasmids particles, into which certain specific DNA sequences, namely those for cos sites are inserted. Since these cos sites enables the DNA to gel packed in lambda particle, cosmids allow the packaging of DNA in phage particle in vitro, thus permitting their purification. Like plasmids, these cosmids perpetuate in bacteria and do not carry the genes for lytic development. The advantages of the use of cosmids for cloning is that its efficiency is high enough to produce a complete genomic library of 10/6 ---10/7 clones from a mere 1 micro g of insert DNA. The disadvantages, however, is its inability to accept more than 40–50 kbp of DNA. Bacteriophage P1 system and F- factor based vectors can allow cloning of DNA segments, as large as 100 to 1000 kbp length. These are therefore preferred.

5.12. PHAGEMIDS AS VECTORS Phagemids are prepared artificially by combining features of phage with plasmids, as the name suggests. One such phagemids, which is commonly used in molecular biology laboratories, is pBluescript 11 KS, which is derived from pUc19 and 2961 bp long. The KS designation indicates the orientation of polylinker, such that the transcription of lac Z gene proceeds from the restriction site for KpnI to that for SacI. The detailed structure of pBluescript II KS. It may be noted that it has the following features: (i) a multiple cloning site (MCS) flanked by T3 and T7 promoters to be read in opposite directions on the two strands, (ii) an inducible lac promoter (Lac I), upstream of lac Z region, which

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complements with E. coli and provides the facility for selection of chimaeric vector DNA using the criterion of white colonies (iii) fl (+) and fl(-) origins of replication derived from a filamentous phaGE FOR RECOVERY OF SENSE (+) and antisence (-) strands of lac z gene, when host is coinfected with a helper phage; (iv) an origin of replication derived from plasmid, and used in the absence of helper phage; (iv) a gene for ampicillin resistance for antibiotic selection of chimeric phagemid vector.

5.13. P1 CLONING VECTORS FOE CLONING LARGE DNA SEGMENTS The bacteriophage P1 cloning vectors can allow cloning of 100 kbp long DNA segments with an efficiency of 10 raise to power 5 clones per micro g of insert DNA. Therefore, in their capability, they fall between YACs and cosmids. The vector with insert DNA is amplified in E. coli and several micrograms of cloned DNA can be recovered from 5–10 ml of exponential phase of E. coli cells. Two P1 cloning vectors with their essential elements.

F-Factor Based Vector (Which Behave Like a Phage) F-factor based vectors have recently been developed for cloning large DNA segments in E. coli. The cloning of large DNA segments is achieved by a method called chromosomal building, in which through repeated recombination size of cloned segments can be increased. These bacterial vectors will complement the YAC vectors for cloning segments larger than 100 kbp in length and offers some advantages over YAC system. These vectors have already been used for cloning large DNA segments from the bithorax gene of drosophila.

5.14. PLANT AND ANIMAL VIRUSES AS VECTORS A number of plants and animal viruses have also been used as vectors both for introducing foreign gene into cells and for gene amplification and expression in host cells. In the latter case, we may be interested only in getting increased quantity of the gene product and may not be interested in the integration of the foreign gene in the host genome. Some of the viruses that are commonly used as vectors will be described in this section.

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Plant viruses (cauliflower mosaic virus or CaMV and germiniviruses) Cauliflower mosaic viruses, tobacco mosaic virus (TMV) And geminiviruses are three groups of viruses that have been used as vectors for cloning of DNA segments. CaMV infects particularly the members of Cruciferac and has a double stranded DNA molecule, 8kbp in size, whose sequence is now known. Following infection, the virus spreads systematically throughout the plant in a very high copy number reaching up to 105 virus particles per cell. These features make Ca MV a suitable vector for transformation of higher plants although there are instance of use of other viruses also as vectors leading to the production of transgenic plants. Geminiviruses comprise a group of single stranded DNA plant viruses causing important diseases in cassava, maize and other cereals. They replicate via double stranded DNA forms and have been subdivided into two groups, one infecting monocots and transmitted by leafhoppers and the other infecting dicots and transmitted by whitefly. The dicot geminiviruses have two DNA segments in their genome, DNA A AND DNA B, both are essential for infection by mechanical inoculation. DNA A carries all genes responsible for replication. It has been shown that deletions in coat protein gene do not destroy infectivity of the virus, which means that long deletions in this region can be used to insert foreign DNA to obtain gene constructs that will be extremely useful as amplification and expression vectors in plants. Further, since the coat protiens are the most abundant protein synthesized by the virus, promoters of coat protein genes can be used for high level expression of cloned genes. CLV and TGMV have already been tested for their ability to allow multiplication and expression of a cloned gene.

Animal viruses A number of animal viruses are also used as vectors, either for the delivery of nucleic acid into the cultured cells followed by its integration with the host genome or for the amplification and high level expression of foreign gene, using the promoters from virus genes. These cloned genes can have a variety of uses including gene therapy in mammalian cells and synthesis of important proteins by cloned genes in a cell cultures.

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5.15. TRANSPOSONES AS VECTORS Transposones of Higher Plants (Ds, Ac or Mu 1 of maize) Ac and Ds are popular transposons in corn, and were earlier known to represent a transposon with short terminal repeats enclosing a long DNA segment, which measures more than 4500bp in Ac and about 400bp in Ds. Each possesses genes including the gene for transposase enzyme responsible for transposition. Part of this region can be deleted and the transposon can be used for cloning of forign DNA segment in much the same way as in other cases.

Transposons of Drosophila P element of Drosophila consist mainly of 31bp terminal inverted repeats enclosing a 3 kilobase proteins coding region, coding for transposase and transposase derived from other complete P element help their mobilization. In these deleted P elements, gene for transfer can be inserted to yield recombinant P element, which can be microinjected into the fertilized egg, along with normal P element. The inserted gene is transposed onto an embryonic chromosome.

5.16. ARTIFICIAL CHROMOSOME (YAC AND MAC) VECTORS FOR CLONING LARGE DNA SEGMENTS We know that in plasmids, sequences up to 10–15 kbp; in lambda phage sequences up to 22kbp and in cosmids sequences up to 40kbp can be cloned. Yeast artificial chromosome vectors, mentioned earlier allow cloning of sequences that are several hundred kilobase pairs long. These long molecules of DNA which may represent whole chromosomes can be cloned in yeast by ligating them to vector sequences that allow their propagation as linear artificial chromosomes. These long molecules of DNA which may represent whole chromosomes can be cloned in yeast by ligating them to vector sequences that allow their propagation as linear artificial chromosomes. These long DNA molecules can be generated and allow construction of comprehensive libraries in microbial hosts. With the isolation of mammalian telomere and centromere, mammalian artificial chromosomes (MACs) will also be produced further. YACs have two disadvantages. (i) Cloning efficiency is low (1000 clones/ micro g DNA against 106–107 clones/micro g DNA for cosmids), thus making it impractical to generate complete genomic libraries through the use of YACs; (ii)

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It is not possible to recover large amount of pure insert DNA from the individual clones; selective amplification of YACs DNA has recently allowed this problem to be overcome. Both these problems have been overcome bacteriophage P1 system.

5.17. PROMOTERS Promoters from non-plant sources express poorly in plants and promoters of non-animal sources express poorly in animals. Therefore, to obtain correct expression, genes transferred to plants should be linked to plant-specific promoters and those transferred to the plant should be linked to the plantspecific promoters and those transferred to animals should be linked to animalspecific promoters. These promoters can be interchanged so as to confer the transferred gene, a specific pattern of expression. To illustrate this some example of promoters used in plants and insects will be discussed.

5.18. NOPALINE SYNTHASE (NOS) PROMOTER FROM T-DNA This promoters 200 bp long ad contains several DNA sequence motifs which direct the expression of linked gene. The upstream element from –97 to –130 when duplicated, increases the expression three fold, suggesting that this may be an enhancer. The nos promoter is the most active in basal regions of plants, its activity slowing down in vegetative parts at the onset of flowering, even though in the flower its activity is increased.

Dual promoter of mannopine synthase (mas) genes 1 and 2 These promoters are present in a T-DNA fragments 467 bp long. The expression seems to be developmentally regulated and the linked genes seems to be developmentally regulated and the linked genes to be most active in the basal region of the plant. Expression is also induced on wounding. Since they are closely linked, if a marker gene is linked to one of these two promoters and a foregin gene is linked to the other promoter, selection for high expression of marker gene will lead to high level of expression of the foregin gene. •

35S RNA promoter of CaMV. This is the most extensively used promoter in a wide variety of plants. The promoter is 343 bp long and contains a strong transcriptional enhancer, which on duplication

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•

results in 10 fold increase in expression, even at a distance of 2 kbp. High expression is mainly observed in leaf tissue. Polyhedrin promoter from baculovirus: Baculovirus can synthesize polyhedron protein in infected cells, so that polyhedron makes up to 50% of the total protein in the cell, giving up to 1g of protein per liter of insect cell culture. In view of this, the promoter has been used to construct expression vector to allow high level of expression of any gene under the influence of this promoter. This is being used for developing biopesticides or even for production of specific chemicals in industry.

5.19. EXPRESSION CASSETTES The literal meaning of cassette is ‘a device containing film such as magnetic tape for insertion into an equipment like tape recorder, VCR, etc.’ Therefore by expression cassette, we mean gene constructs, which allow the insertion of foregin genes, either as transcriptional or translational fusions, behind specific promoters. pRT plasmids are typical in this connection and have been derived from Puc18/19. They consist of a series of vectors, which differ in polylinker sequences, each flanked by 35S promoter of CaMV on one end and a sequence for Poly A addition on the other end. A variety of marker genes have been inserted into these cassettes and their expression studied, both in protoplasts and in stable transgenic tissues.

Baculovirus, an expression vector system for insect cells Development of baculovirus expression vector system is based on its life cycle which involves the following steps; (i) in early phase virus particles are bubbled from infected cells and spread the virus; (ii) in the late phase (2–5 days), occluded virus units accumulate in the host nucleus and get embedded in protein polyhedral; (iii) insect is decomposed releasing polyhedral; (iv) polyhedral are ingestedby another insect, where polyhedral are dissolved releasing virus for multiplication. The use of baculovirus as a cloning vector involves following steps: (i) desired gene is cloned in a transfer vector under the control of polyhedron promoter; (ii) chimeric transfer vector is contransfected with wild type virus into the host cell; (iii) polyhedron gene of wild type is replaced by cloned gene through recombination; (iv) recombinant virus is plaque purified, since they will have polyhedral occlusion bodies; (v) the selected recombinant virus is multiplied and used foe expression of heterologous protein.

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5.20. VIRUS EXPRESSION VECTOR FOR MAMMALIAN CELLS A number of viruses have also been used as transfer or expression vectors for mammalian cells. These includes the following: (i) The first viruses used for mammalian cells included DNA viruses like papoviruses and adenoviruses, but since their capacity for foreign genes is small, they cannot be used for the expression of many genes which are fairly large in size; (ii) retroviruses are very useful vector, since they can infect broad variety if cell types and can be used for cloning large genes; however, being single stranded RNA, they cannot replicate without being integrated so that we will need replicating cells for expression or alternatively high-titer for fully differentiated cells; (iii) adeno-associated virus (AAV) overcomes the limitations associated with retroviruses even though, like retroviruses, it is an integrating virus, with 5kb single stranded DNA; it is nonpathogenic, and can be concentrated to high titer; (iv) herpes virus, is another important non – integrating virus of large size (150 kb), which can be used for expression pf large intact genes. Several of the above viruses have already been use and showed expression of cloned genes, but they need to further characterized for their long-term usage.

5.21. BINARY AND SHUTTLE VECTORS For the purpose of gene transfer in higher plants, a number of binary vectors were developed. These were based on the pPCV series of plasmids. These vectors contain a conditional mini-RK2 replicon, which is maintained and mobilized by trans-acting functions derived from the plasmid RK2 replicon. The plasmid RK2 was introduced into both E. coli as well as Agrobacterium to facilitate replication of binary vectors in both these hosts, so that the vector can be maintained and shuttled between both—hence the name shuttle vector also. Similar binary or shuttle vectors for maintaince and transfer between E. coli and yeast cells have also been designed (Figure 5.4).

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Figure 5.4: Shuttle vector.

5.22. AC-DS ELEMENTS The activator (Ac) and dissociation (Ds) elements are simple transposons of maize. It has 4.565 kb long autonomous sequence which transcribes 3.5 kb long GC-rich un-translated leader sequence. Four introns are spliced out from the primary transcript and 3.312 nucleotide long mature MRNA is obtained. Consequently 807 amino acids long protein is translated. Within the Ac element the terminal inverted repeats (TIR) of 11 bp are located at both the ends which play a key role in transposition. The protein synthesized by Ac is called transposase which causes transcription of Ac elements. The Ds (dissociation) is the defective element of Ac family. Due to deletion, non-autosomal elements are obtained from the autosomal elements. It activates the trans-acting transposase. The 11 bp long TIR is common to all Ds elements. The enzyme transposase cannot be expressed by the Ds element. But the transposase that is produced by Ac element is diffused and trans-acting. It causes trans- position of Ds elements. By detecting part of the region transposons can be used as vector for cloning a foreign DNA.

5.23. P ELEMENT The transposons of Drosophila are known as P elements. The size of different P elements vary The largest clement is 1907 bp long containing 31 bp long terminal inverted repeats at both the ends The complete elements move autonomously due to expression of transposase enzyme. The other P elements lack internal gene that produce transposase. The terminal and sub-terminal sequences cause transposition in them. A foreign DNA can be inserted into the transposase

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element to develop recombinant P elements which can be microinjected into the fertilized egg along with normal P element The normal P supplies transposase to the recombinant P element. Here the non-autonomous element acts as a transformation vector. Consequently, the foreign gene inserted into embryonic cell is trans- posed onto an embryonic chromosome of Drosophila. Gierl et al. (1989) have reviewed the transposable elements of maize. A recombinant P element was prepared by inserting DNA fragment of rosy gene (and a marker gene for identification of transgene). It was inserted into a plasmid. Similarly a P element, lacking the ability for integration was inserted into a plasmid to encode transposase. Both the plasmids were co-injected into the embryo of Drosophila melanogaster mutant in rosy gene. It was found that 50% progeny of D. Melanogaster were rosy progeny. This shows the successful transfer of a foreign gene into a cell b h P element (Gierl et al., 1989).

5.24. EXPRESSION VECTOR In a number of cloning vectors described earlier in this chapter, the main utility has been to clone a gene or a DNA sequence. In other words, they are used for obtaining millions of copies od the cloned DNA segments, for further use in genetic engineering experiments or for further basic studies. With this objective in mind, the cloned genes in this vector need not express themselves either at the transcription level or at the translation level. But, when the cloned gene is used for transformation to generate transgenic plants or animals or the production of microbes to be used in industry, than the cloned gene must be expressed. Sometimes, a high level of expression od gene is desirable, if the product of cloned gene is to be recovered as a commercial product. The objective can be achieved through the use of promoters and expression cassettes.

6 Techniques of Genetic Engineering

6.1. GENE CLONING It is an essential process in the success of the genetic engineering were insertion of a specific fragment of foreign DNA into a cell, through a suitable vector, so that it can replicate independently and transferred to progenies during cell division (Figure 6.1).

Figure 6.1: Gene cloning.

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6.2. CLONING IN PROKARYOTES The DNA fragment to introduced into the host cell is called insert DNA, desired DNA, target DNA or foreign DNA. Such gene/DNA fragment is taken out from an organism or gene library, i.e., a random collection of cloned DNA fragments in vectors that consist of all genetic information of that organism concerned. However, getting a desired DNA is not so easy because the cellular DNA is very large and the position of desired DNA is not accurately known. Libraries of such cloned fragments prepared.

6.3. STRATEGIES OF RECOMBINANT DNA TECHNOLOGY There is no single method of recombinant DNA technology, but it involves several steps. Maniatis et al. (1976) have described the basic techniques of gene cloning. The principal steps of gene cloning: (i) (ii) (iii) (iv) (v) (vi)

Isolation of DNA of known function from an organism (A). Enzymatic cleavage (B) and joining (C) of insert DNA to another DNA molecule (cloning vector) to form a recombinant DNA (i.e., vector + insert DNA) molecule (D). Transformation of a host cell, i.e., transfer and maintenance of this rDNA molecule into a host cell (E). Identification of transformed cells (i.e., cells carrying rDNA) and their selection from non-transformants. Amplification of RDNA (F) to get its multiple copies in a cell. Cell multiplication (G) to get a clone, i.e., a population of genetically identical cells. This facilitates each of clones to possess multiple copies of foreign DNA.

6.4. GENE LIBRARY A gene library is the collection of different DNA sequences from an sequence has been cloned into a vector for ease of purification, storage and analysis. There are two types of libraries on the basis of source of DNA used: (i) genomic library (where genomic DNA is used), and (ii) cDNA library (where CDNA or MRNA). A gene library should contain a certain number of recombinants with a high probability of containing any particular sequence.

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6.5. GENOMIC LIBRARY It is a collection of clones that represent the complete genome of an organism. All fragments of DNA inserted into vectors for further propagation into suitable host represents the entire genome of an organism. For construction of a genomic library the entire genomic DNA is isolated from host cells/tissues, purified and broken randomly into fragments of correct size for cloning into a suitable vector. There are two basic ways of fragmenting genomic DNA randomly: physical shearing (e.g., pipetting, mixing or sonication) and partial restriction enzyme digestion, (by using limiting amount of restriction enzyme the DNA is not Using these methods genomic DNA is broken randomly into smaller fragments. A total of 6 random DNA fragments obtained after physical shearing. The vector isolated from bacterium is also digested with the restriction enzyme which digests genomic DNA. The fragments of genomic DNA are inserted into the vector. Each vector consists of different fragments of DNA. The recombinant DNA molecules are transferred into bacterial cells or bacteriophage particles are assembled. The genomic library for organisms with smaller genome size (e.g., E. coli) can be constructed in plasmid vector. Only 5,000 clones (of the average size of 5,000 bp) results in 99% chance of cloning the entire genome of 4.6 x 10°bp. In addition, libraries from organisms with larger genomes are constructed using phage, Cosmid, BAC or YAC vectors.

6.6 CDNA LIBRARY The mRNAS are highly processed representatives of genes which express under specific conditions. The MRNAS cannot be directly cloned because they are unstable hence MRNAS are converted into CDNAS. The made from library complementary or copy DNA (CDNA) is called cDNA library. The CDNA library represents the DNA eukaryotic of only organisms, prokaryotic ones. Because genomic DNA of eukaryotes contains introns, regulatory not regions, repetitive sequences Therefore, establishment of library genomic eukaryotes is not meaningful. The cDNA library can be constructed by using mRNA because mRNAS are the highly processed, intron-free representatives of DNA only coding having sequence.

6.7. ISOLATION OF MRNA It is much difficult to isolate a specific mRNA if it is in low amount in the cell. The

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majority of mRNA sequence in eukaryotic cells contains a long polyadenylated tract (i.e., about 100A residues) at their 3’ termini (Kates 1970). By this virtue they can be separated from other types of RNAS. Therefore, MRNA binds with an oligo-dT cellulose affinity column or poly-U sepharose from which it can be eluted. Its integrity can be studied using gel electrophoresis which allows mRNAs to be size-fractionated by recovering chosen regions of the gel lane. Commercial preparations of both these substrates are now available. The mRNA preparations can be enriched for the desired molecules by fractionation according to their size by using sucrose density gradient centrifugation (Boffey, 1987).

6.8. PREPARATION OF CDNA. 6.8.1. Reverse Transcription Reverse transcriptase is required for the synthesis of DNA copy of an MRNA molecule. The mRNAs are reverse transcriptase enzyme and four deoxynucleotide triphosphates (DNTPS) i.e., DATP, DGTP, DCTP and DTTP. The oligo-dT primer binds to polyadenylated tail that contains -AAAA(n) residues and provides free – OH site for reverse transcription. Reverse by (b) treated with oligo-dT primer, transcriptase adds complementary DNTPS oneto-one free 3’ -OH site of primer, forms single strand of DNA resulting in RNADNA hybrid. Cellular DNA and total RNA inhibit reverse transcriptase activity. Therefore, it is necessary that MRNA must be in pure form before cloning. The oligo-dT segments contain between 10–20 nucleotides in length that hybridize to the poly A tract on mRNA. The oligo-dT primer ensure the initiation of cDNA synthesis at 3, terminus of RNA-DNA hybrid. Cellular DNA and total RNA inhibit reverse transcriptase activity.

6.9. OLIGO-DG PRIMER Oligo-dG primer is added in the reaction mixture and (e) Ade temperature is maintained to about 55°C. This facilitates the binding of dG to oligo-dC tails formed on cDNA. Template for the synthesis of double stranded cDNA in the presence of Now cDNA acts as a DNA polymerase I or AMV (Avian myeloblastosis virus) reverse transcriptase.

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6.10. SYNTHESIS OF SECOND STRAND OF CDNA Klenow DNA polymerase I and DNTP precursors are added to the reaction mixture. This enzyme adds complementary nucleotides to the cDNA one by one to 3’ -OH site of primer. Consequently, from one cDNA, double stranded DNA clone is synthesized the length of which is similar to that of the mRNA molecule initially used. This is used for cDNA cloning and construction of cDNA library and in gene cloning experiments. The double stranded cDNA is identical to the gene which codes for mRNA (only in prokaryotic genes). However, the majority of eukaryotic genes contain introns (the non-interrupt their coding sequences. Methods of removing CDNA in eukaryotes have been described earlier. CDNA can be radioactively labeled by nick translation and used as a identification of DNA fragments containing the gene. By using these procedures a cDNA clone bank is built up which hybridization probe.

6.11. INSERTION OF DNA INTO VECTOR The DNA thus isolated as above or procured from DNA or gene bank is fragmented by using the specific restriction enzyme to vector (e.g., PBR322) is also treated with the same restriction enzymes so that the cohesive ends generated may have complementary residues similar to foreign DNA (Figure 6.1B). The plasmid contains amp and tet marker genes to confer resistance against antibiotics, ampicillin and tetracycline respectively. Some time after cleavage by restriction enzymes and ligation of foreign DNA fragment with the plasmid a few important genes are destroyed in develop specific cohesive ends. Example, BamHI destroys tet genes. The sticky ends can forms a phosphodiester linkage between free 5’ phosphoryl and 3’ OH group is added along with ATP at 4–10ºC for a long incubation time. A number of products highly heterogeneous mixture of recombinant DNA molecules together with parental plasmids are formed from this reaction which are highly heterogeneous mixture of recombinant DNA molecules together with parental plasmids. For insertion of double stranded cDNA into a cloning vector it is necessary to add to both termini single stranded DNA sequence which should be complementary linear vector. In order to get efficient formation of recombinant DNA molecules, addition of sticky to a tract of DNA at the termini of ends on both termini is necessary. There are two methods for generation of cohesive ends on the double stranded cDNA, use of linkers and homopolymer tails.

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6.12. USE OF RESTRICTION ENZYME LINKERS Linkers are the chemically synthesized double stranded DNA oligonucleotides containing on it one or more restriction sites for cleavage by restriction enzymes, e.g., Eco RI, Hind II, Bam HI, etc. Linkers are ligated to blunt end DNA by using T4 DNA ligase. Both the vector and DNA are treated with restriction enzyme to develop sticky ends. The staggered cuts, i.e., sticky ends are then ligated with T4 DNA ligase with very high efficiency molecules are produced.

6.13. USE OF HOMOPOLYMER TAILS For the first time Jackson et al. (1972) applied this method to construct a recombinant DNA by inserting phage A DNA into SV40 DNA by using dA-dT homopolymers. Using terminal transferase the synthesis of homopolymer tails of the defined at both 3’ termini of double stranded DNA and vector can be done. In the presence of length precursor DATP, terminal transferase helps to add poly-dA at 3’ termini of vector. Likewise the same enzyme adds poly-T at 3’ termini of DNA molecule, when precursor TTP is present. The linearized vector having tails is incapable of recircularization, unless ligated to a double stranded DNA fragment. The vector and DNA tails are annealed.

6.14. TRANSFER OF RECOMBINANT DNA INTO BACTERIAL CELL Several methods have been developed for introduction of recombinant DNA molecule into host cells. According to types of vectors and host cells, the methods are gene transfer into host cells are briefly discussed.

6.15. TRANSFORMATION Once a mixture of recombinant DNA is obtained it is allowed to be taken up by the suitable bacterial cells. Originally the transformation procedure was developed by Mandell and Higa (1979). The strains of E. coli usually do not have restriction systems, hence it degrades foreign DNA. To escape from degradation exponentially growing cells are pretreated with CaCl, at low temperature; thereafter DNA is mixed up. The event of entering the plasmid containing foreign DNA fragment into a bacterial cell is known as transformation.

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6.16. TRANSFECTION Transfection is the transfer of foreign DNA into cultured host cells mediated through chemicals The charged chemical substances such as cationic liposomes, calcium phosphate are taken and mixed with DNA molecules. The recipient host cells are overlayed by this mixture. Consequently the foreign DNA is taken up by the host cells. For detail see transfer in preceding section. DEAE dextran liposome-mediated gene For the first time phage A was used to transfer the foreign DNA into E. coli cell, therefore. it is often termed as transfection (a hybrid of transformation and infection). The efficiency of transformation is not high as it is influenced by bacterial strain and size of foreign DNA. It has been found that the efficiency of this process could generate about I0 tr ug) of cloned circular plasmid (e.g., PBR322). It has not been possible to achieve efficiencies of over 10°transformants per g plasmid. If found that the efficiency of this process could generate about 10 transformants per mierogram (ug) of cloned cireular plasmid (e.g., pBR 322). It has not been possible to achieve efficiencies of over 108 transformants per g plasmid. If linear DNA is transformed it is almost completely insufficient in transformation.

6.17. SELECTION (SCREENING) OF RECOMBINANTS After the introduction of recombinant DNA into suitable host cells, it is essential to those cells which have received the recombinant DNA molecules. This process is called screening or selection. Selection of recombinant DNA cells is based on expression or non-expression of certain characters or traits. The vector or foreign DNA present in recombinant cells expresses the characters, while the non-recombinants do not express the traits. Following which are used mainly for selection of recombinants in E. coli are some of the methods.

6.18. DIRECT SELECTION OF RECOMBINANTS The above features ease the direct selection of recombinant cells. If the cloned DNA itself codes for resistance to the antibiotic ampicillin (amp) the recombinants can be allowed to grow on minimal medium containing ampicillin. Only such recombinants will grow and form colony medium that contain amp gene on its plasmid vector. Following this procedure you cannot say whether the recombinants growing on such medium contain relegated plasmid vector or

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contain recombinant plasmid plus foreign DNA fragment. Because amp gene is present in both the recombinants on pBR322.

6.19. INSERTIONAL SELECTION INACTIVATION METHOD This is more efficient method than the direct selection, In this approach one of the genetic traits is disrupted by inserting foreign DNA Antibiotic resistance genes acts as a good insertion inactivation system. As discussed in earlier section that plasmid PBR322 contains two antibiotic resistance genes, one for ampicillin (amp gene) and the other for tetra- cycline (ter” gene). If the target DNA is inserted into tet” gene using BamHI, the property of resistance to tertracycline will be lost. Such recombinants would be test-sensitive. When such recombinants (containing target DNA in tet gene) are grown onto medium containing tetracycline, they will not grow because their tetR gene has been inactivated. But they are resistant to ampicillin because amp gene is functional. On the other hand the self-ligated recombinants will show resistance to tetracycline and ampicillin. Therefore, they will grow on medium both the antibiotics. To ascertain the presence or absence of tetR gene in the inserted DNA fragment in plasmid replica plating is done from the master plate. Bacterial colonies of master plate are gently pressed with sterile velvet so that a few cells of each colony may adhere on it, which is then pressed on other plate containing the nutrient medium amended with tetracycline. Plates are incubated for the growth of bacterial colonies. The appearance of colonies is compared with a master plate and those colonies that fail to grow on a plasmid which had insert DNA in the tet gene of plasmid and had destroyed tet gene replica plate can be said to have.

6.20. BLUE-WHITE SELECTION METHOD It is also a powerful method used for screening of recombinant plasmid. In this method a reporter gene lacZ is inserted in the vector. The lacZ encodes the enzyme B-galactosidae and contains several recognition sites for restriction enzymes. Beta-galactosidase breaks a synthetic substrate, X-gal (5-bromo-4chloro-indolyl-B-D-galacto-pyranoside) into an insoluble blue colored product. If a foreign gene is inserted into lacZ, this gene will be inactivated. Therefore, no blue color will develop, because B-galactosidase is not synthesized due

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to inactivation of lacZ. Therefore, the host cells containing rDNA will form white colored colonies on the medium containing X-gal, whereas e other cells containing non-recombinant DNA will develop the blue colored colonies. On the basis of colony color the recombinants can be selected.

6.21. COLONY HYBRIDIZATION (NUCLEIC ACID HYBRIDIZATION) TECHNIQUE After the growth of cells on nutrient medium it is likely that a few cells may have specific DNA as desired among several thousand cells. How is it A portion of host DNA possible to pick up those cells which have the desired DNA. To make it Separation of strand easy, the “colony hybridization technique” has been developed by Grustein and Hogness (1975). It is suitable for use with plasmids. An analogous hybridization technique, has been given for use with phages (Benton and Davis, 1977). Single stranded DNA Hybridization plaque method, network bases 32 p-DNA Probe hybridizes the of (a) DNA Probes: The colony hybridization technique is based on Hybridization of desired DNA with a DNA probe the availability of a radiolabeled DNA probe. A DNA probe is the radiolabeled (2PO) fragment of DNA molecule (20–40 bp) which is complementary at least to one part of desired DNA Generally probe DNA is labeled with 3PO while I emits y-rays. In addition to using radioactive isotopic elements, isotopic sulfur or fluorescent molecules may also be used. Therefore, a DNA probe recognizes A, T, G and C nucleotides and combines with the complementary sequences of the target DNA. A and T of the probe combine with T and A of target DNA and vice versa, Similarly, G and C of probe combine with C and G of target DNA and vice versa. The use of DNA probe in searching the functional target DNA is given in nucleic acid hybridization technique small or some times with 121. The 3P liberates B-particles. The small sized DNA probe easily catches the target DNA for hybridization than the long sized probe. Besides, a 32p-DNA than the target DNA molecules. Moreover, the DNA probe is very specific to its target DNA; this specificity is called stringency.

6.22. IN VITRO TRANSLATION In vitro translation is now used as a method to confirm the identification of recombinant clones. Nagata et al. (1980) have used primary translation screened by isolating the interferon gene from total leukocyte poly A+RNA. Translation of poly A+RNA is done when it is microinjected into 00cytes from

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the toad, Xenopus laevis with the result of secretion of interferon into culture medium. Interferon is detected by its antiviral activity. It is supposed that the number of interferon RNA may be between 10 to 10, therefore about 104 transformants must be repeated to get at least single clone. Thereafter, DNA is immobilized on nitrocellulose or diazobenzyloxymethyl cellulose paper to isolate complementary MRNA by nucleic acid hybridization as described earlier. The RNA can be eluted from the cDNA-RNA hybrid and microinjected into oocytes and the ranslation products are detected by immunoprecipitation techniques.

6.23. IMMUNOLOGICAL TESTS The immunological techniques are the final test analogous to colony hybridization technique as described earlier. It is an alternative screening procedure which relies on expression, and generally applicable approach to potentially a very powerful method since the only absolute requirement is that the required MRNA encodes a protein for which a suitable antibody is available (Williams, 1981). Two in situ techniques given by Broome and Gilbert (1978), and Erlich et al. (1978) are in current use. In the immunological test, instead of radiolabeling of DNA molecules, antibodies (immunoglobulins) are used to identify the colonies or plaques developed on master plates that synthesize antigens encoded by the foreign DNA present in plasmids of the bacterial clones. For this purpose a special vector, known ds expression vector, is designed where the foreign DNA is transcribed and translated within the bacterial eel (Glover, 1984). The growth medium containing specific anti-serum may help detection of viable immunoprecipitate (precipitin) around the colonies or plaques.

6.24. BLOTTING TECHNIQUES DNA, RNA and proteins are separated by blotting techniques. These are described in the following sections.

6.25. SOUTHERN BLOTTING TECHNIQUES A method, developed genes in a DNA restriction fragment is called as Southern blotting technique. Southern blots can easily provide a physical map of restriction sites within a gene located normally and reveal the number of copies of the gene in the genome, and the degree of similarity of the gene when compared with

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the other complementary genes. a molecular biologist E.M. Southern (1975) for analyzing the related on a chromosome. The procedure starts with digestion of DNA population by one or many restriction enzymes (Figure 6.11). Consequently, DNA fragments of unequal length are produced. This preparation is passed through agarose gel electrophoresis which results in separation of DNA molecules based on their size. DNA restriction fragments present in gel are denatured by alkali treatment. Gel is then put on top of the buffer saturated filter paper. Upper surface of the gel is covered with nitrocellulose filter and overlaid with dry filter paper. The dry filter paper draws the buffer through the gel. Buffer contains single stranded DNA. Nitrocellulose filter binds DNA fragments strongly. After baking at 80°C, DNA fragments are permanent and fixed to the when come in contact o1 nitrocellulose filter. Then the filter is placed in a solution containing radiolabeled RNA or denature DNA probe of known sequences. These are complementary in sequence to the blot-transferred DNA. The radiolabeled nucleic acid probe hybridizes the DNA on nitrocellulose filter. The filter is thoroughly washed to remove the probe. The hybridized regions are autoradiographically detected by placing the nitrocellulose filter in contact with a photographic film. The images show the hybridized DNA molecules. Thus, the sequences of DNA were recognized following the sequences of nucleic acid probe.

6.26. NORTHERN BLOTTING TECHNIQUE Southern blotting technique could not be directly applied to the blot transfer of mRNA separated by gel electrophoresis, because RNA was found not to bind with nitrocellulose filter Alwine’s et al (1979) devised a technique in which RNA bands are blot transferred from the gel to chemical reactive paper. An aminobenzyloxymethyl cellulose paper prepared from Whatman filter paper No. 540 after a series at uncomplicated reactions, is diazotized and rendered into the reactive paper and, therefore, he comes available hybridization with radiolabeled DNA probes Th hybridized bands are found by antaradiography Thus, Alwine’s method extends that of Southern method and tor this reason it has been grunt the jargon Northern blotting. “There is not this northern or western like Southern.” These blot transfers are reusable because of the firm covalent bonding of RNA to the reactive paper. Effective in binding the DNA as well Small fragments of DNA can most effectively be transferred to the diazotized paper derivative to nitrocellulose. These techniques were more advanced and demonstrated that MRNA bands can also be blotted directly on nitrocellulose paper and appropriate condition.

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6.27. WESTERN BLOTTING Towbin et al. (1979) developed the western blotting technique to find out the newly encoded protein by a transformed cell. Its working principle lies on antigen-antibody reaction: hence is an immuno detection technique. In this method radiolabeled nucleic acid probes are not used. This technique follows the following steps: (Extraction of protein from transformed cells. (I) Separation of protein by using SDS-PAGE (sodium dodecyl sulfate polyacrylamide electrophoresis) where SDS acts as solvent for electrophoresis. Transfer of electrophoresed gel in a buffer at low temperature (40°C) for half an hour (h) Blotting of proteins on nitrocellulose filter paper of nitrocellulose filter, Whatman filter and coarse filter in transfer buffer; (i) Soakind of nitrocellulose filter, Whatman filter and coarse filter in transfer buffer; (ii) Placing of Whatman filter paper on a cathode plate followed by stack of coarse filter Whatman filter, electrophoresed gel, nitrecellulose filter, Whatman filter paper, coarse filter stack, Whatman filter and anode plate transferred; (iii) Putting the complete set up in transfer tank containing sufficient transfer buffer. (iv) Application of an electric field (30 V overnight for 5 hours) to cause the migration of proteins from the gel to nitrocellulose filter and binding on its surface. (The nitrocellulose filter has exact image of pattern of proteins as present in the gel. This type of blotting is called western blotting.) (v) Hybridization of proteins by using radiolabeled antibodies (I123- antibodies) of known structure, isolated from the rabbit. (vi) Washing of nitrocellulose filter with a wash solution (Tris-buffered saline + tween 20) to facilitate the removal of unhybridized antibodies. (vii) Detection of hybridized sequences by autoradiography. The dots of diagram shows the presence of desired protein.

6.28. RECOVERY OF CELLS Colonies complementary to nitrocellulose signal are recovered from the master plate 2d cultured in nutrient broth in order to get the sufficient bacterial clones containing recombinant plasmid. However, in some instances the foreign DNA is cleaved enzymatically from the vector molecules. Sometimes aim lies to achieve expression of gene within the bacterial call if it encodes for valuable polypeptides.

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6.29. EXPRESSION OF CLONED DNA Factors taken into account for expression are: (i) Supply of prokaryotic promoter for expression of eukaryotic genes, (ii) Supply of ribosomal binding sites for the cloning vector, (iii) Removal of introns from mRNA obtained from eukaryotic genes, and (iv) Inhibition of gene responsible for degradation of foreign protein within the bacterium The genes of eukaryotes differ in different ways from those of E. coli. Therefore, the eukaryotic genes must be modified in such a way that they could resemble the prokaryotic, genes and express in E. coli. To begin with transcription in prokaryotes the binding of bacterial RNA polymerase to a promotor region of the DNA immediately before (or upstream of) the gene is essential.

6.30. SHINE-DALGARNO SEQUENCE Prokaryotic mRNA, have a sequence upstream from the initiation codon that play a role in attachment of the 30S ribosomal subunit to the MRNA. This sequence (about 3–9 bases long) is about 3–12 bases upstream from the initiation codon which is also complementary near the 3’ terminus of 16S TRNA (Shine and Dalgarno, 1975). The name of this sequence was coined as the Shine-Dalgarno sequence after the name of the discoverer. For the expression of eukaryotic gene within E. coli, presence of a Shine-Dalgarno sequence in bacteria is to a sequence.

6.31. DETECTION OF NUCLEIC ACIDS There are two approaches for labeling the nucleic acids to be used as DNA probe: radioactive labeling and non-radioactive labeling.

6.32. RADIOACTIVE LABELING Methods for in vitro introduction of radioactivity in nucleic acids was developed during 1970s through metabolic labeling. In this method large amount of radioactivity required. However, was this method was time consuming and laborious. The methods developed later on utilized phage T4 polynucleotide kinase (which transfers y-PO of ATP to 5’-OH terminus in DNA or RNA) or E. coli DNA polymerase I (which replaces normal nucleotides). The nucleic acids

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are labeled in vitro by using the following methods: a normal nucleotide of double stranded DNA with α – 32P labeled nucleotide.

Nick Translation This method is used to label the double stranded DNA probes. If there a is no nick, a nick is made on double stranded DNA by using DNase (in a buffered solution containing DNTPS) which exposes 3’-OH group on one strand of DNA. All the four nucleotides are alpha position of phosphate group. (c-32P-dNTPs) at radiolabel generally only one nucleotide, i.e., a- P-dCTP is radiolabeled, whereas the other nucleotides are unlabeled. E. coli DNA polymerase I is used in this method. This enzyme shows 5’ 3’ hydrolyses the nucleotides from 5’ side of the nick. Exocatalytic activity and either removes or Nucleotides are eliminated from 5’end and simultaneously added at 3’ side resulting in movement of the nick along the DNA. Therefore, this method is called nick translation. The reaction mixture includes probe DNA to be labeled. 2-P-dTP,IHUK u s 0e the other three DNTPS, DNase 1 and E. coli DNA polymerase I. It is incubated at 14°C for 2–4 hours. The hybridized DNA is separated from the rest of DNTPS and used to hybridize the DNA.

6.33. RANDOM PRIMED RADIOLABELING OF PROBES This technique and Vogelstein (1983, 1984). The restriction fragments purified by gel electrophoresis from denatured linear or circular DNA are used to prepare probes. In this method primers are used for DNA synthesis but DNA nicking is not done. The purified DNA to be labeled is mixed in a buffer and heated at 100°C so that it may be denatured. Then it is put on ice to lower down the temperature. The reaction mixture (containing Klenow fragment of E. coli, DNA Polymerase I, one radiolabeled nucleotide, e.g., O- P-dCTP and three unlabeled nucleotide triphosphates) is incubated at 37°C for 30 minutes to 16 hours. Since, Klenow DNA Polymerase I lacks 5’3’ exonuclease activity, the radiolabeled probe is synthesized by primer extension but not nick translation as described earlier. The hybridised DNA is separated from unincorporated nucleotides and used as probe. Probes Developed by PCR: This method is advantageous because very small amount of template DNA can be used. The method of operation is the same as described. In this method the reaction mixture includes linear template DNA (to be labeled), amplification buffer Taq polymerase, primers, one radiolabeled and three unlabeled dNTPs. The hybridised DNA is isolated and used as probe.

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6.34. NON-RADIOACTIVE LABELING Mostly, non-radiolabeled DNA probes are also used to detection of desired DNA. There are three systems of non-radioactive routes such as using horseradish peroxidase, biotin probe and steptavidin probes as described below.

Horseradish peroxidase (HRP) method The HRP is a plant-derived enzyme which covalently linked to DNA. Chromogenic is substrates (such as chloronaphthol) or chemiluminescent substrates (that emit light after reacting with DNA (probe enzyme, e.g., luminol) are used for detection of HRP-linked DNA. Chloronaphthol yields an insoluble purple product in the presence of peroxide and HRP. Consequently, presence of DNA is detected. Similarly, HRP oxidizes luminol and emits luminescence by using X ray film; hence luminescence can be detected.

6.35. DIGOXIGENIN (DIG) LABELING SYSTEM Digoxigenia is a cardenolide steroid derived from a plant Digitaria as a hapten. The presence of digoxigenin can he detected by an antibody associated with an enzyme (anti digoxigenin-alkaline phos- phatase conjugate). The probe is labeled with digoxigenin (11) DUTP which is a nucleotide tri- phosphate analog. This analog containing digoxigenin moiety is incorporated into DNA by nick translation or random prime labeling. The DNA acts as probe. Anti DIG (antibody to digoxigenin) conjugated with alkaline phosphatase detects the DIG labeled probe through enzyme-linked immunoassay. A chromogenic substrate [such as 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitrobutane tetrazolium chloride (NBT)] is added which reacts with alkaline phosphatase and produce purple blue color (Figure 6.14). A chemiluminescent substrate such as dioxetane is also used with digoxigenin streptavidin systems. There are several derivatives of digoxigenin which emit light after enzymatic activation by alkaline phosphatase. The denatured DNA present on Southern blots is hybridized by labeled probe. The membrane is placed in detection buffer containing antidigoxigenin (20 pg/m) and bovine serum albumin (BSA) (5% w/v) and incubated at 37°C for 1 hour. After incubation membrane is washed thrice. Enzyme activity of alkaline phosphatase is detected by using BCIP (0.17 mg/ml) and nitrobutane tetrazolium chloride (NBT) (0.33 mg/ml) activation purple/blue color develops.

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6.36. BIOTIN-STREPTAVIDIN LABELING SYSTEM Biotin is vitamin H and avidin is found in egg white. Principle of this system is based on the interaction between biotin and the glycoprotein avidin. A biotincontaining nucleotide analog (biotin-141ATP) or probe is prepared through nick translation or random priming methods. Streptavidin is conjugated to alkaline phosphatase. The biotinylated probes detected by specific binding of streptavidin conjugated alkaline phosphatase. Thereafter, a chromogenic or chemiluminescent substrate is added that reacts with alkaline phosphatase and detects the hybrids. The intensity of color is proportional to the amount of biotin present in hybrid DNA. This method detects the hybrids much faster than the radioactive probes. But very small number of probes (20 nucleotides) consists of only a small number of biotinylated sites. Therefore, intensity of signals is limited or biotin as dye.

6.37. GENE CLONING IN EUKARYOTES Most of studies on gene cloning have been carried out in bacteria. In addition, many difficulties are associated with them, for example, (i) correction of introns of eukaryotic MRNAS, (ii) failure of transfer of equal number of plasmids into daughter cells during cell division and yield of two types of cells, one with plasmid and the second without plasmid, post-translational modification (e.g., inhibition of proteolysis, addition of oligosaccharides to specific sites on the polypeptide chain, the glycosylation), (iii) threat for hazardous effects and (iv) expression to ensure the presence of a stable plasmid in the bacterium when applied commercial applications. With these prospects the attraction of the use of eukaryotic cells is obvious. In recent years, considerable efforts have been made for the improvement of crop plants through genetic engineering. Many works on various aspects of building up of vectors and expression of insert genes in eukaryotic cells are in progress. Some of them are briefly described in this. • •

Plant cells large number of plants ranging: There is a single cell to multicellular forms. Success has been achieved in some plants. Yeasts: Generally bacterial cultures are on a guitable for the production of polypeptides.

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6.38. TRANSFORMATION IN FILAMENTOUS FUNGI Transformation through plasmid of E coli was first described in protoplasts of S. cerevisiae (Himnen et al., 1978) and then in N. crassa. However, the situation in filamentous fungi is different from the others as thcre is no convincing evidence for autonomously replicating plasmids. But sequence having functions similar to ARS have been isolated method of transformation is similar to for plant cells, Following arc the examples where transformations in protoplasts have been carried out: A. nidulans, A. niger, A. oryzae, Cephalosporium acremonium, Coprinus cinereus, Glomerella cingulata, Gaeumannomyces gruminis, Mucor sp. Penicillium chrysogenum, Septoriu nodorum, etc.

Application of transformation of fungal protoplasts Mainly the transformation approaches are used for (i) the analysis of promoters and construction of expression vectors, and (ii) biotechnological application involves the construction of vector which have different pieces of promoters. These can be cut by restriction enzymes and joined with DNA ligase. By this method changes in promoter region are also possible and this can be achieved through mutagenesis. Thus, a strain can be improved in the expression of foreign genes in fungi. Analysis of promoter overproduction of the products (Peberdy, 1989).

6.39. GENE TRANSFER IN DICOTS BY USING AGROBACTERIUM TI-DNA AS VECTOR It is not possible to transfer Ti-plasmid into plant cell because of its large size, most likely up to 235 Kb. Therefore, T-DNA is excised and used as foreign DNA for expression in plant cells. From bacteriological point of view opine production and development of gall are not required for the successful integration and expression of DNA. Hence, replacement of nos and ops genes with a foreign DNA fragment retaining the opine synthase promoter for expression of DNA DNA fragment has no harm. Schell and Van Montague (1983) undertook some experiments where genes for resistance to kanamycin and methotrexate expressed in cultured callus cells. Generally speaking, promoters for the expression of foreign DNA. It has become possible that specific tissues of plants when they are with specific regulated promoters.

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6.40. GENE TRANSFER IN MONOCOTS The limitation of Ti-plasmid is that it is specific only to the dicot plants. A. tumifaciens does not induce tumor in monocot plants. However, most of the major crops including cereals are monocots. Hooykaas Van et al. (1984) discovered that A. tumifaciens could transfer T-DNA into certain monocots with the consequences of expression of opine gene within the plant cell without inducing the tumor. This discovery made it possible that T-DNA can be expressed into cells of monocot plants.

6.45. Plant Cell Transformation Plant cell suspension culture coupled with DNA transfer techniques has become a new field of gene manipulation in plants. Cell suspension cultures have been the traditional source of plant material for biochemical selection of plant cell mutants, such as (d) numerous cell lines resistant to various amino analogs (Maliga, 1980). Suspension of protoplasts constitutes an identical plant material from one laboratory to other. The approaches made for plant protoplast transformation are (i) Introduction of DNA sequence via integration into T-DNA of Ti-plasmid of A. tumifaciens. (ii) Transfer of naked DNA into protoplast including bacterial spheroplasts and microinjection and (iii) Transformation with plant viruses carrying inserts, e.g., Gemini and caulimoviruses.

6.41. PLANT CELL TRANSFORMATION BY ULTRASONICATION Ultrasonication is done for various biological experiments. But in some plants this technique has been done for successful gene delivery The Biotechnology Research Center, Beijing (China) has used this technique for gene transfer in plants like wheat, tobacco and sugarbeet. When the cultured explants were sonicated with plasmid DNA carrying market genes such as cat, nptll, and gus, and sonicated calli transferred o selective medium, shoots were grown successfully. The calli in control set of experiment which were not sonicated with plasmid did not grow on selective medium but they died. Transgenic tobacco plants were obtained at a frequency of 22%.

6.42. LIPOSOME-MEDIATED GENE TRANSFER Liposomes are microscopically small sized particles. They contain a

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phospholipid bilayers which are concentric in nature. Liposomes enclose aqueous chamber and can entrap water soluble molecules. Therefore, they are called lipid bags. Many plasmids are enclosed in them. By using polyethylene glycol (PEG) they may be stimulated to fuse with protoplast. In several plants like carrot, tobacco, petunia., etc. this technique has been used for successful transfer of genes. Due to endocyclosis of liposomes, DNA enters the protoplast. It gets adhered first to protoplast surface and get fused with protoplast t the site of surface. Consequently plasmids are released inside the cell. There are several advantages in using this technique such as (E) low toxicity, (i) protection of DNA/RNA from cleanses that lyse them, (ii) long stable storage of DNA fragments in liposome, (iii) applicability in various cell types, and (iv) high level of reproductivity.

6.43. ANIMAL CELLS Culture of animal cells on a large scale is more difficult and expensive than plant cells, bacteria, yeast, etc. This is why the commercialization of natural products of animal origin is limited. However, most of the animal products have been produced by using bacteria as a biological tool. In India, Dr. K. Chandrashekharan, Center for Cellular and Molecular Biology (CCMB) Hyderabad emphasized the use of recombinant DNA technology for the introduction of foreign DNA into mammalian cells. This would help in curing certain genetic defects. Some of the examples of gene cloning in animals (a) Animal A variety of animal viruses are known, for example, simian virus 40 (SV40), adinovirus, retrovirus (ssRNA), vaccinia virus, etc. They can increase the efficiency of animal cell transformation if they are used as vector. A special feature of viruses is that they contain the strong promoters which can of the viruses. The most commonly used virus is SV40 which contains a circular DNA of about 5.2 Kb. In addition to the region, the DNA contains early genes and late genes. Early genes are required for DNA replication, whereas late genes encode coat protein of the virus. DNA fragment and phage, the amount of DNA to be packed into virus capsid is limited. The genome has 3 identified non-essential regions; hence the use of non-defective viral vectors is limited. The defective virus vectors have missing genes whose infection process has been helped by viral DNA constituting functional copies of missing genes. If the cos cells (the host monkey cells) are used as host, they defective replication origin growth of SV40. Recombinants containing foreign DNA inserted into early region can be propagated without the need for helper virus (Glover, 1984). In this way, the recombinant and helper can be separately recovered from the host.

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6.44. ELECTROPORATION Electroporation (using electric field) is now used to transfer the foreign DNA into the fragile cells. Brief pulses of high voltage electricity (about 350 V) are applied to protoplasts suspension containing naked or recombinant plasmids. The electric pulses induce the formation of large pores in the cell membrane. These pores give a passage through which the foreign DNA can enter into the protoplasts and thus, increase the transformation frequency. The transformed protoplasts are cultured for about one month. These develop mcrocalli which ere plated on solid medium containing selective markcr (e.g., kanamycin). After 37–45 days, the calli are analyzed for the presence of differences in transformed cells. By using electroporation method, genes of choice have been successfully transferred in protoplasts of wheat, rice, petunia, sorghum, maize and tobacco. Moreover, transformation frequency can be improved by using linear DNA instead of circular one, by giving heat sock to protoplast at 45°C for 5 minutes, by adding PEG and using 1.25 kV/em voltages.

6.45. PARTICLE BOMBARDMENT GUN It was developed by Prof. Stanford and coworkers of Comell University (USA) in 1987. As the term denotes, it shoots foreign DNA into plant cells or tissue at a very high speed. This technique is also known as particle bombardment, particle gun method, biolistic process, microprojectile bombardment or particle acceleration. This technique is most suitable for those plants which hardly regenerate and do not show sufficient response to gene transfer through Agrobacterium, for example, rice, wheal corn, sorghum. chickpea and pigeonpea. The apparatus consists of a chamber connected to an outlet to create vacuum. At the top, a cylinder is temporarily sealed off from the rest of chamber with a plastic rupture disk. Helium gas flows into the cylinder. A plastic microcarrier is placed close to rupture disk.

Pollen transformation through particle bombardment Genetic improvement in alfalfa has been made by plant tissue culture methods. This method is time consuming, requires special techniques and efficiency of getting stable regenerate is low. Ramaiah and Skinner (1997) produced transgenic alfalfa through direct delivery of DNA into pollen grains by particle bombardment method. Plasmid PBI121 bearing GUS reporter gene was into pollen grains. The microprojectile bombarded liquid pollen suspension and tripped recipient female flower. Pollinated flowers set seeds in about a month.

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Thirty per cent of plants derived from fertile seeds showed integration of GUS plasmid. It was confirmed by Southern analysis. After 10 vegetative generations some were allowed to fertilize the tagged transgenic plants lost the integrated GUS plasmid, whereas in few others, copies of GUS gene decreased due unknown reasons.

6.46. MICROINJECTION Microinjection is a technique of delivering foreign DNA into a living cell (a cell, egg, oocyte, embryos of animals) through a glass micropipette. One end of a glass can be transformed by a plasmid carrying the early region of SV 40 with a cos cells express wild type large T antigen and can support the micropipette is heated until the glass becomes some forms to very fine tip at the heated end. The tip of the pipette attains to about 0.5 mm diameter which resembles an injection needle. The process of delivering foreign DNA is done under a powerful microscope. Cells to be microinjected are placed in a container. A holding pipette is placed in the field of view of the microscope. The holding pipette holds a target cell at the tip when gently sucked. The tip of the micropipette is injected through the membrane of the cell. Contents of the needle are delivered into the cytoplasm and the empty needle is taken out.

6.47. DIRECT TRANSFORMATION Like plant cells, the mammalian cells can be transformed by the foreign DNA fragments. For transforming the mammalian cells, it is necessary to precipitate the DNA with calcium phosphate and mix the cells to be transformed. DNA molecule passes through cell membrane and integrates randomly with mammalian chromosome. Using this technique selective marker can be linked up with DNA fragment to be cloned and can be introduced into mammalian cells. The transformed cells are, thereafter, separated from cell line after plating them on selective medium. Many techniques transformed cell line. Following these biochemical processes. Perucho et al. (1980) successfully isolated chicken thyamidine gene (tk gene) by ligating with PBR322 and transforming the take (deficient of thyamidine gene) mouse.

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6.48. GENE DNA SEQUENCING The structure of DNA (e.g., gene insert, a recombinant plasmid or entire genome) can be analyzed by determining the nucleotide sequences. In molecular cloning, the information of nucleotide sequences is essential. In 1965, Robert Holley and his research group at Cornell University completely sequenced nucleotides of tRNA (tRNA for yeast alanine). In 1977, the following two methods were ONA developed. Allan Maxam and Walter Gilbert developed a chemical method sequencing. In this method, end-labeled DNA is subjected to base specific cleavage reaction before gel separation. In routine sequencing of DNA this method is not commonly followed. In the same year (1977) Frederick Sanger and co-workers developed It is also called dideoxynucleotide chain termination method because dideoxynucleotides are used in an enzymatic method of DNA sequencing G Jodder of molecules.

6.49. MAXAM AND GILBERT’S CHEMICAL DEGRATION METHOD This method is not very popular because it is time consuming and labor intensive. In this method the DNA molecule can be radiolabeled at either 5’ end by using polynucleotide kinase, or 3’ end by terminal transferase. One end of radiolabeled double stranded DNA is removed by using endonuclease. A base is modified chemically followed by cleavage of sugar-phosphate backbone of DNA. No specific reaction for the four bases is carried out, except specific reaction to G only and purine specific reaction which removes A or G. A difference in these reactions indicates the presence of A.

6.50. CLEAVAGE OF PURINE The mixture is separated in four sets, each treated with a different reagent which degrade only G or C or A and G or C and T. In one set, DNA is treated with acid followed by dimethyl sulfate. This causes me thylation of A, (at 3’ position and G (at 7’ position). Subsequently, addition of alkali (-OH) and pyrimidine results in DNA cleavage and removal of purines (A or G).

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6.51. CLEAVAGE OF PYRIMIDINE Similar to the cleavage of purine, pyrimidine (C or T) is a cleaved in the presence of 1–2 M NaCI solution. It works only with C. Differences between these two indicate the presence of T in the DNA sequence. Cleavage of pyrimidines (C or T) through hydrazine hydrolysis. Partial chemical cleavage of DNA fragments as done above generates the populations of radiolabeled molecules extending from radiolabeled terminus to the site of chemical cleavage. These fragment different sizes that represent unique pairs of 5 and 3’ cleavage products in the random collection. A complete set is formed by these products the length of each number is short by nucleotide. These can be separated by gel electrophoresis. The fragments containing labeled terminus can be observed by autoradiography of the gel. Following the order of fragments obtained from different digestions the sequence of nucleotides is deduced and interpreted.

6.52. SANGER METHOD (DIDEOXYNUCLEOTIDE CHAIN TERMINATION METHOD) Twice Nobel Prize winner Frederick Sanger powerful method for et al. (1977) developed DNA sequencing that utilizes single stranded DNA as template. This method is also called a dideoxynucleotide chain termination method. The requirements are: with free 3’-OH ends to primer start DNA synthesis, DNA polymerase and DNTPS. Shows the presence of free 3’-OH group at 3’ end in DATP and no 2,’3’-OH in 2,’3’ddATP In 2,’3,’ ddATPa hydrogen atom is attached at 2’ and 3 carbons instead of -OH hydroxyl group. If any of four ddNTPs binds, the chain elongation is terminated. Because ddNTPs do not have free 3’-OH end which is required for chain elongation. Therefore, no phosphodiester bond will be formed.

6.53. AUTOMATIC DNA SEQUENCERS Automatic DNA sequencing machines were Sanger’s method. In this new method a different fluorescent dye is tagged to the DDNTPS. Using this technique a DNA sequence containing thousands of nucleotides can be determined in a few hours. Each dideoxynucleotide is linked with a fluorescent dye that imparts different colors to all the fragments terminating in that nucleotide. All four labeled ddNTPs are added to a single capillary tube. It is a refinement of gel electrophoresis which separates fastly. DNA fragments of different colors are

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separated by their respective size in a single electrophoretic gel. A current is applied to the gel. The negatively charged DNA strands migrate through the pores of gel towards the positive end. The small developed during 1990s. It is an improvement of sized. DNA fragments migrate faster and vice versa. All fragments of a given length migrate in a single peak. The DNA fragments are illuminated with a laser beam. Then the fluorescent dyes are excited and emit light of specific wavelengths which is recorded by sequences are read by determining the sequence of the colors emitted from specific peaks as they pass the detector. This information is fed directly to a computer which determines the sequence. A tracing electrogram of emitted light of the four dyes is generated by the computer. Color of each dye represents the different nucleotides.

6.54. SITE-DIRECTED MUTAGENESIS Now, techniques have been developed the specific portion in the genome in a view to get some novel products of enormous value. Mutations can he done by directing insertions or deletions to a site on the DNA but they These minor changes may have some single amino acid is altered in a improve its properties. For such modifications point- mutations, i.e., changing of single nucleotide, is done on specific portion in the gene. Thus, the most suitable method that could bring about point-mutation in a gene to mutate specific are unlikely to be of use. Importance. if a protein in order to oligonucleotide directed mutagenesis. This is known as technique has potential for protein engineering, the engineered enzymes would be more better than the wild type ones. Change in a single nucleotide base pair is called ‘point mutation.’ Some useful properties in proteins (like stability in subtilisin) that results in substitution of selected amino acids. Using this technique specific point of a gene can be mutated. Therefore, this method has been used to understand the function of many genes. Moreover, this technique can only be used when nucleotide sequence of gene is known.

6.55. METHODS OF MUTAGENESIS When it has been decided which base is to be Site-directed mutagenesis using short synthetic DNA. Changed chemically (A) the oligonucleotide sequence has de- sired mutation at specific site. The oligonucleotide corresponds to the mutated nucleotide and its neighboring regions, mainly between 15 and 20 nucleotides in length. A single stranded clone of the wild type gene is produced by using an M13 phage-based vector. The oligonucleotide is allowed to hybridize with the single stranded clone. The hybridization is performed under the conditions. The

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oligonucleotide will base pair with wild type complementary sequence (B) and acts as primer to produce double stranded DNA by using DNA polymerase (C).

7 Genetic Engineering for Human Welfare

7.1. PRODUCTION OF CHEMICALS AND CLONED GENES 7.2. HUMAN PEPTIDE HORMONE GENES In the human body, peptide hormones are secreted after encoding by peptide hormone gene in the specialized cells, for instance insulin and other Human Growth Hormones (hGH).

7.3. INSULIN This peptide hormone i.e., insulin is secreted by the Islets of Langerhans pancreas which catabolizes glucose in blood. Insulin is a boon for the diabetics whose norm function for sugar metabolism generally fails.

7.4. SOMATOTROPIN Somatotropin, the hGH, is secreted by the anterior lobe of pituitary glands which consists of 191 amino acid units. Its secretion regulated by two other hormones (somatostatin and growth hormone releasing hormone) produced by hypothalamus. Deficiency of somatotropin in about 3 % cases is of hereditary. It has been estimated to about 1 child in 5,000. Turner’s syndrome is one of the chromosome most common disorders in girls and it is characterized by

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short stature and non-functioning of ovaries affecting approximately 1 in 2,500 live female birth. The extraction of somatotropin pharmaceutically from the pituitary glands could not meet annual demand of this hormone. Somatotropin was achieved through gene cloning procedures.

7.5. SOMATOSTATIN Somatostatin, a 14 residue polypeptide hormone is synthesized in the hypothalamus. It is the first polypeptide which was expressed in E. coli as part of the fusion peptide (Itakura et al, 1977) which inhibited the secretion of growth hormone, glucagon and insulin. It does not contain any internal methionine. Eight single stranded DNA segments were synthesized chemically which were annealed in an overlapping manner to form a double stranded DNA (the synthetic gene). It had single stranded projections at the each end as the same are formed by Eco RI. The synthesized gene contained 51 base pairs which were terminated by two non-sense (stop) codon and preceded by a methioine codon as below: ATG- (42 basepairs encoding somatostatin) TGATAG

β-endorphin β-endorphin (30 am acid long neuropeptide with opiate another growth hormone which was expressed in genetically engineered E. coli cells. Shine et al. (1980) integrated DNA sequences of B-endorphin, obtained from mRNA, adjacent t plasmid. The mRNA contained large precursor of protein that consisted of, besides to B-glactosidase genes on 6-endorphin, the hormones a-melanotropin, corticotropin, B-lipotropin and B-melanotropin. The 8-endorphin is cleaved from C-terminus of the precursor peptide. In this way, the transformed hicteria produced an insoluble fusion protein between B-galactosidase and B-endorphin. B-endorphin can be removed from the hybrid protein by tripsin which cleaves only at arginin residue. Before doing so, internal lyzines are protected from trypsinization by citraconylation as below: NH, -B-glactosidase-B- melanotropin B endorphin COOH citraconylation Trypsin B-galactosidase + B- endorphin 2. Human Interferon Genes (HIG). For the first time, Isaacs and Lindenmann isolated the interferon in 1957. Definition and nomenclature of interferon have been recommended by a committee of experts (Anonymous, 1980). Interferon is defined as “a protein which exerts virus non-specific antiviral activity, at least in homologous cells through cellular metabolic procedure involving the synthesis of both RNA and protein.” Thus, interferon is secreted by human cells just to resist the immediate invasion by normal cells.

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7.6. HUMAN INTERFERON GENE (HIG) Interferon definition and nomenclature have been recommended by a committee of experts as “a protein which exerts virus non-specific antiviral activity, at least in homologous cells through cellular metabolic procedure involving the synthesis of both RNA and protien.” Interferon is used to cure many viral diseases such as common cold and hepatitis. It is species specific. In man there are three classes of interferon: (i) Alpha interferon (IFN-o), or (ii) Beta interferon (IFN-B), (iii) Gamma interferon (IFN-y) or immune interferon (by lymphocytes of blood) and lymphoblastoid interferon by transformed leukocytes.

7.7. VACCINES FOR HEPATITIS B VIRUS Hepatitis B virus (HBV) is wide spread produces several chronic liver disorders such as Fulminant chronic hepatitis, cirrhosis and primary liver cancer. HBV DNA is a double stranded circular molecule of about 3Kb size and has a l single stranded gap which must be required with an restriction enzyme for DNA cloning (Glover, 1984). After infection in human beine. HRV a multiply and infect a large number of cells and even does not grow in cultured cells. This probe has been explained to be due to hinderance of its molecular characterization and development vaccines. Plasma of human has been detected to have varying amount of antigens. Three type viral proteins are recognized to be antigenic: (i) viral surface antigen (HBSA). (ii) viral antigen (HBcAg), and (iii) the e-antigen (HBeAg).

7.8. RABIES VIRUS (RV) Rabis virus causes hydrophobia in animals and humans in many countries like South America, Africa, Asia (including India and Pakistan). Researches are being done to synthesize vaccines by inducing genetically engineered E. coli cells. However, attempt has been made to isolate MRNA encoding viral protein from RV infected cells. The genes coding for the production of rabies virus glycoprotein coat has been successfully transferred to E. coli. This is the first step towards the production of antirabies virus vaccines stimulates antibody production in diseased animals. In 1984, Wistar Institute in Philadelphia developed a new genetically engineered vaccinia virus by inserting a small piece of foreign DNA. The genetically engineered virus synthesized antirabies

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vaccines for animals. The recombinant vaccines virus did not cause rabies in those animals that received rabies genome but encoded antigenic molecules and activated the immune system against rabies infection. This new vaccine can be administered orally in animals, and could be decreased the risk of human death due to bites of animals receiving rabies virus (Peters, 1993).

7.9. VACCINES FOR POLIOVIRUS Poliovirus is a causal agent of poliomyelitis in human beings. First attempt to describe the complete structure of poliovirus RNA was made by Kitamura et al. (1981).

7.10. VACCINES FOR FOOT AND MOUTH DISEASE VIRUS (FMDV) Foot and mouth disease (FMD is a serious disease caused by Aphthovirus.) The primary control measure of the disease has been the slaughter of FMDV – infected animals. FMD control is vaccination Vaccines are produced by inactivation of virus grown in bovine language epithelium. A detailed study of FMDV reveals that it contains a single stranded RNA covered in a capsid of for polypeptides, for example, VP1, VP2, VP3 and VP4 where only VP1 has a little immunogenic activity However, the nucleotide sequence encoding for VP1 was identified on the single strand RNA genome and cloned on double stranded PBR322 in E. coli About 1,000 molecules of V per bacterial cell were synthesized (Kupper et al., 1981).

7.11. VACCINES FOR SMALLPOX VIRUS Small pox is a very serious disease of humans in the countries. The vaccinia virus (the cowpox virus) can be used as the basis of the small vaccine by uring the recombinant DNA technology. The genome of vaccinia virus is altered inserting the foreign genes which encode the pathogenic antigens. When a person is immunized with a vaccine in preparations, the foreign genes enter the body cells of the patient’s receiving.

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7.12. MALARIA VACCINES According to WHO estimates 4 million people are at the risk or developing malaria and about 500 million cases occur each year resulting in one million of each year, mainly of children of 5 years age and pregnant women. In addition, development or resistance against drugs by the species of Plasmodium, and insecticides by mosquitoes have bee reported. Therefore, the threat of malaria is still increasing for humans. Therefore, for control of malaria use of vaccines and vector control programs would be successful. Much work going on at Indian Institute of Immunology (New Delhi) and ICGEB on development of malari vaccine by using modern methodologies. All kind of vaccine development through recombinant antigens, synthetic peptides and direct use of DNA are being attempted. All these attempts indicate that development of malaria vaccine is a large complex process. However, progress towards the development of malaria vaccine has been slow due to several reasons, one of which has lacked the in vitro correlates and the suitable animal models for malaria vaccine trials.

7.13. DNA VACCINES For the first time Wolf et al. (1990) injected naked DNA into the muscles of mice which led to expression of encoded marker protein. Thereafter, there has been a surge to use this approach to generate DNA vaccines against a variety of infectious diseases. Thus DNA vaccines are giving hope of a third vaccine evolution.

7.14. GENES ASSOCIATED WITH GENETIC DISEASES This effect In December 1993, a team of International Scientists at French Academy of Sciences, Paris developed the world’s first map of human genome. For over a decade, genetic scientists have been attempting to map the genes. So far, the physical map existed only for 2% of human genome, and the present map covers about 90% of genome. To produce the map they pieces and grew each piece in and fingerprinted to detect overlapping sections. These sections were pieces back together to get the map. This will help the scientists to discover genetic diseases. In human beings there are many cases where certain genes responsible for encoding enzymes are missing which result in genetic diseases. Some of them are briefly described in the following sections.

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7.15. PHENYLKETONURIA GENES In diseased persons when phenylalanine fails to get converted into tyrosin, disturbances in metabolism result in mental retardness. It is possible to cure this disease by using recombinant DNA techniques in early period of pregnancy cut human DNA into a yeast cell for clones. The clones were then cut into fragments used as guides.

7.16. UROKINASE GENES Urokinase is involved in dissolution of blood clots. Urokinase has been synthesized in huge quantity by using genetically engineered bacteria with urokinase genes.

7.17. THALASSEMIA GENES It is a condition in which synthesis of A- and B-globin chains are reduced and the excess chains precipitate and cause hemolytic anemia and spleen enlargement. Human globin genes have been identified and sequenced. It has been found that A- and B-globin genes are closely linked. Human globin genes (cDNA) has also been developed and cloned. However, much work has to be done to cure this disease.

7.18. HEMOPHILIA GENES Hemophilia is sex linked disease in human where blood clotting does not takes place normally due to deficiency of clotting factor VIII: C. By using gene cloning techniques the clotting factor VIII: C gene was cloned which expressed in mammalian cell lines and produced the protein VIII: C responsible for blood clotting.

7.19. ENZYME ENGINEERING The genes encode enzymes; the changes in gene certainly bring about alteration in enzyme structure. In addition to methods available for gene manipulation alteration of genes by site-directed mutagenesis for enzyme engineering has

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become much famous. Thus, site directed mutagenesis produces single amino acids substitutions in the primary structure of enzymes.

7.20. COMMERCIAL CHEMICALS 7.21. PREVENTION, DIAGNOSIS AND CURE OF DISEASE Prevention of many genetic diseases in early period of pregnancy will help the mothers to g birth to the healthy babies. Moreover, certain viral [e.g., polyo, AIDS (acquired immune deficien syndrome)] and protozoan diseases can be prevented from infection in humans. (1) Prevention of Disease For preventive measures several immunogenic polypeptides (vaccines) and proteins (antibodies have been chemically and biologically synthesized. Moreover, it is expected that in near future immunogenic compounds would be available in market and more informations would be gathered on cloned genes for viral (AIDS), bacterial (cholera) and protozoan (malaria) diseases. (2) Diagnosis of Disease Secondly, genetic engineering techniques have solved the problem of conventional method for diagnosis of many diseases. DNA probe, monoclonal antibodies, and antenatal diagnosis some of the available methods used as a tool to diagnose a particular disease.

7.22. PARASITIC DISEASE Probes used for diagnosis pathogens contain the most specific DNA sequences of genetic material of parasite. The specific lies in such a way that the other related species or strain do not contain those sequences. The unrelated unspecific sequences of parasite are first recognized by using DNA hybridization technique. Then a DNA sequence, not present in any species, is identified, cleaved by using restriction enzymes, and inserted into a cloning vector (plasmid). The bacterial cells are transformed by the recombinant vector. The transformants are multiplied. Finally, they are retrieved from the host cells. The DNA sequences of the parasite, n DNA fragmented, are labialized. Following r and used as a probe. The probe can also be chemical.

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7.23. MONOCLONAL ANTIBODIES Antibodies are proteins synthesized in blood against specific antigens just to combat and give immunity in blood. They can he collected from the blood serum of an animal are heterogeneous and contain a mixture of antibodies for polyclonal antibodies). Therefore, they do not have characteristics of specificity, If a specific lymphocyte, after isolation and culture in vitro, becomes capable of producing a single type of antibody which bears specificity against specific antigen, it is known ‘monoclonal antibody.’ Due to the presence of desired immunity, monoclonal antibodies used in the diagnosis of diseases. Major difficulties with antibody secreting cells are that they cannot be maintained in culture. But myeloma cells (bone marrow tumor cells due to cancer) grow indefinitely to produce a huge quantity of identical cells (clones) and also produce immunoglobulins in the same amount. The immunoglobulins are infact monoclonal antibodies.

7.24. GENE THERAPY There are many diseases which can be cured by using specific medicine synthesized bjochemically. Now-a-days techniques have been developed biochemicals, for example insulin, interferon, somatotropin, somatostatin, endorphin, human blood VII, immunogenic proteins, etc. Several companies viz, Eberstadt & Co. (New York), E. Lilly (USA), National Pituitary Agency (USA), Kabi Vitrum AB (Sweden), Genetech Co (USA), Biogen (Switzerland), Hybritech (USA), Astra Research Center (India), etc. are producing However, after 1975, a remarkable advancement in recombinant DNA technology has occurred and accumulated such knowledge that has made possible to transfer genes for treatment of human diseases. Several protocols have been developed for the introduction and expression of genes in bumans, but the clinical efficiency has to be demonstrated conclusively. Success of gene therapy depends on the development of better gene transfer vectors for sustained, long term expression of foreign gene as well as a better understanding of gene physiology of human diseases (Rangarajan produce therapeutic or trying to produce on mass scale to make available at low cost.

7.25. TYPES OF GENE THERAPY All the gene therapies that can be done in humans and classified into the

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following four type: a) b) c) d)

Somatic gene therapy Germ –line therapy Enhancement genetic engineering Eugenic genetic engineering

7.26. METHODS OF GENE TRANSFER A variety of gene transfer strategies have been developed during the last decade for the treatment of human diseases which can be grouped into the two categories: (i) (ii)

Virus mediated Non-viral approaches.

7.27. GENE THERAPY SUCCESS The success of gene therapy depends on gene delivery mechanism as well as the choice of target tissue. Rangarajan and Padmanaban (1996) have discussed different conditions leading to success of gene therapy: •

•

Cell types capable of dividing in vitro (e.g., myeloblasts, hepatocytes, keratinocytes endothelial cells, etc.) are amenable to in vitro and in vivo gene therapy, both in vivo methods are preferred for cell types such. The function of gene products also govern the selection of tissues, for example in case of the haemophilia a gene can be delivered in any tissue provided with the gene product is released into blood stream. In addition, in case of cystic fibrosis, the gene should be delivered to specific cell types where introduction of correct gene is required.

7.28. DNA PROFILING DNA is the master molecule of all life forms. It constitutes the blue prints of every livin organism through which characters are passed generation to generations. However, every living differs only due to organism nucleotide of chromosome sequences. The nucleotides form codes; therefore, this coded genetic information can be profiled to produce most authentic identity card of any organism. The complicated technology that facilitates the identification of

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individuals at genetic level is known as DNA fingerprinting or more specifically DNA profiling. This genetic analysis is based on identifying tiny segments of the hereditary material which testify the unique molecular signature which cannot be altered.

7.29. METHOD OF DNA FINGERPRINTING In this technique DNA is isolated from blood stains, semen stains or hair roots of disputed children or any suspected person and the same from parents, close associates or relatives of suspected criminals (based on the cases). Since hairs contain less amount of DNA, it can be produced in a large amount by using polymerase chain reaction (PCR). RBCS do not contain DNA, therefore, WBCS are the source of DNA. Thus the DNA isolated is cut with restriction enzyme and subjected to Southern blotting. The DNA bands appearing on membrane are with 32P-DNA probe, washed in water to remove the hybridized complementary DNA sequences develop images (prints). Identical prints that contain specific DNA sequences appearing confirmed. Probability of two persons having similar sets of base pairs in the same sequence of VNTR of DNA is one in 30–300 million individuals hybridized unhybridized DNA, and passed through X-ray identified and thus identity is on two X-ray films are for.

7.30. APPLICATION OF DNA PROFILING (a)

(b)

Setting up of Genetic Databank: Realising the potential of DNA profiling genetic databank had been/being set up thoughout the world. Databases of the genetic fingerprints of criminals have been set up in many parts of the USA and Britain. Forensic Science Services Birmingham (Britain) has set up the world’s first National DNA Database, where about 5 million record would be available. It has collected samples of tissues and DNA of suspected individuals has been profiled. Reuniting the Lost Children: BNA profiling technology helped in reuniting the lost children with their respective parents or vice versa who were separated during war, violence or tural disasters. For example, in Argentina many people were abducted during military rule 976–1983). They were presumed to be died. But an Arzentinian Human Right Group (the randmother’s of the Plaza de Mayo) was the opinion that the children were still alive and adopted military personnel after killing their natural parents. In

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1987, a genetic databank storing blood Samples of grand parents and relatives of the lost children was set up. By matching the DNA profile one or more grand parents with that of lost children about 40 missing children have been united with their families (Chawla, 1998) Solving disputed problem of parentage, identity of criminals, rapists, etc: To carry out the test a small portion of blotted blood is punched out from which blood cells are isolated nd information is taken from computer databank. The first case from preserved sample was done P identify a soldier who was burnt to death in a car accident. In India, a disputed parentage has been solved. In June 1988, a four-year-old female baby named axmi was stolen from Chennai by a missionary and renamed as Merry by the so-called parents. After FIR and taking police help the matter was not solved.

7.31. IMMIGRANT DISPUTE More-or-less, in every country, there is the problem of immigration. Ultimately, the identity is not confirmed whether they have crossed the borders or are the resident of that country. Therefore, the problem of immigrants can be solved through DNA databank.

7.32. ANIMAL AND PLANT IMPROVEMENT Transgenic Farm Animals Production of transgenic animals through microinjection techniques will play a significant role in veterinary sciences as well as for human welfare. A farm animal can produce milk containing tons of gram of protein per liter, many times more than a liter of bacterial culture. The possibility of producing human pharmaceutical demonstrated when lactating mice secreted the active tissue plasminogen activator (t-PA) in their milk. The t-PA is an enzyme which dissolves the blood clot responsible for coronary artery blockage that results in heart failure. John Mc Pherson at Genzyme Co., in collaboration with K. Elbert and colleagues of Tuft University produced the first transgens goat by using a goat beta-casein promoter gene linked to t-PA gene. The transgenic goat produce t-PA in quantities as compared to cell culture derived material. But goat peptide is slighty different from the culture derived t-PA. The goat peptide has two chains of peptide, whereas single chain peptide was found in culture derived t-PA. Population of the country is rapidly

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increasing day by day. However, the most urgent need to the nation is to meet the food demand of the people.

7.33. POLLUTION OF ABATEMENT For detoxification and degradation of toxic chemicals, enzymes are encoded by specific genes present on plasmids. Chakraborty and co-workers (1979) succeeded in isolating the microbial culture which could be used in a number of organic chemicals, toxic in nature, such as salicylate, 24-D. 3 chlorobenzenes, ethylene, biphenyls, 1, 2, 4-trimethylbenzene, 2, 4, 5-trichlorophenoxy acetic acid, etc. Genes responsible for degradation of environmental pollutants, for example, toluene, chlorobenzene acids, and other halogenated pesticides and toxic wastes have been identified. One separate plasmid is required. It is not like that one plasmid can degrade all the toxic compounds of different groups. The plasmids are grouped into four categories: • OCT plasmid which degrades, octane, hexane and decane, • XYL plasmid which degrades xylene and toluenes, • CAM plasmid that decompose camphor, and • NAH plasmid which degrades naphthalene. Dr Anand Mohan Chakrabarty (an Indian born American of genetic engineering called as scientist) produced a new product strains into a single cell of P. putida. This superbug is such that it can degrade all the four types of substrates for which four separate plasmids were required superbug (oil eating bug) by introducing plasmids from different strains into single cell P. putida.

8 Animal Biotechnology

8.1. INTRODUCTION The most important areas of today’s research which have potential economic value and prospects of commercialization are the cell and tissue culture based on the production of vaccine, monoclonal antibodies, etc.

8.2. HISTORY OF ANIMAL CELL CULTURE Alex carrel used tissue and embryo extract as culture media. The fibrin clot of plasma served as an anchor for cell attachment and the extract provided growth factors and nutrients. A major innovation to get cell suspension was the use of trypsin for cell disaggregation from tissue explants. This allowed single cell culture. Thus the technique of cell cultures differs tissue culture. The use of biological fluids and extracts was a contaminated major problem because of getting contaminated. In 1907, Ross Harrison made first attempt to culture animal cells, and cultivated embryonic nerve cells of a frog by using hanging drop method. Thereafter, this method was extended and a wide range of mammalian cells were cultured in vitro. However, after supplimenting with embryo of chick and plasma, cells proliferated well. These provide nutrients and proliferation factors. Moreover cell culture method was extract quite improved after the discovery of antibiotics during 1940s. Such studies provided an impetus for culture of animal cells in large scale. During this period many human carcinoma cell lines (e.g., HeLa cell line) were isolated and grown in culture. Alexis Carrel, the famous physiologist kept the chick embryo heart alive and its beating continued in vitro for about three months. Animal cell

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culture studies resulted in total stop of using monkeys for multiplication of animal viruses.

8.3. REQUIREMENTS FOR ANIMAL CELL AND TISSUE CULTURE 8.4. ANIMAL CELL GROWTH IN CULTURE Unlike plant and microbial cells, the animal cells can grow only to a limited generations even in best nutritive media. This growth also depends on the sources of tissue isolated. The special features of different cell cultures are briefly discussed. (i) Neuronal cells constitute the nervous system. In culture the neuronal cells cannot divide and grow. The cells that form connective tissue (skin) is called fibroblast. Fibroblast can divide and grow in culture to some generations, and after completing several generations, they die. It means that all normal animal cells are mortal. (ii) In culture the animal cells divide and grow. Consequently, they fill the surface of the container in which they are growing. Thereafter, they stop further growth. This phenomenon is termed as contact inhibition, i.e., inhibition of further cell growth after reaching the wall of container. The environment of cell growth in culture differs from that of in vitro, for example: • • •

Absence of cell-cell interaction and cell matrix interaction, Lack of three-dimensional architectural appearance, and Changed hormone and nutritional environment. The way of adherence to glass or plastic container in which they grow, cell proliferation and shape of cell results in alterations. • In culture the cancer cells apparently differ from the normal cells. Due to uncontrolled growth and more rounded shape, they loose contact inhibition. Therefore, they are piled on each other. These features are exploited by cancer specialists, i.e., the cancerologists to identify cancer cells from the normal cells. The requirements for animal cell and tissue culture are the same as described for plant cell, tissue and organ culture Desirable requirements are: (i) air conditioning of a room; (ii) hot room with temperature recorder; (iii)

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microscope room for carrying be installed; (iv) dark room glassware and culture media, and should out microscope work; (v) plastics-shelving; (vi) sealed container and drawers; and (vii) specialized chemicals.

8.5. SUBSTRATES FOR CELL GROWTH There are many types of vertebrate cells that require support for their growth like anchorage-dependent cells e.g., plastic, glass, palladium types may be used • Plastic as a substrate Disposable plastics are cheaper substrate. • Glass as a substrate Use only sterilized chemicals (radiations, dry heat fin oven) and by heat (in autoclaved test tubes, slides, coverslips flasks, rods, moist palladium.

8.6. CULTURE MEDIA Culture of animal cells and tissue is rather Culture utter synthesize certain chemical constituents from inorganic more difficult than that of microorganisms and substances. However and chemical of media used for culture of animal cell and he culture media provide the optimum growth factors (e.g., pH, osmotic pressure) (a) Natural Media: Natural media are the natural sources of nutrient and proliferation of animal cells and tissue. It is used since long time but now available in market in the form of liquid microbes).

8.7. GLASSWARE, EQUIPMENTS, AND CULTURE MEDIA The thoroughly washed glassware and equipments and carefully prepared culture media are millipore filter paper. The sterilized materials are removed sterilized by heat, steam or when temperature cools down and used according procedure adopted.

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8.8. EQUIPMENT REQUIRED FOR ANIMAL CELL CULTURE The equipment required for animal cell culture are given below.

8.9. LAMINAR AIR FLOW (LAF) LAF hood acts as animal cells, Culture manipulation in aseptic conditions protects from contamination by any microorganisms such as bacteria and fungi. The contaminated bacterial/fungal cells grow more rapidly than the cultured animal cells. Therefore, the growth of animal cells fails to occur in the presence of contaminants. The working area of LAF hood is first made sterile by using 70% ethanol. Manipulation of any cell is done by keeping the LAF in ON position aseptic working table for inoculation of Blower The LAF hood performs two functions: provides a sterile environment for cell manipulation (i.e., protects tissue culture from operator) and (protects the operator from the potential infection risk from the culture. There are different types of LAF hoods, airflow hoods (cabinets) are commercially available. In this apparatus, sterile airflows inside the space of cabinet which maintains the sterile conditions required for all transfer work (Figure 8.1). Different types of laminar airflow hoods are available on the dishes Flask sis of nature of the cells and kinds of organisms and tissue culture as below.

8.10. CO2 INCUBATORS The CO, incubators provide the suitable environmental conditions to the growing animal cells. A silicon gasket is used on the inner door which makes the chamber of incubator airtight. It separates the internal environment from the external environment. The external environment possesses the microbial contaminants, while the internal environment remains always contaminationfree. The filtered air, i.e., a high efficiency particulate air (HEPA) is injected inside the chamber which maintains the internal environment of chamber sterile. The relative humidity inside the chamber remains high; therefore, correct osmolarity is maintained and medium is protected from desiccation.

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8.11. CENTRIFUGES There are different types of centrifuges based on speed. A low speed centrifuge is needed for most of the cell cultures. The separated beads of cells are disrupted simply by a gentle breaking action. Commonly cells are centrifuged at 20°C. The motor evolves heat which rises the temperature. Therefore, use of low temperature for centrifugation is preferred so that the cells should not be exposed to high temperature.

8.12. INVERTED MICROSCOPE Use of an inverted microscope is important to observe cell cultures in situ. Because, the cells are found on bottom of the tissue culture vessel (e.g., Petri plates). The culture medium remains above the growing cells in plates. If such plates are put over the stage of an ordinary microscope, the growing cells at bottom cannot be observed. Therefore, the inverted microscope is used for such purpose.

8.13. CULTURE ROOM All types of cultured plant tissues are incubated under the conditions of well controlled temperature, humidity, illumination and air circulation. The culture room should have light and temperature control systems. Generally temperature is maintained at 25 ± 2°C and 20 98% relative humidity and uniform air ventilation. Generally cultures darkness each for a period of 12 hours.

8.14. DATA COLLECTION (OBSERVATION) The cultures are monitored at regular intervals in the culture room for the growth and development of cultured tissues. Observation is also made under aseptic area in laminar airflow.

8.15. ISOLATION OF ANIMAL MATERIAL (TISSUE) During the experimental work, attempts should be made that animal materials

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are not contaminated. When glassware and media are sterilized, animal materials should be handled. Before that a balanced salt solution (BSS) is required. This solution consists of 1000 units of penicillin and 0.5 mg of streptomycin washed in BSS aseptically to avoid contamination. The tissue to be cultured should be properly sterilized with 70 per cent ethanol, and removed surgically under aseptic conditions. Attempts should be made to avoid contamination. Thus, the tissue isolated is either stored in freeze or used immediately.

8.16. ESTABLISHMENT OF CELL CULTURES There are many type of animal cells that can grow in such as tumor cells, pigmented melanoma cells, neuroblastoma cells, steroid producing adrenal cells, growth hormone prolactin secreting cells, teratoma cells capable of differentiating in cold trail conditions pigmented or cartilage cells, etc. On the basis of purpose of experiment, a Sue n is atinuous (immortal) cell line can be developed from cultured tissues. Healthy animal tissues ted are cultured on artificial nutrient media that proliferate and differentiate into able of dividing us mixture of different types of cells.

8.17. CELL LINES The primary cell culture cannot long time because the cell utilize all nutrients of the medium. Therefore, sub remains viable for culturing needs to be done on another fresh medium. Hence, the primary culture is removed adherent cells are dissociated enzymatically medium and passed into fresh culture flask or by repeated pipetting. Then the cells are diluted with fresh. This results in multiple copies of a proliferating cells. During the course of repeated sub-culture and selection the cell line gets evolved and properly established consisting of rapidly proliferating cells. Thus the unaltered form of cell fine (only for a limited number of generations) is called continuous cell line which propagates in single type of cell with a negligible amount of non-logarithmic ways. This mixture of cells is in single cell suspension which may be used as a primary culture or starter culture. The primary culture (in the form of single-cell suspension is subcultured by transferring into culture dishes/flasks containing special growth nutrients t optimal growth conditions Consequently, some cells attach to the surface and proliferate to yield single cell line, inspite of being damaged in suspension. Therefore, while subculturing the suspension should be diluted with fresh medium at certain ratio and transferred into a flask (Anon,

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1988 IS maintain remain culturing cells are medium Primary Cell Cultures: The first step in establishing cells in culture is to dissociate organs single cell suspension. It is done by mechanical or enzymable methods. The cells are transferred into special glass or plastic containers containing culture medium. Under these conditions, maintenance of growth of such cells is called primary cell culture.

8.18. HYBRIDOMA TECHNOLOGY Blood consists of two major components, various kinds of cells and the fluid called serum Serum consists of heterogeneous population of antibodies. Different antibodies are ferent types of B-lymphocytes. Hence, serum consists of different types of antibodies which are often called polyclonal antibodies. The antibodies bind to specific domains of antigens (called antigenic produced by dif determinants or epitopes) and neutralize them.

8.19. MONOCIONAL ANTIBODIES PRODUCTION OF MONOCLONAL ANTIBODIES (MOAB) The mouse MOAB have revolutionized the field of biology and more specifically immunology. The mouse MoAb have been used in human patients with varying level of success who were suffering from leukemia, lymphoma, melanoma and colorectal cancer. Clinical trials have indicated several limiting factors such as: (i) heterogenecity of tumor cells (not all malignant cells carry relevant antigen), circulating free antigens (they bind Fab on antibody molecules and thus block MoAb from binding to the target cells), (ii) antigenic modulation (antigen modulated off the cell surface as a consequence of binding of MoAb to the cancer cell as in leukein. This problem can be overcome by applying introduced into hemster kidney cells.

8.20. APPLICATIONS OF MONOCLONAL ANTIBODIES The monoclonal antibodies are used in the four main ways, i.e., disease diagnosis, disease treatment, passive immunization and detection and purification of biomolecules. However, their exploitation for various uses is based on two

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features: (i) exacting the specificity of antibody binding, and (ii) presence of similar structure of all antibody molecules.

8.21. DISEASE DIAGNOSIS Using MoAb medical conditions and diseases of sufferers can be diagnosed. One of the approaches is the ‘antibody- sandwich’ strategy which is also called ELISA (enzyme-linked immunosorbent assay). The circulatory antigens in blood can be assayed using ELISA or radioimmunoassay (RIA) technique. By using ELISA you can test HIV, hepatitis, typhoid, herpes, etc. For each test, separate ELISA kit is used (Figure 8.1).

Figure 8.1: ELISA testing.

9 Plant Biotechnology

9.1. INTRODUCTION The beginning of plant biotechnology or plant tissue culture was made as early as 1898, when a German botanist, G. Haberlandt cultured successfully differentiated individual plant cells, which was isolated from different plant species. Further progress in cell culture research was made although embryos culture, roots and other tissues was successfully achieved in this period. The discovery of the importance of auxins and B-vitamins, the foundation of plant tissue culture was laid down by three scientists (Nobecourt, White and Gautheret) even though small pieces of tissue and not individual’s differentiated cells could be grown in cultures during 1934–1939. To boost up these areas ICGEB in a workshop was held at New Delhi from September 18 to 20 1985, recommended the need of more research in developing countries on plant cell culture, differentiation, regeneration and transformation in tropical grain legumes, woody legumes and cereals. This was led to improve growth under stress condition, pest and disease resistance, improved nutritional quality, nitrogen fixation and the control of partitioning within plants. Totipotency: The living cells of multicellular organisms are capable of independent development, when provided with suitable conditions, this statement given by White, 1963. According to Morgan, 1901 coined the term totipotency to denote this capacity of cells to develop into an organism by regeneration. However the concept of totipotency is important for tissue culture.

9.2. HISTORY The experiments on wound healing in plants through spontaneous callus

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formation on decorticated region of elm plants was coined by historically Henri-Louis Duhamel du Monceau in 1756. But the science is advance related to cell and tissue culture after propounding the cell theory by Schleiden and Sachwann (1839). Trecul in 1853 observed callus formation in a number of plants. Hamberlandt is regarded as father of tissue culture. The organ culture was made attempts from 1902–1930. The isolated embryos of some crucifers and successfully grew on mineral salts and sugar solutions by Hanning in 1904. The successfully regenerated a bulk callus, buds, roots from a poplar trees on the surface of medium containing IAA which proliferated cell division by Simon 1908. The cultured plant material maintains its morphological identity, more or less with the same anatomy and physiology as in vitro of the parent plants by Doods and Roberts, 1985. The cultured tobacco tumor tissue from the hybrid Nicotiana Glauca and N. Langsdorffi by White (1939). R.P White (USA), Gautheret (France) and Nobercourt (France) independently cultured tissues excised from several plants on the defined nutrient media for a long period in 1930. The cultured cambium tissue of carrot on Knop’s solution supplemented with other chemicals in trace amount in 1939 by Gautheret. The Van Overbeek in 1941 and coworkers used coconut milk for embryo development and callus formation in Datura. Miller in 1955 was eventually a potent cell division factor from degraded DNA preparations was isolated and identified named as Kinetin. Miller and skoog in 1957 was advanced the hypothesis of organogenesis in cultured callus by varying the ration of auxin and cytokinin in the growth medium. The shoot was formed with keeping the ratio of kinetin higher and root developed when ratio was lower. A cell can be propagated by subculturing by Muir in 1953 developed a successful technique for the culture of single isolated cells which is commonly known as paper-raft nurse technique. The Pfizer Inc. New York USA in 1952 got the US patent and started producing industrially the secondary metabolites of plants. The first naturally production of natural product shikonin by cell suspension culture was obtained. According to Guha and Maheshwari in 1966 developed techniques for the production of the vast numbers of embryos from culture of pollens and sporogenous tissues of anther. The methods to double the chromosomes number

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in microcpors of Nicotiana and Datura and collected seeds from the homozygous diploid plants within 5 months by Nitsch in 1974. To develop the following techniques of cell culture and somatic genetics of mainly grasses and cereals has emphasized by Vasil in 1982. The tissue culture was started in India during the mid 1950 at the Department of Botany (University of Delhi) by Panchanan Maheshwari. It is regarded as father of embryology in India. Sipra Guha Mukherjee and S.C Maheshwari in 1964–1967 for the first time developed the haploid through anther and pollen culture at the University of Delhi. The discovery haploid production was a land mark in the development of plant tissue culture. The recombinant DNA technology made possible to transform artificially cultured plant cells by introducing foreign genes.

9.3. REQUIREMENTS FOR IN-VITRO CULTURES 9.4. TISSUE CULTURE LABORATORY The plant cells culture and tissues in vitro is not easy task. It requires all the nutrients and physiochemical factors in maintained in a laboratory. For the plant tissue culture a good laboratory have required nutrient medium preparation, sterilization, cleaning and storage of supplies, aseptic condition for working the living materials, a controlled environmental conditions for growth and development of culture, observation and evaluation of culture as hoped, recording the observation made during the experiment by White in 1963. The most basic facilities that an individual’s needs for tissue culture such as • • •

Washing and storage facilities Media preparation room Transfer area

9.5. WASHING AND STORAGE FACILITIES A separate are is required which should have large sink area with provision for hot and cold running water, distillation apparatus, washing machine, pipette washer, drier and cleaning brushes, keeping and weighing the chemicals and putting glassware.

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9.6. MEDIA PREPARATION ROOM An area is required for preparation of media. In such space there should be provision for bench space for chemicals, labware, culture vessels, closures and miscellaneous equipment required for mediapreapartion and dispensing

9.7. TRANSFER AREA The transfer of plant tissue was done in open laboratory bench under clean and dry atmosphere conditions. Later on closed plastic box was constructed which consisted of UV tube. UV light from the tube and use 95% ethanol helped to maintain sterile conditions inside the space of plastic box.

9.8. NUTRIENT MEDIA According to White in 1934 observed the unlimited growth of isolated root tissues when provided with nutrient medium containing inorganic salts, sucrose, vitamins, growth hormone and few amino acids.

9.9. INORGANIC CHEMICALS It includes macronutrients such as nitrogen, phosphorus, potassium, calcium, magnesium and sulfur in the form of salts in large amount and microelements such as boron, molybdenum, copper, zinc, manganese, iron and chloride.

9.10. GROWTH HORMONES There are the several hormones are known to be stimulate the biological activity in cultured materials. Cytokines promote the cell division and regulate growth and development similar to kinetin. Auxin resembles indole acetic acid (IAA) and stimulates shoot elongation. Gibberellins are used in apical meristem. The most widely used cytokines are adenine, kinetin, zeatin, benzyladenin and auxins are IAA, NAA, 2,4-D. The induction of callus the amount of kinetin should be 0.1 mg/l. Structural formula of cytokines and auxins are given in Figure 9.1.

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Figure 9.1: Structure of Growth Hormones (a) auxins, (b) cytokinin, (c) Gibberellin A3.

9.11. ORGANIC CONSTITUENTS The organic compounds serve as a organic carbon and energy such as Sucrose, D-glucose, peptone, yeast extract, coconut water, malt extract, tomato juice, etc.

9.12. VITAMINS There are number of vitamins which are required for culture such as Vitamin B1, Vitamin B2, Vitamin B6, Vitamin C, Vitamin H, Vitamin B12.

9.13. AMINO ACIDS The most widely amino acids are used such as L-asparatic acid, L-asparagin, L-glutamic acid, L-glutamine and L-arginin.

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9.14. SOLIDIFYING AGENTS Most commonly agar a polysaccharide obtained from a seaweed, i.e., a red alga, Gelidium amansii is used as solidifying or gelling agent.

9.15. PH The pH affects the uptake ions optimum pH between 5.0 to 6.0 is required for growth and development of cultured tissues. The pH should be maintained before the sterilization process or medium.

9.16. MAINTENANCE OF ASEPTIC ENVIRONMENT The contaminants produced toxic metabolites which inhibit growth of cultured plant tissues. Therefore each stem must be handled aseptically and with great care.

9.17. STERILIZATION OF GLASSWARE Glassware should be sterilized in hota air oven at 160–180°C for 2–4 hours.

9.18. STERILIZATION OF INSTRUMENTS The metallic instruments should be sterilized such as scalpels, forceps, needles, spatulas, etc. dipping them in 75% ethanol followed by flaming and cooling. It is called incineration.

9.19. STERILIZATION OF CULTURE ROOMS The floor and room are washed first with detergent then 2% sodium hypochlorite or 955 ethanol. Large surface area is sterilized by exposure to UV light.

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9.20. STERILIZATION OF NUTRIENT MEDIA The nutrient media are properly dispensed in glass container plugged with cotton or sealed with plastic closures and sterilized by autoclaving at 15 psi for 30 minutes that gives 121̊ C for 30 minutes. During autoclaving plant extract, vitamins, amino acids and hormones are denatured so therefore use Millipore filter paper which has 0.2 µm pore diameter for sterilization.

9.21. STERILIZATION OF PLANT MATERIALS The plant materials have microbial contaminates so wash with sodium hypochlorite, hydrogen peroxide, mercuric chloride or ethanol should be used to make plant material sterilized.

9.22. METHODS OF PLANT CELL, TISSUE ORGAN CULTURE BASIC STEPS There are the basic steps which follows: • Preparation of suitable nutrient medium. • Sterilization of explants • Inoculation • Incubations • Regeneration • Hardening • Plantlet transfer

9.23. TYPES OF CULTURE There are different types of culture which are produced from plant material such as Explant culture, organ culture, cell or suspension culture, callus culture, and protoplast culture (Figure 9.2).

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9.24. EXPLANT CULTURE

Figure 9.2: Regeneration of plant through plant tissue culture. There are different varieties of seeds plants such as trees, herbs, grasses which exhibit the basic morphological units such as root, stem and leaves. There are various stages and differences in cells, tissues and their topography. Parenchyma is the most versatile of all types of tissue. They are capable of division and growth. However, development of a tissue is characterized by three types of cell growth: cell division, cell elongation and cell differentiation. The healthy part of the plant should be used for culture purposes. Explant culture are the cultures of plant material. Any part of plant may be explant such as young and healthy pieces of stem, leaf, stem hypocotyl, cotyledons, etc. Explant cultures are generally used for induction of callus or regeneration of plant.

9.25. CALLUS FORMATION The callus develops be infection of microorganisms from wounds due to stimulation by endogenous growth hormones, the auxins and cytokinins. However, it has been artificially developed by adopting tissue culture techniques. It is governed by the source of the explant, nutritional composition of medium and environmental factors. Explants of meristematic tissues developed cells

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more rapidly than thisn three walled and lignified cell of tissue. Callus is formed through three developmental stages: induction, cell division and differentiation.

9.26. ORGANOGENESIS Roots, shoot and leaves are the organs that are induced in plant tissue culture. Since embryo is an independent structure and does not have vascular supply, it is not supposed to be the plant organ. It starts with stimulation caused by the chemicals of medium, substances carried over from the original explants and endogenous compounds produced by the culture by Tomas and Davey (1975). Skoog and co-workers gave the concept of regulation of organogenesis by a balance between cytokinin and auxin. High ration of auxins and cytokinin stimulated the formation of root in tobacco callus, but a low ratio of the same induced shoot formation.

9.27. ROOT CULTURE The root is culture in liquid medium. It has several advantages over solid media. The techniques of root culture give certain important information’s such as nutritional requirements, infection by Rhizobium and nodulation, physiological activities, for example, production of alkaloids, nicotine, etc. by given Dodds and Roberts, 1985. According to White in 1934 reported first time successful organ culture, e.g., potentially unlimited growth of excised tomato roots. Subsequently, roots of several species of gymnosperms and angiosperms have been successfully cultured.

9.28. SHOOT CULTURE In the shoot culture apical meristem is cultured (the region of shoot apex laying to leaf primordium). It is clearly differ from shoot apex by having shoot apex and a few leaf primordial. The culture of relatively large tips (5–10 mm long sections), this technique is also known as meristem culture, Meristemming and mericlones (Murashige, 1974).

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9.29. MICROPROPAGATION According to Murashige in 1974 has described the procedure of development of micropropagation into three different development stages such as • • •

Establishment of explant aseptically Multiplication of propagules by repeated subcultures on a specific nutrient medium. Rooting and hardening of plantlets and planting into soil.

9.30. CELL-SUSPENSION CULTURE It is prepared by transferring a fragment of callus (500 mg) to the liquid medium (500 ml) and agitating them aseptically to make them cells free. It is difficult to have suspension of single cell. However the suspension includes single cell, cell aggregates residual inoculum and dead cells (Dodds and Roberts, 1985). According to King (1980) has described that a good suspension consists of a high proportion of single cells than small cluster of cells. It is more difficult to have a good suspension than to find out optimum environmental factors for cell separation.

9.31. BENEFITS OF CELL CULTURE Benefits for cell culture such as: • • • • •

Suspension can be pipetted. They are less heterogeneous and cell differentiation is less pronounced. They can be cultured in volumes up to 1.500 liters. They can be subjected to more stringent environmental controls. They can be manipulated for production of natural products by feeding precursors by Kurz and Constable, 1979.

9.32. SOMATIC EMBRYOGENESIS It can be initiated into two ways: •

Inducing embryogenic cells within the preformed callus;

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• Directly from pre-embryonic determined cell (without callus). Which are ready to differentiate into embryoids by Sharp et al., 1980. In the first case, embryoids are initiated in callus from superficial cell aggregates where cells contain a large vacuole, dense cytoplasm, large starch granules and nucleus by Mc Willian et al., 1974. Two nutritional media of different composition are required to obtain embryoids. First medium contains auxins to initiate embrogenic cells. Second medium lacks auxin or reduced level of auxin is needed for subsequent development of the embryonic cells into emryoids and plantlets. In both the cases reduced amount of nitrogen is required (Ammirato, 1983). The embryogenic cells pass through three different stages such as globular heart shaped and torpedo shaped to form embryoids. These embryoids can be separated and isolated mechanically by using glassbeads. When embryoids reach torpedo stage they are transferred to filter paper bridge and sucrose on which whatman no.1. Filter paper is placed to make a bridge by Dodds and Roberts, 1985.

9.33. SOMACLONAL VARIATION The genetic heterogeneity of cells in a population represents continuity of gentotypes, whereas phenotypically the population is represented as a discrete sum of subclones. After cloning a single cell, from the population of the strain of Dioscorea deltoidea, there developed subpopulation that defined in teir growth rate and sapogenin content. The mass occurrence somaclonal variants, increase in the resistance, productivity and vital force of the plant habe been explained by Carlson, 1983. This would be due to the dominance of nonlethal mutations that lead to heterozygosis with a wild type allele which is thus phenotypically expressed as hybrid heterosis. There are some somaclonal variation in some plants such as Brassica spp., Nicotiana tabacum, Oryza sativa, Saccharaum officinarum and Zea mays.

9.34. PROTOPLAST CULTURE Protoplast is the biologically active and most significant material of cells. When cell wall is mechanically or enzymatically removed the isolated protoplast is known as naked plant cell. The plant cell wall act as physical barrier and protects cytoplasm from microbial invasion and environmental stress. It consists of a complex mixture of cellulose, hemicellulose, pectin, lignin, lipids, protein, etc. For dissolution of different components of the cell wall it is essential to have

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the respective enzymes. There are number of stages like isolation of protoplasts, regeneration and protoplast fusion and somatic hybridization.

9.35. ISOLATION OF PROTOPLASTS • • • • •

Surface sterilization of samples (Leaf) Rinsing in suitable osmoticum Plasmolysis of cells Peeling of lower epidermis Isolation and purification of protoplasts

9.36. REGENRATION The protoplast regenerate a cell wall, undergo cell division and form callus. The callus can also be subcultured. Embryogenesis begins from callus when it is placed on nutrient medium lacking mannitol and auxin. The embryo developed into seedlings and finally mature plants (Figure 9.3).

Figure 9.3: Isolation of protoplasts.

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9.37. PROTOPLAST FUSION AND SOMATIC HYBRIDIZATION According to Schieder in 1982 discussed four major aspects of protoplast fusion as: •

• • •

Production of fertile amphidiploid somatic hybrids of sexually incompatible species is achieved. Induced fusion of protoplasts from two genetically different lines of species must result in a variety of homo as well as heterokarytic fusion products. Selection of a few two somatic hybrid colonies from the mixed population of regeneration protoplast is a key step in successful somatic hybridization technique. Production of heterozygous lines within one plant species which normally will be propagated only vegetatively, e.g., potato. Transfer of only a part of genetic information from one species to another using the phenomenon of chromosome elimination. The transfer of cytoplasmic genetic information from one to a second line or species. It has been possible to transfer useful genes, e.g., nif genes, disease resistance genes, rapid growth genes from one species to another, thereby to widen the genetic base for plant breeding (Figure 9.4).

Figure 9.4: Purification, culture and regeneration of protoplasts

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9.38. FUSION PRODUCT Fusion of cytoplasm of two protoplasts results in coalescene of cytoplasms. The nuclei of two protoplasts may or may not use together even after fusion of cytoplasms. The binucleate cells are known as heterokaryon or heterocyte when nuclei are fused the cells are known as hybrid or synkaryocyte and when only cytoplasm fuse and genetic information from one of the two nuclei is lost is known as cybrid, i.e., cytoplasmic hybrid or heteroplast (Doods and Roberts, 1985) (Figure 9.5).

Figure 9.5: Cybrid/Hybrid production.

9.39. METHODS OF SOMATIC HYBRIDIZATION • •

• • • •

Isolation of protoplast from suitable plants. Mixing of protoplast in centrifuge tube containing fugigenic chemicals, i.e., chemicals promoting protoplast fusion such as polyethylene glycol (PEG) 20% W/V, sodium nitrate (NaNO3), maintenance of high pH 10.5 and temperature 37ºC. Wall regeneration by heterokaryotic cells. Fusion of nuclei of heterolaryon to produce hybrid cells. Plating and production of colonies of hybrid cells. Selection of hybrid, subculture and induction of organogenesis in

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the hybrid colonies. Transfer of mature plants from the regenerated callus.

9.40. SELECTION OF SOMATIC HYBRIDS AND CYBRIDS After the fusion protoplasts of media regenerate cell walls and undergo mitosis. This results in mixed population of parent cells, homokaryotic fusion product and hybrids. Hybrid cells should be differentiated from other cells. There are various selection methods used for selection of fusion products. Selection methods are dependent on: • Physical properties of fused cells. • Biological properties of fused cells. • Biological properties of colonies formed from fused cells. • The somatic hybrids cannot be identified. He first time P.S Carlson and co-workers produced first intersperct somatic hybrids between Nicotiana glauca and N. langsdorfii in 1972. The most of somatic hybridization experiment, selection procedure includes full of chlorophyll-deficient (non-green) chloroplasts of one parent with the protoplasts of the other parents. This help in visual selection of heterokaryon under microscope.

9.41. ANTHER AND POLLEN CULTURE A male reproductive organ is diploid in chromosome numbers. As a result of microsporogenesis, tetrads of microspores are formed from a single spore mother cell. They are known as pollen grains after release from tetrads by Bhojwani and Bhatnagar, 1974. Haploid plants are very useful in direct screening of recessive mutation because in diploid or polyplod screening of recessive mutation is not possible, development of homozygous diploid plants following chromosome doubling of haploid plant cells (Figure 9.6). In China, the most widely grown wheat is a doubled haploid produced through homozygous diploid lines. Anther cultures of rice are also successfully grown. Haploid plants have been produced in tobacco, wheat and rice through pollen culture.

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Figure 9.6: Anther culture. At present more than 247 plant species and hybrids belonging to 38 genera and 34 families of dicots and monocots have been regenerated using anther culture technique. They include economically important crops and trees such as rice, wheat, coconut, rubber trees, etc. by Maheswari et al., 1983.

9.42. CULTURING TECHNIQUES Anthers are superficially sterilized and washed with double distilled sterile water. They are excised from the flower buds and their proper developmental stages are determined under microscope. On confirmation of stages such as (a) anther are directly transfer on nutrient agar or liquid medium where induction of embryogenesis occurs (b) the pollen grains are aseptically removed from the anthers and cultured on liquid medium (Reinert and Bajaj, 1977). Methods of another culture.

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9.43. IN-VITRO ANDROGENESIS In vitro androgenesis is the formation of sporophyte from the male gametophyte on artificial medium. It is most commonly found in family Solanaceae and Poaceae (Graminae). Success of in vitro androgenesis is based on adjustment of development stage of pollen, minerals of culture medium and growth regulators as well as thermal shock or other treatments. Methos of in vitro androgenesis is given in Figure. Pollen grains are isolated from excised anther and extracted in a beaker. Well-collected pollens are washed properly and centrifuged and decanted. They are inoculated in liquid medium, then subcultured in solid MS medium. In culture pollen grains can be induced to produce callus or embyo from which whole plants are regenerated in one month. This technique is most successful in Brassica, Datura, petunia, etc. Mainly there are two ways of in vitro morphogenesis of pollen grains, direct and indirect figure.

9.44. DIRECT ANDROGENESIS It is also called pollen-derived embryogenesis. Here pollen directly acts as a zygote and therefore passes through various embryogenic stages similar to zygotic embryogenesis. When pollen grains has reached globular stage of embryo, then the plants. Direct androgenesis is very common in many plants of the family Solanacease and Brassicaseae by Prakash and Giles, 1987.

9.45. INDIRECT ANDROGENESIS In indirect androgenesis the pollen grains, instead of normal embryogenesis, divide erratically to develop callus. Indirect androgenesis has been found in barley, wheat, Vitis, coffea, etc. Possibility of pollen morphology from the divinding pollen varies. The haploid callus, embryo or plantlets may originate from the continued division of vegetative cell of the pollen, multiple division of generative cells, division products of both generative and vegetative cells by Sangwan, 1981.

9.46. MENTOR POLLEN TECHNOLOGY Pollination is a natural phenomenon which occurs in many plant species. However, there are certain plants where pollen grains remain viable but unable

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to set seed on its own pistil i.e, they are self incompatible. Similarly, in several other plants, besides being viable they even fail to germinate on stigmas of another species, i.e., they are interspecific in compatible. After the birth of recent mentor pollen technology the incompatibility problem of pollen grains can be overcome by altering the fertilization ability. The pollen which has been purposefully treated is called mentor pollen. Hence the mentor pollen defined as the compatible pollen which has been treated in many ways to inhibit its fertilization ability and retain is power to stimulate incompatibility pollen to accomplish fertilization. The mentor pollen itself may or may not germinate on stigmas. As a result of this treatment, hybridization between closely related species or genera and itself pollination in a cross pollinating plants could be achieved.

9.47. EMBRYO CULTURE Embryo culture has also been done for the production of haploid plants. It is used for the recovery of plants from distinct crosses. It is useful when embryos fails to develop due to generation of embryonic tissues. It is being used extensively in the extraction of haploid barley Hordeum vulgare from the crosses H. vulgare × H. bulbosum. Das and Barman (1992) developed the method of regeneration of tea shoots from embryo callus. It produced somatic embryoids within 8 weeks of culture in the second medium which differentiated into buds after 2 weeks. Several shoots with 4–6 leaves developed after 16 weeks of culture.

9.48. EMBRYO RESCUE Viable hybrids are produced as a result of sexual crosses between two varieties of the species. However, if sexual crosses are done between the species of the same genus or between two different genera, production of hybrid is rather difficult because of several barriers arising either during pollination, fertilization or embryogenesis. It has been observed that in some cases in spite of successful pollination and fertilization, embryo does not develop. This due to inherit deficiencies or incompatibility between the developing embryo or endosperms. In such cases, immature embryos are dissected out from the fruit (seed) and grown artificially on medium which differentiate into shoot, root and plantlets. This technique of growing immature embryo is termed as “embryo rescue.” Embryo rescue technique is very useful in wide hybridization, complete growth of embryo in plant, breaking dormancy of certain seeds where dormancy

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period is very long. By using embryo rescue technique wild varieties can be crossed with cultivars. As compared with cultivars, the wild species have greater resistance to pests and pathogens and produce grains of better quality.

9.49. TRIPLOID PRODUCTION The triploid primary endosperm (fusion of the second male gamete with two polar nuclei). Some examples of triploid plants raised from endosperm cultures such as Rice (Oryza sativa), Maize (Zea mays) and Barley (Hordeni vulgare). The triploids of poplar (Popu’s tremuloids) have better quality pulpwood. Therefore, it is important to the forest industry.

9.50. PROTOPLAST FUSION IN FUNGI The protoplast fusion in fungi is the improvement of strains to be used for commercial purposes. For example, two strains of Cephalosporium acremonium were crossed and Cephalosporin C was improved. To improve the citric acid production in A. niger were unsuccessful (Peberdy, 1989). The protoplast techniques are applied in the following four major areas to get the crosses: • • • •

Between different species and genera. Between apparently incompatible starin of the same species. Between different starins or isolates of a species primarily for breeding. Between isogenic starins which may provide an opportunity for genetic mapping.

Intraspecific protoplast fusion It is the cross between the same species in an individual which involves the isogenic strains or the non-isogenic ones e.g., Absidia glauca, Pleurotus ostreatus, Fusarium graminearum, Treesi, etc.

Interspecific protoplast fusion It is the crosses between two different species It is important in the area where new products are to be produced, e.g., S. cerevisiae × S. fermentali, Pleurotus ostreatus × P. florida, etc.

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9.51. APPLICATION IN AGRICULTURE There are number of application in agriculture which has solved the problem of incompatibility of plants and scope of production of new verities within short period of time. • • • •

Improvement of Hybrids Production of Encapsulated Seeds Production of Diseases Resistant Plants Transfer of nif gene to Eukaryotes

9.52. BIOETHICS IN PLANT GENETIC ENGINEERING There are new varieties of production of plant due to mutation. Cultivation of genetically modified crops by the farmers is increasing fast thought out the world. It will support healthcare and industry and provide food, feed and fiber security at global basis. The major concern about the genetically modified plant or crops foods is given below such as: • • • • • • •

Genetically modified crop may bring about changes in evolutionary pattern. Effect of genetically modified crops on biodiversity and environment. Transfer of transgene from genetically modified food to pathogenic microbes. Risk of change in fundamental vegetable nature of plants. Effect of genetically modified on non-target and beneficial insects and microbes. Pollen transfer from genetically modified plants. The risk of transfer of allergies.

10 Industrial Biotechnology

10.1. TECHNIQUE OF MICROBIAL CULTURE It is a multistep process and requires media formulation ionization, environmental control and operation of bioreactor, etc. These steps are discussed in chapter.

10.2. GROWTH MEDIA Micro-organisms require several nutrients (e.g., carbon, nitrogen, phosphorus, minerals oxygen for growth and yield. The nutrient formulations which support optimum microbial g and yield are called growth medial. On the basis of purity of chemical compounds used, media grouped into the following three types:-Synthetic Media: Microbes are cultured on a small scale in laboratory on artificially devised nutrient media by using pure chemicals. Such media are called synthetic media Czapek Dox agar medium for isolation of fungi. Semi-synthetic Media: The media which contain pure form of chemicals as well as complex compounds are called semi-synthetic media; for example nutrient broth, brain hear broth, etc. (i) In the media the complex compounds are beef extract, yeast extract, peptone, net casein digest. Now these media are commercially available. (ii) Natural Media: The media prepared by using the natural complex compounds are called natural media, e.g., soybean extract broth, V8 juice broth, soil extract broth, etc.

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10.3. SOURCES OF NUTRITION There are different sources of nutrients required by different types of microorganisms are given below: (i)

(ii) (iii)

Carbon Sources: The carbon sources used for large scale microbial culture in le are sugarcane molasses, beet molasses, vegetable oil, starch, cereal grains, whey, glucose, lactose, malt, hydrocarbons, etc. Nitrogen Sources: The nitrogen sources are corn steep liquor, slaughter-house urea, ammonium salts, nitrate, peanut granules, soybean meal, soya meal, yeast extract, dis soluble, etc. Growth Factors: There are certain micro-organisms which are not capable of synthesizing vitamins or amino acids. Therefore, to achieve optimum growth, media are supplemented with growth factors

Trace elements Micro-organisms also require certain trace elements (e.g., Zn, Mn, Mo, lime importance in Microorganisms 367 Fe, Cu, Co, etc.) in trace amount. Because these are associated with stimulation of metabolism in earymes (metaloenzymes) and proteins (leg-hacinoglobin). •

•

•

Inducers, repressors and precursors: The catabolic enzymes are induced only in the presence of inducers. For example. yeast extract induces strepiomycin. Production of catabolic enzymes and secondary metabolites is repressed by the presence of certain compounds in the culture medium Antifoams: Protein sources in culture medium (i.e., Products of medium are produced by micro-organisms) cause foaming. Mostly foaming creates problem in microbial process. Therefore, check the foaming problem some antifoams, i.e., fatty acids are added in the culture medium Water: Water is the most important component of the living cells. Because all metabolic activates occur in cytosol. Water-soluble ionic forms of nutrients are absorbed by cell. In laboratory single or double distilled water is used for preparation of culture medium. But for large stage production clean water should be used for consistent composition is required. Dissolved chemicals and pH of water are measured. Water is also needed for ancillary activities for example, cleaning, washing rinsing, cooling, heating, etc.

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10.4. PROCEDURES OF MICROBIAL CULTURE The following sections discuss below about the procedures of microbial cultures.

10.5. STERILIZATION Sterilization is a process of completes eradication of micro organisms from a given place or source. For its scale culture of micro-organisms in laboratory in culture tubes or flasks (in 100 1000 m), the growth media are sterilized by autoclaving at 15 psi (i.e., pound per square inch) for 15–20 minutes. At this pressure, temperature reaches to about 120°C. According to requirement to pressure cooker or an autoclave is used. However, for large fermentation thousand to millions of liters of culture medium is used Large size fermentor and huge amount of medium are utilized by using steam. Besides, if medium is sterilized in a separate vessel, the fermentor must be sterilized by steam, passing the sterilized medium into it.

10.6. CONTROL OF ENVIRONMENTAL CONDITIONS FOR MICROBIAL GROWTH The success of fermentation produces biomass and products depend on the defined environmental conditions that existed fermentor. Therefore, temperature, pH, agitation Oxygen concentration, etc. should have maintain the process through careful monitoring of the fermentation. Microbial growth is significant 0Nuenced by pH of the medium and temperature. Bacteria prefer neutral pH, while acidic pH favor and growth of yeast and fungi. Therefore, pH of the growth medium should be maintained as required by the microbe to be used in fermentor.

10.7. AERATION AND MIXING Aeration and mixing are done in laboratory by keeping the flasks shakers. Hence, it is called shake culture.

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10.8. VESSELS FOR MICROBIAL CULTURES Micro-organisms are grown on according to requirement. For example, in laboratory to single microbial culture is grown and maintained on slant of culture tubes. They are also grown in a simple Erlenmeyer flasks of different volumes (100–1000 ml). Growth in laboratory grown cultures can be improved simply by designing the flask using shakers at controlled temperature as given below:) Baffle Flasks: When the sides of the flask are indented or V-shaped notch is produced Such flasks are called baffle flasks. The V-shaped notch or indentation increases turbulence of the agitated culture medium. This increases the efficiency of oxygen transfer and improves growth of the micro-organisms in the culture.

Shakers There are different types of shakers used in laboratory. Shakers continuously agitate (100–120 throws/minute) the culture medium and transfer oxygen. This results in an improvement in microbial growth.

Fermentors are the closed Fermentors (bioreactors) vessels, which are used for production of products (cell mass and metabolites) on a large scale. Fermentors are of different capacities. Small-scale fermentors (10–100 liters) are used by scientists in research laboratories for optimization of different parameters of microbial growth and production of products. Besides, large-scale fermentors (thou- and to million liters capacity) are used in industries for production of commercial products.

10.9. TYPES OF MICROBIAL CULTURES Differences lie in micro-organisms with respect to their growth and production of products. Hence, the micro-organisms are cultured in different types of vessels in various ways. Therefore, to get the desired product, micro-organisms are grown as a batch, fed-batch or continuous cultures.

Batch culture Batch culture is the simple method. A de- selected microbe is grown in a closed culture sys- tem on a limited amount of medium of microbial culture. The laboratory grown micro-organisms in ordinary flask is basically a batch culture.

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Continuous culture A continuous culture is that culture where a depletion of nutrients, rather than by accumulation of toxic products; it is prevented steady exponential phase for growth of culture retards due to addition of fresh medium to the fermentor and removal of spent medium and microbial biomass from it as a result of which the exponential phase of culture is prolonged.

Fed batch culture Basically it is the batch culture which is fed continuously with fresh medium without removing the original culture medium from the fermentor. It results in continuous increase in volume medium in the fermentor. In fed-batch culture the nutrients should be added at the same rate as are consumed by the growing cells. Therefore, excess of nutrient addition should be avoided. In batch culture when hi concentration of substrate inhibits microbial growth, the fed-batch culture is preferred over the former. Hence, in a fed-batch culture substrate is fed at such a concentration that remains below the toxic level. This activity accelerates the cell growth. A high cell density is achieved in fed-batch they are compared to fed-batch culture. Fed-batch culture is an ideal process for production of intracellular metabolites in maximum amount. For example, alkaline protease used in biological detergents is produced by the species Bacillus Batch feeding of nitrogen sources (e.g., ammonia, ammonium ions and amino acids there substrates at low concentrations and induces protease synthesis.

10.10. MEASUREMENT OF MICROBIAL GROWTH There are different methods of counting microbial growth. These are based on different parameters of cells such as dry-weight and wet-weight measurement, absorbance, cell plate, density, turbidity, ATP measurement, feasible count, ATPase activity and use of Coulter counter (a)

(b)

Wet Weight Measurement: Measuring cell mass is an easy step of cell growth measurement. A known volume of culture sample from the fermentor is withdrawn and centrifuged. Wet weight of pellets is measured by using pre-weight filter paper. A pre-weighed filter paper of similar size is used to subtract the weight of the wet filter paper. Thus wet-weight of cells is calculated. Dry Weight Measurement: Dry weight measurement of cell material is similar to that of weight. Here dry weight of pre-weighed filter paper containing pellets of microbial cells is measured. Dry

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(c)

(d)

(e)

weight of filter is nullified by subtracting the dry weight of only filter paper of similar size. Thus dry weight of microbial cells can be obtained. For example dry weight of about million cells of E. coli is equal to 150 mg. Dry weight of bacterial cells is usually 10–20% of their wet weight Absorbance: Absorbance is measured by using a spectrophotometer. Scattering of light in S with increases in cell number. When light is passed through bacterial cell suspension, light is aired by the cells. Therefore, transmission of light declines. At a particular wavelength absorbance light is proportional to the cell concentration of bacteria present in the suspension. Thus cell growth bacterial suspension at a particular wavelength at different intervals can be measured in terms of absorbance and a standard graph (between absorbance and cell concentration) can be prepared. Total Cell Count: Cell growth is also measured by counting total cell number of the microbes present in that sample. Total cells (both live and dead) of liquid sample are counted by sing a special microscope glass slide called Petroff-Hausser Counting Chamber. In this chamber ground is marked on the surface of the glass slide with squares of known area rid has 25 large squares, a total area of 1 mm2 and a total volume of 0.02 mm (1/50 mm). All cells counted in large square and total number per ml sample is measured. If I square contains 12 cells, e total number of cells per ml sample will be: 12 cells x 25 squares x 50 x 10 = 1.5 x 10 cells. If there is dilute culture, direct cell counting can be done. However, the cell culture of high density can be diluted. Otherwise, clumps of cells would be created which would create a problem in the counting of bacterial cells Viable Count: A viable cell is defined as a cell which is able to divide and increase cell numbers, The normal way to perform a viable count is to determine the number of cells in the sample is capable of forming colonies on a suitable medium. Here it is assumed that each viable cell ill form one colony. Therefore, viable count is often called plate count or colony count. There are two ways of forming plate count.

10.11. METABOLIC PATHWAYS IN MICROORGANISMS Formation of vegetative cells in micro-organisms takes place only when

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there is continuous supply of energy. The cell components are synthesized by metabolism, which is “the ordered transformation of substances in the cell by a series of successive enzymes reactions through specific catabolic pathways.” The metabolic pathways play dual role: (i) it provides precursors for the cell Bonents and (ii) energy for energy requiring processes (Schlegel, 1986). There is a variety of micro-organism which use organic compounds either in simple form or in very complex form via a number of metabolic pathways.

10.12. GLYCOLYSIS OR EMP PATHWAY It is most widely distributed catabolic pathway which proceeds through fructose-1: 0-bisphosphate (FBP) hence, also known as FBP pathway. Glucose comes in metabolically active form which is phosphorylated phosphate. Thus, glucose-6-phosphate is the starting point of all three lytic mechanisms (Schlegel, 1986). Glucose-6-phosphate is converted into fructose-1:6-bisphosphate which then is cleaved into triose phosphates. All the triose phosphates (pyruvate), and ATP and NADH.

10.13. ENTNER-DOUDOROFF PATHWAY Glucose is converted in its active form as glucose-6-phosphate by hexokinase and converted into glucose-6- are converted into two molecules of pyruvic acid glucose-6-phosphate. It is dehydrogenated to 6- Glucose is converted in its active form as phosphogluconate which removes water and yields 2-Keto3-deoxy-6-phosphogluconate (KDPG).Due to formation of the intermediate product, the KDPG, this pathway is also known as KDPG pathway. The KDPG is then cleaved into pyruvic acid and glyceraldehyde-3-phosphate which is finally oxidized into pyruvic acid. In overall reaction one molecule of glucose yields two molecules of pyruvic acid and one mol of ATP, NAD(P) H, and NA distributed in many bacteria of the genus Pseudomonas.

10.14. PENTOSE PHOSPHATE PATHWAY This pathway forms a loop into the EMP pathway, for example, in heterofermentor lactobacilli. The bacteria do not synthesize aldolase which is needed to convert fructose biphosphate into two molecules of triose phosphate. Therefore,

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breakdown of glucose progresses through pentose phosphate pathway. Glucose-6-phosphate is converted to 6-phosphogluconate via dehydrogenation hydrolysis. The 6-phosphogluconate yields ribulose 5-phosphate as the final oxidation product.

10.15. MICROBIAL PRODUCTS Most of the natural products constituted by carbon, nitrogen, hydrogen, oxygen and phosphorus can be fermented under anaerobic conditions by microorganisms. There are many fermentation products used commercially. A list of some micro-organisms and their products.

10.16. PRIMARY METABOLITES Trophophase many intermediate After inoculation when microbial growth is in exponential or metabolic products nucleotides, proteins, carbohydrates, lipids, vitamins, etc.), or energy yielding catabolism (e.g., acetone, ethanal, butanol, organic acids, etc.). Therefore, the metabolites produced during trophophase produced. These are further needed either in growth (e.g., amino acids, are primary metabolites.’ The concentration of some of the metabolites exceeds many are known as times more than required by the producers. The principal primary metabolites and the respective micro-organisms.

10.17. SECONDARY METABOLITES When the trophophase of growing culture is over, then starts the idiophase. Microbial products other than primary metabolites produced during idiophase by slow growing or non-growing cells of micro-organisms are known as secondary metabolites or idiolites such as toxins, gibberellins alkaloids, and antibiotics. The secondary metabolites play It is produced by a limited number of micro-organisms. when depletion of one or more no role in growth of microorganisms. Nutrients are caused in culture medium.

10.18. ENZYMES Enzymes are naturally occurring biocatalysts which accelerate metabolic

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reactions. Various metabolic activities and production of primary and secondary metabolites are not possible without the involvement of enzymes. Enzymes produced during fermentation are mostly extracellular but a few are intracellular for example asparaginase, invertase and uric acid. Intracellular enzymes may be produced in industries, but with many difficulties. The important extracellular enzymes are amylases, cellulases, invertase, B-galactosidase (lactase), esterase, lipases, and proteases.

10.19. MICROBIAL BIOMASS Microbial cells which produces many commercial products, they serve as main source of biomass. Microbial biomass is exploited as a microbial protein or plays a significant role in supplying the protein in world food shortages.

10.20. SCALE-UP MICROBLIAL PROCESS It influenced by the continuously changing environment when moving inside Microorganisms larger fermentor. To avoid these problems the large scale should be taken as the point of reference. The possible effects should be studied by stimulation of the large-scale variations in a small experimental set up. Limiting factors (e.g., are mass transfer) are scaled down and can be studied in an economic way. The desired micro-organism can be used directly for production of products, because there are Several risks associated with economics, production and quality of products. There must be more benefits on small investments but not vice versa. The laboratory processes need to be validated at the intermediate stage in a pilot plant. The pilot plant acts as a small model of commercial plant.

10.21. DOWNSTREAM PROCESSING When fermentation is over, the desired microbial product is recovered from the growth. Then the product is purified, and processed with equal efficiency and economy. The recovery and purification is called downstream processing. The technology downstream processing is as important as self. The operation of any fermentation production process integrates both the associated technologiesassociated technology with the fermentation process.

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10.22. SEPARATION OF BIOMASS Usually the biomes (microbial cells) are separated from culture medium (spent medium). If the product is biomass (single cell protein or vaccines), then it is recovered for processing and spent medium is discarded. Generally, cell mass is separated from the fermented broth by centrifugation or ultra-centrifugation. When there is no aeration and agitation some of the microbial cells soon settle down in the fermentor.

10.23. CELL DISRUPTION If the deficient product is intracellular (e.g., viruses, some enzymes and recombinant proteins like human insulin) the cell biomass can be disrupted so that the product should be released. The solid-liquid is separated by centrifugation or filtration and cell disruption are discarded.

10.24. CONCENTRATION OF BROTH The spent medium is concentrated if the product is extracellular.

10.25. INITIAL PURIFICATION OF METABOLITES There are several methods for recovery of product from the clarified fermented, e.g., precipitation, solvent extraction, ultra-filtration, ion-exchange chromatography, adsorption and solvent ex vacton. The extraction procedure varies according to physico-chemical nature of the molecule of product, and preference of the manufacturers. Isolation of intracellular microbial as human insulin. Steps of isolation of extracellular microbial metabolite.

10.26. METABOLITE-SPECIFIC PURIFICATION Specific purification methods are used when the metabolite is purified to a very high degree. (i) De-watering: When a low amount of product is found in a large volume of spent medium, the volume is reduced by removing water to concentrate the product. It is done by vacuum drying or reverse osmosis. This process is called de-watering. (ii) Polishing of Metabolites: It is the fi- to the

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step of making the product to 98–100% pure. The purified product is mixed with several cheaper inert ingredients called excipients. The formulated product is packed and sent to the market for the consumer.

10. 27. ISOLATION AND IMPROVEMENT IN MICROBIAL STRAINS There is a large number of micro-organisms found in a variety of habitats such as soil, water, air, volcano, arctic water and hot spring. Each microbe has some peculiar feature. All are not supposed to e novel and useful products. Hence, they are first isolated from their natural habitat. Then the aims are further improved using physico-chemical or molecular biological techniques, so that products could be produced at commercial scale. For example, Thermus aquaticus is a hyperthermophilic clerium which grows on volcano. An enzyme, Taq polymerase, is isolated from this bacterium. It is enzyme which is used in PCR for polymerization of DNA synthesis at high temperature. Isolation of Strains For isolation of micro-organisms, samples, mutrient media are prepared. Such specially designed media for culture of specific microorganinsm are called enrichment culture technique. Equal amount of medium is poured into sterilized Petri plates Known amount of serially diluted samples is poured onto the surface of agar medium. The Petri plates for bacteria, 5–7 days for fungi and 14–21 days for actinomycetes, because growth rate of different micro-organisms different are collected from several sites of a habitat. Accord- optimum temperature desired by the microbe (e.g., are incubated for different period such as 24 hours are put into a BOD incubator at on surface of nutrient medium.

10.28. STRAIN IMPROVEMENT OR MICROORGANISM A microbe isolated as above does not ensure that the product produced by it would be in sufficient quantity. Therefore, the strain of such organisms is improved by using classical (mutation and selection) and modern recombinant DNA technology to get the desired product in sufficient quantity. Penicillin production by Penicillium chrysogenuim is one of the good examples of antibiotics. During early days (1940s) penicillin was among those only officers at Brigadier levels were fortunate to get penicillin treatment but not the common soldiers. Today’s strain of P. chrysogenum is capable of producing 1000 times more penicillin

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than the A. Fleming’s strain. It has been possible through successive mutation and mutant selection of over-producers produced in low amount.

10.29. RECOMBINATION Recombinant is Defined as any process which helps to generate new combinations of genes that were originally present in different individuals. Recombination system may or may not be associated with sexual reproduction among the organisms. There are two approaches which have been made to produce recombinants (organisms having a new combination of genes), protoplast fusion and recombinant DNA technology. Protoplast Fusion: Protoplasts of cell walls. You can produce protoplasts using lysosome (cell wall degrading enzyme) in isotonic solution. Methods have been developed to fuse protoplast of two cells of different microorganisms. In 1982, Tosaka produced high lyzine production strain of Brevibacterium flavum by fusing protoplasts with another B. flavum strain.

10.30. RECOMBINANT DNA TECHNOLOGY (= GENETIC ENGINEERING TECHNIQUE) Recombinant DNA Wi. The first commercial genetically engineered protein (human insulin called Humulin) was persons suffering from diabetes mellitus. The efficiency of ammonia metabolism of Methylophilus methylotrophus (a bacterium used as single cell protein) has been improved by incorporation of a plasmid containing glutamate dehydrogenase gene from E. coli produced in 1982.

11 Environmental Biotechnology

11.1. ENERGY SOURCE Fossil-fuel based industry Energy per se is an integral Component of any socioeconomic development for raising the standard as also improving the quality of life of the people in general of living Khoshoo, 1988). Moreover, energy has played much role in the dawn of human civilization. It is obtained in different forms such as nuclear energy, fossil fuels energy (coal, oil and gas), and nonfossil and non-nuclear energy.

11.2. NUCLEAR ENERGY In recent years, we have much hopes for getting nuclear energy. It is made available through the two processes: (a) nuclear fission, where a nucleus of an element is broken into two nuclei or more and releases sufficient amount of energy, and (b) nuclear fusion, in which case energy is released as a result of joining of two very small nuclei. For getting energy of the first kind, nuclear reactors are set up in the developed and of developing countries like India. Energy is generated from uranium for the peaceful In spite of development of nuclear waste management technology, still there is fear for the disposal of radioactive nuclear waste. Radioactive chemicals are long lived, and if entered into human systems it can cause death also. The second method of nuclear energy generation is still in infantry stage. It may take more than 50 years to be developed.

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11.3. FOSSIL FUEL ENERGY The living plants buried during the carboniferous period (about 330–350 million years ago) have been a source for fossil fuels (coal, oil and gases). Coal is the major reserve, followed by oil and natural gas. It is widely distributed and occurs in high quantity. It may reach its peak of production in another 150 years. Oil stands second to coal; its price is increasing day by day due to high cost of extraction and purification. However, during World War II, oil was cheaper than coal. But its price increased gradually with oil-based economics in most of the developed countries.

11.4. NON-FOSSIL AND NON-NUCLEAR ENERGY In addition to energy sources as described above, the star (sun), planet (earth), satellite (moon) and water and wind are the other of energy owing to which our existence is possible. Global power potential of some renewable resources. Energy from tides (due to moon) and geothermal one (hot interior of the earth) have least Contribution. Recently, in Gujarat, the Central Electricity Authority at the Kutch Tidal Power Project, Navlakhi, is investigating for the possibility of electricity generation from tidal wave energy, along specific areas of the Indian coastline. It is expected that 900 MW electricity can be generated. Investigations for assessment of tidal power potential in the Gulf of Kutch were conducted by the National Hydroelectric Power Corporation (NHPC) in association with National Institute of Oceanography, Geological Survey of India (G.S.I) and Central Water and Power Research Station.

11.5. BIOMASS AS A SOURCE OF ENERGY The peculiar feature of Plants is that they possess various photosynthetic pigments in thylokoids present in cells either in free state or in chloroplasts. The photosynthetic pigments are chlorophyll a, chlorophyll q chlorophyll xanthophylls carotenoids. The presence of these pigments varies with group of the plants. In prokaryotic photoautotrophs where chloroplasts lack.

11.6. COMPOSITION OF BIOMASS Cell wall is constituted by mainly 6 components: (i) cellulose, (ii) hemicellulose.

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(iii) Soluble proteins (iv) water soluble sugars, amino acids and aliphatic acids, (v) ether and alcohol-soluble constituents (e.g., fats, oils, waxes, resin and many pigments), and (vi) proteins. These components build up plant biomass. Proportion of these constituents vary in different groups of plants and even the same group. If the concentration of sugar is high, the biomass will be sugary, e.g., and sugar beet. Similarly, starch present in biomass yields the starchy biomass potato and tapioca. Variation in chemical constituents (cellulose, hemicelluloses and lignin) in some plants.

11.7. TERRESTRIAL BIOMASS Since long terrestrial biomass has been used to fulfill the need of food, feed, vegetables, fiber furniture and cooking purpose as well. Traditionally the need of fire/fuel was fulfilled by trees, remains of agricultural crops, and fossil fuels (coal, petroleum). During the course of time, we totally became dependent on conventional energy sources of fossil fuel and electricity But gradually increasing world wide human population and diminishing stock of fossil fuel have challenged Swaminathan (1980) has emphasized for “photosynthetic model of development” This model is applicable for India and other developing countries. However, the extent and nature of this model may vary with energy demand of that country us to seek out the alternative sources of energy.

11.8. AQUATIC BIOMASS It is obvious that the first life originated in water. Therefore, water bodies support a vast community of plant and animal. Many aquatic plants become troublesome for aquatic animals and human as well such as the aquatic weeds like water hyacinth, Salvinia, Hydrilla, Lemna, Pistia, Wolffia, etc. In addition to higher plants, the lower plants (especially blue-green algae and green algae) have much future prospects, as far as production of biomass conversion of aquatic biomass into biogas/hydrocarbon and abatement of pollution.

11.9. SALVINIA Salvinia, a member of Pteridophyta, is commonly known as water fern. It grows luxuriantly in stagnant water, for example ponds, pools and lakes. S. molesta is the world’s worst weed known So far. As a serious menace it is known only from Africa. Sri Lanka and India. In India, it predominates in Kerala, Kashmir

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and North-East states by S. cucculata, In Kashmir, beauty of Dal Lake is gradually fading due to rapid growth of this weed. Recently biogas production from Salvinia was suggested Kashmir it is represented.

11.10. WATER HYACINTH (EICCHORNIA CRASSIPES) Water hyacinth is the most noxious weed of the world and even in paddy field. It is believed that water hyacinth occupies about 2,00,000 acres d in Bihar and 30.000 acres in West Bengal. It grows luxuriantly at temperature 28–30°C. In world, it grows abundantly in tropical regions in non-saline water in ponds, pools, lakes reservoirs mainly multiplies on domestic sewage. Generally the huge amount of biomass is of no use. Nowadays, cultivation of water hyacinth on sewage for minimizing pollution has been suggested Use of water hyacinth in biogas production.

11.11. WASTE AS A RENEWABLE RESOURCES Waste is the spoilage, loss or destruction of either matter or energy, which is unusable to man. Gradually increasing civilization through industrialization and urbanization, has led to increase in generation of wastes into environment from various sources. Waste generation is, therefore, a necessary outcome of consumption, and also because of insufficient process, general ignorance, wasteful habits and social attitudes.

11.12. COMPOSITION OF WASTES Waste is a general term which includes all types of wastes of constituents and phases. Therefore, composition of waste differs with differing re phases and sources. It may be inorganic, organic or mixed types. Organic wastes play a role in being renewed and becoming a source of energy. Composition of organic materials 1s given under ‘composition of biomass.

11.13. SOURCES OF WASTES Industries generate various types of wastes/by-products which contain sufficient

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amount of energy. Some of the industrial wastes described below bisulphite liquor and lignocellulosic pulp. Paper Mill: The wastes are (i) Chemical Industries: The chemical wastes are maleic anhydride and phthalic anhydride (ii) Oil Refineries: They produced wastes as gas, oil, paraffins (n-alkanes), olefins or other hydrocarbons. (iv) Cotton Mills: Cotton mills produces the cotton seeds and fibers as wastes. (v) Food Industry: Waste materials of food industries are the collagen meat packaging waste and lactoserum (a by-product of cheese industry). (vi) Dairy: Dairy industry is one of the important industries which requires special attention as far as treatment and disposal of wastes are concerned. Dairy wastes contain milk whey, butter milk, unused milk skim, plant washings and traces of detergents. The waste is a dilute solution or suspension containing lactose, protein, dairy wastes serve a food substrate for production cell protein, lactic acid, vitamins, ethyl alcohol and alcohol beverages (Garg (xii) Sugar Mills and Distilleries. Molasses and bagasse the wastes generated from sugar mills.

11.14. NON-BIOLOGICAL PROCESS (THERMOCHEMICAL PROCESS) There are different non-biological routes for biomass conversion into energy VIZ, direct Combustion, gasification, pyrolysis and liquefaction.

11.15. DIRECT COMBUSTION Biomass from plants (wood, agricultural wastes) or animal (cow dung) origin are directly burnt for cooking and other purposes. In recent years “hog fuel” production technology has been developed which is being utilized for generation of electricity. Now-a-days municipal. Agricultural and light industrial wastes are used for conversion into energy by direct burning in refuse fired energy systems.

Hog fuel The mixture of wood and bark waste are burnt directly is collectively termed as hog fuel. It was developed in USA.

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11.16. PYROLYSIS Pyrolysis matter, for example, solid residues, wastes (saw dust, wood chips, wood pieces) in an oxygen- decomposition of organic deficient atmosphere absence of oxygen at high temperature (200–500°C or rarely 90me Products of pyrolysis are gases, organic liquids and chars, depending on the pyrolysis process and temperature of reaction. The condensable liquids separate into aqueous (pyroligneous acid), oil and tar fraction (if the substrate composition of gas is carbon monoxide (28–33 per cent) methane (3–5-18 per cent), higher hydrocarbons (1–3 percent) and hydrogen (1–3 percent During pyrolysis, hydrogen content of gas increases with increasing the temperature (Jahn, 1982).

11.17. GASIFICATION Gasification of carbonaceous material under controlled amount of air or pure oxygen, and high temperature up to around 100ºC. As a result of gasification, high amount of gases Gasification of biomass is done in a gasifier designed in various ways. Success for gasification process is based on its designing. Therefore, design a gasifier is an important factor in controlling gas quality. Gas is used in a controlled manner for irrigation, pumping and electricity generation. The advantages of gasification over coal are: (i) much low oxygen requirements, (ii) practically no steam requirements, (iii) low cost for changing H/CO, ratios which are high in wood no little desulfurization cost.

11.18. LIQUEFACTION Liquefaction involves the production of oils for energy from wood or agriculture and carbon residues by reacting them with carbon monoxide and water/steam at high pressure (4,000 TV in and temperature (350–400ºC) in the presence of catalysts. By this method about 40–50% oil can be obtained from wood, This oil serves a good source of fuel.

11.19. THE BIOLOGICAL PROCESS (BIOCONVERSION) Bioconversion involves the conversion of organic materials into energy, fertilizer, food and chemicals through biological agency. The term biological

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agents mean the microorganisms Le bacteria, Actinomycetes, fungi and algae. In broad sense bioconversion involves two steps: photosynthetic production of biomass, and its subsequent conversion into more useful energy forms (gaseous, liquid Ghosh (1980) has estimated that the average production of waste materials in India is about 1540 x 10 tonnes/year.

11.20. ENZYMATIC DIGESTION This process involves the conversion of cellulosic and lignocellulosic aerials into alcohols, acids and animal feeds by using microbial enzyme, e.g., cellulose, hemicellulase, amylase, pectinase, etc. Degradation of cellulose: It is clear that cellulose is a polymer of B-1,4 linked anhydrous pucose units, comprising of 40–60 per cent of cell wall materials of plants. Microorganisms, which produce celluloses and other enzymes in high amount are recent years, Cellulomonas, Trichoderma reesei, T. viride, and other microorganisms are used for the production of cellulases in high amount.

11.21. ANAEROBIC DIGESTION An aerobic digestion is a partial conversion by microorganisms of organic substrates into gases in the absence of air. The gases produced are collectively known biogas. Anaerobic digestion is accomplished in three stages: solubilization (of complex substrates by enzymes into simple forms, i.e., fatty acid, sugars, amino acids), fermentation (of hydrolvsed organic substrates into simplest forms, e.g., organic acids) and methanogenesis (production of methane from simple substrates by methanogenic bacteria under anaerobic conditions). Anaerobic digestion is carried out in a digester, which is a brick-lined or concrete-lined chamber covered completely to prevent the entry of air.

11.22. AEROBIC DIGESTION Aerobic digestion involves the conversion of organic substrates by microorganisms into useable forms in the presence of air, for example composting (biological decomposition organic wastes/residues under controlled conditions to result in release of C, N, P, K, etc.) and oxidation systems (of sewage in oxidation ponds by bacteria and algae) to produce gases, single cell protein, fertilizers.

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11.23. BIOREMEDIATION Bioremediation is the use of living microorganisms to degrade environmental pollutants prevent pollution. It is a contaminated sites and preventing pollution. However, it has global, regional, application. The basis of bioremediation is the enormous natural capacity of microorganisms degrade organic compounds. This capacity could be improved by applying the GMMS technology for removing pollutants from the environment, restore Japan, academic, industrial and governmental research is tightly coordinated for globe application of environmental biotechnology. Researchers are bioremediation that can affect desert formation, global climate change and the life cycle materials. Attempts are formation. This work is based on developing biopolymers that retain water and reverse des formation (Figure 11.1).

Figure 11.1: Enzymes secretion by microbial cells.

11.24. PHYTOREMDIATION Phytoremediation is the use of plants for environmental remediation and involves removing organic compounds and metals from soils and water. This technology is based on plants that have the ability to tolerate high levels of heavy metals. Phytoremediation involves a number of biological mechanisms including direct uptake, the release of exudates into the rhizosphere (to enhance

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bacterial and fungal processes), and metabolic processes within the root and shoots cells. Selected plants are grown on the contaminated site, where they draw up pollutants and concentrate them within various tissues. The plants are then harvested and may be further treated by burning them in a controlled system. The residue of the plants could be recycled or placed in landfills (Ma et al., 2011). Phytoremediation involves a number of biological mechanisms including direct uptake, release of exudates into the rhizosphere (to enhance bacterial and fungal processes), and metabolitic processes within the root and shoot cells. Selected plants are grown on the contaminated site, where they draw up pollutants and concentrate them within various tissues (Rajkumar et al., 2010). Designing a phytoremediation system varies according to contaminant conditions at the site, the level of clean up required and the plant species to be used (Ali et al., 2013). It is a broad term which involves several different techniques such as, phytofiltration, phytovolatilization, phytodegradation, phytostabilization, and phytoextraction (Figure 11.2) (Ali et al., 2013). Phytoextraction is very useful process in which contaminant in soils are uptake through the roots and translocation into the aerial parts of the plant. Nature has not a tendency that all plant accumulates in equal proportion. Some plants have accumulated in large quantity and these hyperaccumulator plants are the base of phytoremediation technology. There are different steps that involve in phytoextraction process: (i) uptake and bioavailability, (ii) translocate of heavy metals, (iii) sequestration of metals in leaves and vacuoles. High amount of heavy metals concentration accumulates in plant organs is not usually a naturally process for favored reaction somehow it’s the plant capability to uptake more than other plants (Greipsson, 2011) Plant defense system mechanisms play a role for metabolic, physiological and expressional changes under stressful conditions caused by different pollutants. Germplasm of hyperaccumulators is the backbone of this technology. Therefore, understanding the genetics of hyperaccumulation is an important tool for the enhancement of hyperaccumulation efficiency. Phytochelatins (PC) and metallothionines (MT) and heavy metal ATPase (HMA) genes play a crucial role in signaling, uptake, detoxification and accumulation of metal. Their combined role enhances the hyperaccumulation efficiency. This technology helps for the development of plants with the higher potentially to clean our environment by giving a favorable condition (Chaudhary et al., 2018).

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Figure 11.2: Types of phytoremdiation process. Phytochelatins (PCs) are the most important class of metal chelators, it is used to chelate the variety of toxic metals. Phytochelatins are produced in a cell under the stress condition and reaction to the high concentration of heavy metals. Some important PCs-metal complexes have been derivative from different microorganisms, fungi, and plants. The work of PCs-metal complexes is to lower down the binding capacity of heavy metals to the cell wall and the same time to detoxify the cell compartments. It can resist very high concentration of heavy metals without causing toxicity (Chaudhary et al., 2018). In assessment to free metal ions, the PCs-metal are in complex form and much more stable (Figure 11.3). Metallothioneins (MT) are also a group of phytochelatins which binds heavy metals through a thiol group of cysteine and also plays important role in detoxification of heavy metals. These MT have a different mechanism to protect the plant from heavy metals by scavenging of the ROS and sequestration (Huang and Wang, 2010). It regulates the action of metallodrugs, their transcription genes activation and the activity of metalloenzymes under any stress condition. The regulation of MT genes was depending on the type of plant tissue. These genes are activated when plant under a biotic stress such as cold, heat, salt, drought, heavy metal and oxidative stress. Metallothioneins genes helped to keep the plant from metal by their hyperaccumulation and they are expressed in high concentration in hyperaccumulator plants as compared to non-hyperaccumulator plants (Gautam et al., 2012).

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Figure 11.3: Phytochelatins (PCs) genes expressed in various plants under heavy metal stress. The metallothioneins (MTs) are classified on the basis of cysteine arrangement in their structure. There are more than 20 conserved Cys are found in mammals and vertebrates which are known to their tolerance towards Cd ions (Gu et al., 2015). Metallothioneins are classified into four classes first the MT1, it was expressed in a plant named Cicer arietinum and their subclasses MT1a and MT1c in A. thaliana. The second one is MT2, it is also found in plant Cicer arietinum but their subclasses are MT2a and MT2b. And the last one is class MT3, it is found in A. thaliana and Musa. Some other classes include MT4a-Ec-2 and MT4b-as Ec-1 found in A. thaliana and Triticum aestivum (Lee et al., 2014). There is strong structure similarity between GSH and MT3 due to their same biosynthesis precursor molecule which is thiol-rich tripeptides. A powerful inhibitor named buthionine sulfoxamine, inhibit the activity of g-glutamylcysteine synthetase enzyme which leads to the decrease in concentration of g-glutamylcysteine and GSH in cells (Figure 11.4).

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Figure 11.4: Diagram of detoxification, conjugation, and sequestration in the vacuole where the pollutant can do harm to the cell. (Chelators shown are GSH: glutathione; GLU: glutamate; MT: metallothioneins; and PCs: phytochelatins). Significantly progress has been made in their recognition and their role in phytoremediation. Some of the other genes like heavy metal ATPases are also used for phytoremediation, these are HMA2, HMA3, HMA4 (Chaudhary et al., 2016). The expression of these genes is liable for heavy metal uptake, translocation, and sequestration might be allowing the yield of plants that can be effectively exploited in phytoremediation. The most closely connected of the A. thalianais P1B-type genes. The HMA2 is expressed in the translocation of Zn and Cd in A. thaliana, barley, rice, and wheat. In Arabidopsis, the cellular and subcellular patterns of AtHMA2 expression were related to the AtHMA4 gene. The expression of HMA2p-GUS gene was observed for the most part in the vascular tissues of the leaf, stem, and root. HMA2-GFP proteins were also localized in the plasma membrane of the plant cell. Recently, results on the characterization of the HMAs2 gene from the different plants for possible application apply in phytoremediation approaches (Chaudhary et al., 2016).

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The ATPase families of integral membrane transporter proteins that help to uptake transition metals are involved in mediating metal-resistant and metalhyperaccumulating traits. The plants were expressing 35S promoter AtHMA4 as well also the metal transporters such as HvHMA2 (Barabasz et al., 2013). While HMA3 may involve to metal detoxification by sequestering Cd into the vacuole HMA4 acts as a physiological master switch during the process of hyperaccumulation metal, and HMA2 and HMA4 play roles in root to shoot metal translocation (Figure 2.8). It is hypothesized that the roles of metal transporters in plants will be essential for the development to genetically modify plants that accumulate specific metals, with subsequent use in phytoremediation process. The efficiency enhanced of HMA3 and HMA4 is a prerequisite for hyperaccumulation and hyper-resistance in hyperaccumulators plant (Figure 11.5).

Figure 11.5: Heavy metal ATPase (HMA) gene contributes in hyperaccumulation of heavy metals. Hyperaccumulator term was proposed first time by Brooks et al. (1977) in reference to those plants that can accumulate more than their natural favored condition approximately 1000 mg kg-1 of heavy metals. Plants accumulate more and more contaminants and tolerate without showing any symptoms (Memon and Schroder, 2009). Baker and Brooks suggested that the minimum threshold tissue concentration for plants as 0.1% and considered Ni, Cr, Cu, Co, and Pb hyperaccumulators but same as above the experiment was done in case of Mn, and Zn threshold value for plants was established as 1%. Plants accumulate heavy metals in root to shoots which favored that can allow translocation of minerals and sugars as they require a proper ratio maintained between the

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amounts of heavy metals specific in roots to shoots. This process is named as translocation factor (TF). Hyperaccumulator plants need more than the TF value 1 (Tangahu et al., 2011). Same as above another factor discussed here about bioaccumulation factor (BF) value also is required more than 1 for hyperaccumulation (Ahmadpour et al., 2014). Some different 450–500 plants have been identified as hyperaccumulators which include Thlaspi caerulescens that accumulate (Pb, Ni, Cd and Zn), Arabidopsis halleri that can accumulate high levels of heavy metals (Cd and Zn but not Pb), Alyssum bertolonii can uptake (Ni and Co) and some other plants which belong to different families can also participate to accumulate heavy metals such as Caryophyllaceae, Fabaceae, Poaceae, Lamiaceae, Asteraceae, Cunoniaceae and Cyperaceae and many others (Maestri et al., 2010). Plants have specific properties that give us some specific advantages to remediate environment (Meagher et al., 2000). Plants absorb metal particle through roots and root hairs that generate surface area through which pollutants can be extracted from contaminated soil and water. Plant is autotrophs; they take up nutrients directly from environment in gaseous form with the help of photosynthesis process. Heavy metals translocate in roots to shoots and also leave. It depends on plants which are used for phytoremediation purpose and called as hyperaccumulators (absorb more than required) and non-hyperaccumulators when they do not absorb limited amount. Drawbacks are also considered when there is significant reduction of biomass of the plants. Different species are used to remove contaminants from the soil and water but sometimes there is inability of plants mechanism to absorb insoluble form of heavy metals present in soil. This process is dependent on many circumstances like soil pH, water contents and also presence of organic and inorganic substances. Naturally plants have the ability to uptake contaminants due to exist in soluble form in soil and water. However, other types of reaction can also be taken up by the use of different amendments like plant growth promoting bacteria and also chelant-induce hyperaccumulation mechanisms around root (Abollino et al., 2006).

11.25. VERMICOMPOSTING Vermicomposting is the phenomenon of compost formation by earthworms. Obviously earthworms play an important role in the cycling of plant nutrients, turnover of organic matter and maintenance of soil structure. They can consume 10–20% of their own biomass per day. The most important effect of earthworms in agro-ecosystems is the increase in nutrient cycling, particularly nitrogen.

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They ingest organic matter with a relatively wide C: N ratio and convert it to earthworm tissue with a lower C: N ratio. Thus, they affect the physico-chemical properties of soil. It is a sustainable biofertilizer generated from organic wastes

12 Biosafety Guidelines Intellectual Property Rights and Entrepreneurship Development 12.1. BIOSAFETY The growing concerns arising from genetically modified organisms throughout the world the WHO/UNIDO/FAO/UNEP has built up an informal working group on Biosafety. The group prepared in 1991 “Voluntary Code of Conduct for the Release of Organisms into the Environment.” The ICGEB has also played important role in issue related to biosafety and the environmentally sustainable use of biotechnology. The organization is ICGEB has annually workshops on biosafety and on risk assessment for the release of GMOs. It collaborates with the management of UNIDO’s BINAS (Biosafety Information Network and Advisory Service)., aimed at monitoring the global development in regulatory issues in biotechnology. The ICGEB has also provided online bibliographic data base on biosafety and risk assessment for the environmental release of GMOs.

12.2. HAZARDS OF ENVIRONMENTAL ENGINEERING Higher cost of agriculture Ethical Issues Loss of familiarity Risks for agriculture Loss of Biodiversity Reduction of Cultivators Alternation of Nutritional Value Weeds or Superweeds

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Risks for the environment Unpredictable gene expression Instability of transgene Increased use of chemicals in agriculture Resistant of targeted organisms Susceptibility of non-target organisms Risks for human health Pathogens Drug Resistance Antibiotic Resistance Allergies Food Quality Food Safety Toxicity Risks of interaction with non-target organisms Generation of New Line viruses by recombination Horizontal Gene transfer Genetic Pollution through pollen or seed dispersal Transfer of foreign gene to microorganisms, i.e., DNA uptake

12.3. BIOSAFETY GUIDELINES AND REGULATION There are many countries have formulated the biosafety guidelines for rDNA manipulation with the aims: • Minimize the probability of occasional release of GMMs. • Ban the deliberate release of such organisms into the Environment. In India DBT has evolved “the recombinant DNA safety guidelines to exercise powers conferred through the environmental protection ACT 1986 for the manufacture, use, import, export and storage of hazards microorganisms/ genetically engineered organisms, cell, etc. The guidelines are being implemented through the following three mechanisms such as: The genetic engineering approval committee (GEAC) of the ministry of Environment and Forest has the power to permit large scale use of GMOs at commercial level, and open field trials of transgenic materials including agricultural crops, industrial products, health care, etc. (Annual report 1995–96 DBT). The institutional biosafety committees (IBSCs) monitors the research activity at institutional level.

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The review committee on genetic manipulation (RCGM) functioning in the DBT which allows the risky research activities in the laboratories. Main function of institutional biosafety committees (IBSC) • To note • To approve • To see approval of RCGM and to recommend Main function of review committee on genetic manipulation (RCGM) • To recommend generation of appropriate biosafety and agronomic data • To approve • To note Main function of genetic engineering approval committee (GEAC) • Inform decision to administrative ministry to follow PVP/Seed Act • To approve open release to environment • To approve for large scale use. Main function of Indian Council of Agriculture Research (ICAR) • To generate complete agronomic data on transgenics • To recommend suitable transgenics for commercial release Main function of MEC • Recommend safe and agronomically viable transgenics • Inspect facility • Analyze data • Visit trial sites

12.4. INTELLECTUAL PROPERTY RIGHTS The physical objects such as household goods or land are the properties of a person. Similarly, a country ha sits own property. The ownership and the rights on the property of a person is protected by certain laws operating in the country. This type of physical property is tangible. On the other hand, the transformed microorganism, plants and animal and technologies for the production of commercial products are exclusively the property of the intellectuals. The rights of the intellectual must be protected and it does by certain laws framed by a country. However it is important to differentiate between physical property and intellectual property. For example, seed of a plant is tangible asset; it can be sold in market and money can be made from it. But the intellectual property is intangible asset.

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Legal rights or patents provide an inventor only a temporary monopoly on the use of an invention, in return for disclosing the knowledge to the others in a specification that is intended to be both comprehensive to, and experimentally reproducible by a person skilled in the art. The laws are formulated time to time at national and international levels. The USA has declared for adopting a strong and uniform IPR laws thought out the world. Development of the crops variety is another intellectual property right. It is protected by “Plant Breeders rights” (PBRs). The PBRs are available in developed countries but not in India. The plant breeders rights recognizes the impact that farmers and rural communities have contributed a lot to the creation, conservation, exchange and knowledge of genetic/ species utilization of genetic diversity. The intellectual property rights and IPP granted by the government to plant breeders are to exclude others for about 15–20 years from producing or commercializing materials of a specific plant variety. But this variety should be now, novel and never exiting before. In this biotechnology has played a significant role in providing processing, designing and production of valuable commercial products utilizable in many area of the society as well as the country such as medical, agriculture, heath care, industry, environment, etc. The technology transfer in biotechnology requires a minimum amount of technical and legal capability which the developing countries lack at present. • Stages of Protection The intellectual property rights are protected in different ways such as patents, copyrights, trade secrets and trademarks, designs, geographical indications.

12.5. PATENTS It is a special right to the inventor that has been granted by the government through legislation for trading new articles. A patent is a personal property which can be licensed or sold by the person/organization just like any other property. For example Alexender Grahm Bell obtained patent for his telephone. This gave him the power to prevent engine from making or using or selling a telephone elsewhere. In the USA the maximum limit of this monopoly is for 17 years. In India the Indian patent Act passed in 1970 for the “process of patents” but not the “product patent” and the maximum duration of patent is 5 years from the date of grant and 7 years from the date of filling the patent application. The least duration between five years to seven years is applicable for patents. The patents in terms give the inventor the rights to exclude the

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others from making, using or selling his invention as disclosed in “claims” of the patent. Obviously, it is difficult to keep secret the certain inventions such as the fermentation process. Therefore, guidance should be obtained from a qualified patent attorney.

12.6. COPYRIGHTS This is protected by only the expression of ideas. One of the best examples of copyrights is the books. The authors, editors, publishers or both the publisher and author/editor have copyrights. The materials of the books cannot be reprint or reproduced without written permission form copyrights holders. The copyrights protected the expressed materials like materials in printed, recorded video and taped forms. Biotechnological materials subject to copyrights include database of DNA sequences or any published forms. In India copyrights act passed or was amended in 1994 and brought enforce in 1999. It includes computer programme, tables and databases. The computer programme is defined as set of works expressed in words, schemes, codes and any other form of including a machine readable medium capable of causing computer to perform a particular task or achieve a particular result. The Ministry of Human Resource and Development look after copyright act in India.

12.7. TRADEMARKS It is an identification symbol which is used in the course of trade to enable the public to distinguish on trader’s goods from the similar goods of the other traders. The public makes use of these trade works in order to choose whose goods they will have to buy. If they are satisfied with the purchase, they can simply repeat their order by using the trade mark, for example KODAK for photography goods, IBM for computers, Zodiac for readymade cloths, etc. The laws vary in every countries. Through agreement it is ensured that the trademark of one country must be protected in another country. Therefore, there are number of multinational companies spend large amount of money to maintain their trademarks throughout the world.

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12.8. WORLD INTELLECTUAL PROPERTY ORGANIZATION (WIPO) It is one of the specialized agencies of the United Nations. It has provided that the intellectual property shall include rights relating to the following: artistic, literary and scientific works, performance of artists, phonograms, broadcast; innovation in all field of human endeavor; scientific discoveries; trademarks; service marks and commercial names; industrial designs; protection against unfair competition and all other rights resulting from intellectual activity in the area of industrial scientific literary or artistic fields. The intellectual property rights is protected and governed by appropriate national legislation.

12.9. GENERAL AGREEMENT OF TARIFFS AND TRADE (GATT) AND TRADE RELATED IPRS (TRIPS) It was framed in 1948 by developed countries to settle the disputes among the countries regarding share of world trade. It is decide by tariffs rates and quantitative restrictions on imports and exports. For a long time benefits from GATT was achieved only by developed countries. In 1988, the US congress enacted a law “ the Omnibus Trade and Competitiveness Act (OTCA). The USA become powerful to investigate the laws related to trade and check them within the desired period, the US takes action against that country. In 1992, the US gave warning to India to change some of its laws of IPR, patents and copyrights. The certain inhibition was acted in India to sign on GATT draft. Therefore, there was much debate throughout the country on this issue and bad intension of the US. The scientist, politicians, and professionals was argued that the total package of Trips must guarantee for economic and technological subjugation of the country. There are many groups and organizations that have rejected this draft of suggestion and opposed the decision taken by the government.

12.10. BIODIVERSITY BILL 2002 The Biodiversity Bill Act was passed in 2002 for the preservation of biological diversity in India and provides mechanism for sharing equitable benefits arising out of the use of traditional biological resources and knowledge. Under this act “variability among the living organisms from all sources and the ecological complexes of which they are part and includes diversity within in species or

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between species of ecosystems.” In the biological resources as “ plant, animals and microorganisms or parts therefore their genetic material and by products with actual or potential use or value but does not include human genetic material.

12.11. GEOGRAPHIC INDICATOR BILL The geographical indication bill or tag is granted through the TRIPs agreement. It is used on products which correspond to a specific geographical location or origin. The use of a geographical indication as a type of indication of source may act as a certification that the product possesses certain qualities is made according to traditional methods or enjoys a certain reputation due to its geographical origin. The geographical indications are generally traditional products produced by rural marginal or indigenous communities over generations that have gained a reputations on the local, national or international market due to their specific unique qualities.

13 Biotechnology Practical

13.1. STERILIZATION TECHNIQUES (EXPERIMENT 1) 1. Objective Performing different methods of sterilization used in biotechnology.

2. Principle Maintaining the sterile environment during the transfer or culturing of cell/ tissues of microbes plants and animal cells is most important. The concept of sterilization, for making the materials free from the any type of contamination was given by L. Pasteur. Thus sterilization is a process of making an article. surface or medium free from any type of microorganisms that contaminate the object and provide unwanted results. However, sterilization is one of the most important steps for cultivation isolation and study of purified cells or tissues in the laboratory. The other important things to be sterilized are the surgical tools, culture vessels, nutrient media, and plant materials. Some other methods used to make these sterile are disinfection and incineration. (a) Disinfection: Disinfection is a similar process of killing the harmful microorganisms especially the objects but not the culture media. Disinfection of table tops, equipment and others surface are usually done by using glycolic acid compounds, carbolic acid and formaldehyde, ethanol, etc. (b) Incineration: It is a process of killing of microorganisms by using flame,

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therefore, it is called flame sterilization. It is done by keeping the inoculation needle over the flame of Bunsen burner till it become red hot. Thus, the microorganism present on the surface of needle are destroyed. Beside, physical and chemical methods are used for sterilization.

I. Physical methods of sterilization There are several physical methods of sterilization of materials and objects. These are briefly described below: (a) Moist Heat: Culture media (liquid and agar), water, glassware, surgical blades and scalpels are sterilized by using moist heat, i.e., steam under pressure. It is done by using an autoclave, and also by a pressure cooker (used for cooking purposes at home). (b) Dry Heat: Dry heat sterilization is carried out by using a hot air oven. Glassware, glass syringe, forceps, scalpel, pipettes, flasks metallic instruments. Petri dishes, etc. are sterilization in an oven at 150°C for 1 hour, or 250°C for 30 minutes. (c) Radiation: Normally ultraviolet (UV) radiation is used in inoculation chamber or laminar air flow. Expose the working area under UV radiation before 2 hours to start the work. The source of UV radiation is UV lamp or UV tubes enclosed in quartz because glass will not transmit UV radiation. Care should be taken not to see the UV radiation with naked eyes. Otherwise. any abnormality may occur in eyes. (d) Membrane Filtration: Sterilization of heat sensitive substances like enzymes, antibiotics, amino acids could not be done by autoclaving because these may be denatured and non-functional. Hence, these are sterilization through various types of filters which may retain bacteria. Millipore membrane filters are commonly used for this purpose. It is commercially available, e.g., Nalgene, Billipore and Whatman (England) membrane filters. It is made up of cellulose acetate and/or cellulose nitrate that contains small sized pores of varying diameter (0.22, 0.45 and 0.5 um), But membrane filter of 0.22 um is usually preferred Millipore membrane filter is placed inside the filtration assembly which is made up of autoclavable plastic materials, stainless steel or glass. The whole assembly with millipore filter paper is sterilized by autoclaving before use. In such cases, the solutions to be Sterilized usually are passed through membrane filters by negative pressure applied through suction or centrifugal force. The filtrate so obtained is collected is sterile container; the filtrate become microbe-free.

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II. Chemical sterilization (a) Alcohol: Ethanol (70%) or propane (70%) is used to sterilization working table top, inoculation chamber, hands, materials to be used, glass apparatus, etc. (b) Aldehyde: Generally laboratory or chamber is fumigated with formaldehyde when the number of contaminates gets increased. (c) Inorganic chemicals: There are certain chemicals toxic to any organism such as salts of copper, mercury, etc., HgC1, solution (0.1%) is most commonly used as disinfectants for seeds, explants satorals For the same purpose, other chemicals used are sodium hypochlorite (NaOCl) (10%). The materials to be disinfected in the solution are kept in Hg, solution for 5–10 minutes (or naked hypo-chlorite). Soon take out the materials, transfer in to sterilized distilled water and washed properly. Again repeat the process of washing for 5–8 times to remove the traces of chemicals.

3. Requirement • • • • • • • •

Autoclave (for moist heat sterilization), laminar air flow (fitted with UV tube), Oven (tor dry heat sterilization Seed/leaves/root pieces/plants of any plants Sprit, Ethyl alcohol (70%), mercuric chloride (0.1% HgCl,). sodium hypochlorite/calcium hypochlorite (10%) (for chemical sterilization) Vacuum pump, filtration unit and Whitman filter paper (0.22 m) membrane filtration sterilization) Sterile distilled water, glassware. cotton plugs, Bunsen burner, absorbent cotton, plastic ware Petri dishes, etc. Surgical blades, holders, forceps, etc. Plant seedling (10 days old)

4. Procedures •

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Sterilization of Glassware and Object/Tools: For complete sterilization Syringe glasswares, forceps, surgical blades holder, needles, scalpel, and metal instruments are kept in an oven at 150C for 1 hour or 250°C for 30 minutes. Sterilization of Nutrient Media: medium media (broth or medium agar), water and other waste (millipore filter) are sterilized by an autoclave (high pressure steam using moist heat) at 15 per inch square) for 20–30 minutes which gives 121 C Glassware must be wrapped with aluminum foil and flasks containing nutrient medium,

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must be plugged with cotton and then wrapped with aluminum foil. After sterilization, the materials should not be taken out immediately. You must wait for 15–20 minutes to lower down the temperature to about 60 after releasing the pressure. The suitable containers material should be taken out. Surface Sterilization: For surface sterilization of seeds and other parts of a plant (leaves, bark, roots) these are washed by tap water. Then it is dipped in disinfectant chemical solutions first in 70% ethanol (ethyl alcohol) for 30 seconds, then in 10% sodium or calcium hypochlorite for about 10–20 minutes. Time of sterilization varies with the materials used. The material should be thoroughly washed (6–10 times) with double distilled water (5–8 times repeat this process). Sterilization procedures to be performed under aseptic conditions (laminar airflow). Sometimes seeds are dipped only in 10% Na/Ca hypochlorite for 5–10 minutes of sterilization without any other treatment. Membrane Filtration Method: Membrane filtration method is used only for those chemicals which are heat sensitive/unstable at high temperature (e.g., enzymes, antibiotics, amino acid, vitamins Commonly, Whatman filter (0.22 um) is used for sterilization. These substances are passed through an ultra-filtration membrane filters using vacuum pump.

5. RESULTS If culture is not autoclaved properly, it will be contaminated by microorganism. Due to excess autoclaving, culture media turns into brown color. Surface sterilization of seeds with chemicals for long time affect the viability of seeds and other plant parts.

13.2. MEDIA PREPARATION (EXPERIMENT 2) 1. Introduction The growth of microorganisms depends on existing and a favorable growth environment.

2. Principle The culture media are nutrient solutions used in laboratories to make

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microorganisms. For the successful cultivation of a given microorganism, it is essential to understand its nutritional requirements and then provide the essential nutrients in the correct form and proportion in a culture medium. The general composition of a medium is as follows: Culture media could be classified according to consistency, composition and function.

I. Consistency Changing the concentration of solidifying or gelling agents such as agar, gelatine (liquid media do not contain such materials) •

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Cultures in liquid media (or broth) are usually handled in tubes or flasks and incubated under static or shaken conditions. This way, homogenous conditions are generated for growth and metabolism studies (e.g., with the control of optical density and allowing sampling for the analysis of metabolic products). Semisolid media are usually used in fermentation and cell mobility studies, and are also suitable for promoting anaerobic growth. Solid media are prepared in test tubes or in Petri dishes, in the last case; the solid medium is called agar plate. In the case of tubes, medium is solidified in a slanted position, which is called agar slant, or in an upright position, which is called agar deep tube. Solid media are used to decide colony morphology, isolate cultures, detail and isolate bacteria (e.g., using dilutions from a mixed bacterial population in combination with spreading), and for the finding of specific biochemical reactions (e.g., metabolic activities associated with diffusing extracellular enzymes that act with insoluble substrates of the agar medium).

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Chemically-defined (or synthetic) media are collected only of pure chemicals with defined quantity and quality. Complex (or non-synthetic) media are composed of complex materials, e.g., yeast extract, beef extract and peptone, therefore their chemical composition is poorly defined. On the other hand, these materials are rich in nutrients and vitamins.

III. Function •

All-purpose media do not contain any special additives and they aim to support the growth of most bacteria.

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Selective media enhance the growth of certain organisms while inhibit others due to the inclusion of particular substrate. Differential media agree to identification of microorganisms usually throughout their unique physiological reactions. In the detection of common pathogens, most practical media are both selective and differential. Enrichment media hold specific growth factors that agree to the growth of metabolically fastidious microorganisms. An enrichment culture is obtained with selected media and incubation conditions to isolate the microorganisms.

IV. Basic Bacterial Cultivation Techniques Enrichment media help the growth of a specific microorganism against the others present in the sample by its specific nutrient utilization capacity or other unique metabolic properties. Only those microorganisms can grow in the enrichment medium that can use or tolerate the components of the selective medium.

V. How To Make Enrichment Cultures It is very valuable technique developed by some gaints in microbiology in the early 1900s. The certain growth media is used to favour the growth of a particular microorganism over others, enriching a sample for the microorganism of interest. The following requirement for enrichment culture and procedure are given below.

3. Requirements • • • • • • •

Garden soil Gasoline-containing enrichment broth cellulose-containing enrichment broth HgCl2-containing enrichment broth sterile chemical spoons Laboratory scales shaker incubator Glass spreader (alcohol for sterilization) Bunsen burner Incubator

4. Procedure •

Measure 1 gm of the garden soil into the flasks containing different enrichment broths.

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For better airing place the inoculated flasks into a shaker incubator at 28°C for three days. Prepare 10-fold dilution series from the enrichment cultures and from sample. Spread the surface of agar plates having the same composition as the enrichment broths from the individual dilution series. Incubate the cultures at 28°C for three days. Perform germ count estimations and colony morphology examinations after the incubation period.

5. Precautions • • •

Sterile chemical spoons Sterile glass spreader Sterile broth should be used

6. Viva Questions • • • •

What is culture media? What is the significance of this test? What is the difference between nutrient agar and enrichment broths? What the meaning is of spread and streak method?

13.3. ISOLATION AND CHARACTERIZATION OF UNKNOWN BACTERIA (EXPERIMENT 3) 1. Introduction The identification of unknown bacteria which involve preliminary microscopic examination of the gram-stained preparation for its categories of two broad groups which wound later from the basis of selection of biochemical tests to be performed. The first place to identify unknown culture is to be determining what genus it fits into. The purpose of this experiment is to characterize to identify an unknown bacterial culture by using previously studied staining, cultural and biochemical methods.

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2. Principle The identified unknown bacteria from microscopic observation or culture method to detect the morphology, arrangement of cells, cultural characteristics on agar and in broth; the gram stain and other staining reaction the absence or presence of motility and also perform the biochemical test.

3. Requirements • • • • • • • • • • • • • • • • • • • • • •

Unknown bacterial culture Litmus milk Staining reagent for gram-stain Acid fast stain Spore stain Phenol red fermentation broths one of each of dextrose lactose and sucrose Marker Microscope Immersion oil Blotting paper Nutrient gelatin deep tube SIM agar deep tube MR-VP broth Tryticase nitrate broth Urea broth Trypticase soy agar plate Starch agar plate Tributyrin agar plate Reagents for the detection of starch hydrolysis, indole, nitrites Lens paper Clean glass slide Bunsen burner

4. Procedure •

Pour the nutrient agar media into the petri plates and streak the unknown bacterial culture on the surface of dried agar plates for isolation.

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Incubate one inoculated plate at 37oC and other plate at room temperature. Inoculate two trypticase soy agar slants by means of a streak inoculation. Incubate the plates for 24 hrs at 37oC. Keep the culture plates into the refrigerator as a stock culture and other culture to be used as a working culture. Perform a gram staining reaction from the 24 hrs old culture. After observing the staining and morphologic characteristics and perform the biochemical test. Incubate all the inoculated test tubes, slants for 24 to 72 hours at 37oC. Examine the microscopic results and biochemical test such as cell shape, motility, cell arrangement, cell size, Endospores, gram reaction, stain reaction, diameter, growth, margin, elevation, form, color, consistency, sediment in broth, flocculent (Table 13.1).

5. Precautions • •

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Sterile distilled water should be used. Aseptic condition should be maintained.

Table 13.1: Biochemical Characteristics (Observation Table)

Tests Fermentation of dextrose, lactose, sucrose and mannitol Gelatin liquefaction Starch hydrolysis Lipid hydrolysis Casein hydrolysis Litmus milk reactions

Results +

Results -

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Methyl red test H2S production Voges-Proskauer test Catalase test Urease activity Oxidase test Nitrite test Indole test Citrate utilization

6. Viva Questions • • •

What are bacteria? What is the principle of this test? Why we performed methyl red test?

13.4. BACTERIAL GROWTH KINETICS (EXPERIMENT 4) 1. Objective Determination of bacterial growth kinetics.

2. Principle Depending upon nutritional status bacteria exhibit different growth patterns. Bacteria take time to adjust in the environment. Freshly inoculated nutrient broth. This gap of time is called lag phase. Therefore, it uses the nutrients of the medium and multiply very fast showing exponential or the phase of growth. Then the growth becomes stagnant. This stage called stationary phase. After a few days, nutrients of the medium start diminishing; therefore, growth rate

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retards which is called deceleration phase. There comes a stage when there is no increase or decrease in number of microorganisms. This phase is called stationary phase. Finally, due to the continuous accumulation of toxic metabolites, there is death of the bacterium. This phase is called death phase. The latter two phases metabolites of the bacteria. In laboratory, bacterial growth can be demonstrated by plotting a graph between in microbial number (measured as optical density by spectrophotometer), The density of all is absorbed by the optical concentration which is actually the cell concentration with time duration Absorbance is a logarithmic value and is used to plot a graph of bacterial growth.

3. Requirements • • • • •

Pure culture of Escherichia coli Nutrient broth medium Erlenmeyer flask (250 ml) Spectrophotometer with accessories Incubator, shaker, inoculating needles, sterilized pipettes (1 ml)

4. Procedure • • • •

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Procure 1 ml or fully grown culture of E. coli from the broth culture prepared earlier. By serial dilution technique dilute the culture to get 1x10 cells/ml (it is approximate when fully grown at least 1x10 cells/ml), Dispense 100 ml of double strength nutrient broth medium in each of 250 ml Erlenmeyer flask and autoclave at 15 psi for 30 minus. Inoculate the sterilized medium by using a calibrated Pasteur pipette for each flask with one drop (about 0.03 ml/drop) of the diluted broth culture. Place the flask on a shaker adjusted at 15 rpm at controlled temperature of 30t 1°C for 48 hours. Switch on the spectrophotometer at least 20 minutes before taking OD (optical density) so that it can get stabilized. Withdraw 2 ml of the broth culture at every 4 hour intervals for 20– 24 hours and measure absorbance (OD using a spectrophotometer at 600 nm wavelength) Un inoculated growth medium is treated as blank of absorbance (on Y-axis) against time (hours) on X-axis.

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5. Results A sigmoidal growth curve of E. coli is obtained OD of bacterial suspension Increases with increasing time.

13.5. CELL VIABILITY ASSAY (EXPERIMENT 5) To measure cell viability by dye-exclusion method.

1. Principle Determination of the cell viability is essential to get cultures of microbes/ animal/plant cells. For determination of cell viability, most commonly to stain (fluorescein diacetate (FDA) or Evan’s blue stain uses Besides, viability is also tested by phase contrast microscope (to observe cyclosis that indicates active cell metabolism), by electrode (measuring O, uptake), by using plants (measuring photosynthesis), and measuring healthy nucleus. a) FDA Method: For the first time, Wildhom (1972) reported this method for determining the cell viability, FDA is a non-fluorescent and non-polar compound which is easily Permeability across the plasma membrane, On the hydrolysis, this compound is broken by the inside of the cell into the fluorescent polar portion which is not permeable across the plasma membrane, it is accumulated in the intact cells in the cytoplasm but not in the dead cells. living cells after FDA treatment show green fluorescence which denotes their viability. b) Evan’s Blue Method: Evan’s blue (0.1% prepared in 0.4 M mannitol/ sorbitol) is also med to determine cell viability. Equal volume of dye and cell suspension is take. Only dead cells take the stain and appear blue colored while the viable cells remain unstained. The percentage of cell viability is calculated by using the following formula. % viability=No. of non-blue cells x 100% viability Total No of cells (blue and non-blue)

2. Requirement • • • •

FDA or Evan’s blue 0.1% Light/compound microscope, microscope slides Mannitol/sorbilol (0.4 M) Freshly dehisced pollen grains of any plant or cultured cells (plants or animals).

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3. Procedure •

Put a small volume of cell suspension or pollen grains in O.1% solution of Evan’s blue and incubate for 15 minutes. • Observe cells under compound light microscope. • Counts the number of blue cells and non-blue cells. • Calculate cell viability by the formula as given above. Note: Freshly dehisced pollen grains should always be used.

4. Results Incubation period with 0.1% Evan’s blue solution play very important role in the accurate assay of cells viability

13.6. ISOLATION OF GENOMIC DNA FROM BACTERIA (EXPERIMENT 6) 1. Introduction A cell genome consist its chromosomal DNA and extrachromosomal DNA present in plasmids, mitochondrial and chloroplasts.

2. Principle The extraction genomic DNA is accomplished by the breaking of cell walls and membranes either with sodium dodecyl sulfate or cetyl trimethyl ammonium bromide for releasing DNA into the extraction buffer followed by the precipitation of cellular debris and unwanted substances such as proteins, carbohydrates by using organic solvents: chloroform, isoamyl alcohol and phenol and finally precipitation of the nucleic acid by using alcohol and isopropanol.

3. Requirements • • • • • •

Bacterial culture 10% (w/v) sodium dodecyl sulfate (SDS) TE buffer (10 mM Tris -1 mM EBTA, pH 8.0) Phenol:chloroform:isoamyl alcohol (25:24:1) Isopropyl alcohol 70% ethanol

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Proteinase K (20 mg/ml) (stored at -20oC) 5 M NaCl CTAB/NaCl solution: Dissolve 4.1 gm NaCl in 80 ml deionized water and add 10 gm CTAB while warming (65oC) and mix till it dissolves completely. Adjust the final volume to 100 ml by the addition of more deionized water. Shaker incubator Microcentrifuge Ice cube/Ice bags

4. Procedure • •

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Inoculate nutrient broth (5 ml) with bacteria and incubate at 37oC overnight with vigorous shaking. Transfer 1 ml culture into a 17 ml microcentrifuge tube, centrifuge at 10,000 gm for 2 minutes in a microcenterifuge and distilled the supernatant. Add 567 µl TE buffer and perform repeated pipetting to resuspend the cell pellet. Add 30 µl SDS (10 w/v) the lysis solution, followed by the addition of 3 µl proteinase K (20 mg/ml), protein degrader, mix the contents and incubate at 37oC for 60 minutes. Add 100 µl of 5 M NaCl and mix the contents well. Add 80 µl of CTAB/NaCl solution mix and incubate at 65oC for 10 minutes. Add equal volume of chloroform: isoamyl alcohol (24:1) and mix gently to precipitate protein and carbohydrates. Centrifuge the sample at 10,000 rpm for 5 mins in the centrifuge for the appearance of white precipitates at the interface of aqueous two layers. Remove the upper aqueous layers that contain DNA into fresh tubes. Add equal volume of phenol: chloroform: isoamyl alcohol (25:24:1) and mix properly. Centrifuge for 5 minutes. Remove upper aqueous upper layer to new tubes. Add 0.6 ml isopropanol and mix gently until white thread like material, i.e., the genomic DNA appears in the tube. Centrifuge at 10,000 rpm for a few minutes.

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Discard the supernatant. Wash the precipitate with 1 ml ethanol (70% v/v) for a few seconds. Centrifuge at 10,000 rpm for 60 seconds to pellet DNA. Carefully discard the supernatant. Place the tube with their cap open at room temperature for 5–10 minutes to air-dry the DNA pellet and to evaporate away the residual ethanol. Extracted DNA pellet can be stored or dissolve the DNA pellet in 100 µl for further use.

5. Precautions • • • •

The reaction mixture should be prepared just before loading sample in gel (freshly prepared). Wear gloves during experiment. Sterile distilled water should be used. Sterile tubes should be used.

6. Viva questions 1. 2. 3.

What is genome? How many chromosomes are known to be occurring? What is a genomic DNA?

13.7. PLASMID DNA ISOLATE FROM BACTERIA (EXPERIMENT 7) 1. Introduction Plasmids are found in bacteria or in eukaryotic microbes. The isolation of plasmids DNA is based on the breaking of the bacterial cell wall and denaturation of cellular proteins and the chromosomal DNA. Plasmids contain 5–100 genes that are not essential for bacterium but may enhance their survival.

2. Principle Plasmids are widely used as vectors in and its isolation is one of the most important steps in genetic engineering. The isolation of plasmid from bacteria involves three steps:

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Growth of Bacterial Culture: Plasmid should be purified from bacterial cultures that have been inoculated with a small transformed colony picked from an agar plate. Usually, the colony is transferred as a small starter culture, which is grown to late log phase. Harvesting and Lysis: Bacteria are recovered by centrifugation and lysed by any one of a large number of methods, including treatment with non ionic or ionic detergents, organic solvents, alkali and heat. Cell-lysis is carried out in the presence of alkali, i.e., NaOH, SDS is also added which helps in the degradation of the cell-wall. Lysis of genomic DNA also takes place. The plasmid DNA is spared since it is smaller in size. Addition of isopropanol precipitates the plasmid DNA. Sodium acetate already present in the solution helps in this process. Plasmid purification: The plasmid DNA thus obtained contains some protein and genomic DNA and RNA as impurities. These are removed by phenol-chloroform extraction and RNase treatment simultaneously.

3. Requirements • • • • • • • • • • • • • • • • •

Bacterial culture Lysis solution A: 50 mM glucose, 25 mM Tris, pH 8.0, 10 mM EDTA Lysis solution B: 0.2 N Sodium hydroxide (NaOH), 1% SDS Lysis solution C: 3 M Sodium acetate (pH 4.8) Phenol: chloroform: isoamyl alcohol (25:24:1) Chloroform: isoamyl alcohol (24: 1) 100% ethanol (ice cold) 70% ethanol (ice cold) TE (10 mM Tris -1 M EDTA, pH 8.0) RNase A (1 mg/ml in TE, pH 8.0) Deionized water Sterile 1.7 ml microcentrifuge tubes (3) Shaker Incubator Eppendorf Microcentrifuge Pipette (P20,P200,P1000) Pipette tips (sterile) Gloves

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Luria – Bertani medium (LB medium) (pH – 7.0), supplemented with 20 µg/ml ampicillin: (Bactotryptone – 10 gm, Bactoyeast extract – 5 gm, Sodium chloride – 10 gm, Distilled water – 1000 ml). Bunsen burner Inoculating loop

4. Procedure •

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Inoculate the bacteria into 2 ml of LB medium containing 20 µg/ml ampicillin (antibiotic) in a 15 ml test tube and incubate overweight with vigorous shaking. Transfer 1.5 ml of culture in a tube and centrifuge at 13,000 rpm for 30 seconds at 4oC in a microcentrifuge and discard the supernatant. Resuspend the cell pellet by adding 100 µl of ice-cold lysis solution A and vortex vigorously and incubate at room temperature for 5 minutes. Add 200 µl of lysis solution B and mix the content gently by inverting the tubes several times. Add 150 µl of ice-cold lysis solution C and mix the contents properly and store in ice for 5 minutes. Centrifuge at 13,000 rpm for 10 minutes at 4oC. Transfer the supernatant to fresh tubes avoids any contamination collected at the interface. Add equal volume of Phenol: chloroform: isoamyl alcohol (25:24:1) and mix properly. Centrifuge at 13,000 rpm for 2 minutes. Carefully remove the upper aqueous phases and transfer to a fresh tube. Add equal volume of chloroform: isoamyl alcohol (24:1) to the tube and vortex to remove residual phenol. Again centrifuge at 13,000 rpm for 2 minutes. Remove the upper aqueous phase and transfer to a fresh tubes. Add 2x volume of 100% ethanol (ice cold) to the sample and incubate at room temperature for 5 minutes. Centrifuge at 13,000 rpm for 10 minutes. Remove the supernatant from the sample and add 1 ml of chilled 70% ethanol and mix the contents by inverting the tube several times Centrifuge again for 10 minutes at 13,000 rpm.

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Discard the supernatant carefully. Centrifuge DNA pellet for 5 seconds at 13,000 rpm. Remove any alcohol with pipette and allow remaining alcohol to evaporate for 15–20 miutes at room temperature. When the alcohol completely evaporates from the tube, add 100 µl of TE buffer (pH 8.0) and pipette up and down gently to dissolve the DNA pellet completely in the buffer. Add 1 µl of RNase (mg/ml) to the sample tube and leave it for 10 minutes at room temperature to degrade RNA left in the sample. The resultant plasmid DNA can be stored at -20oC. Run the sample in agarose gel containing ethidium bromide and check the results for the plasmid DNA by viewing under UV transilluminator.

5. Precautions • • • • • •

Care must be taken when decantating the supernatant after centrifugation? Always wear gloves. Use sterile centrifuge tube. All the solution should be carefully prepared and autoclaved. All the centrifuge operations should be done for proper time. Plasmid DNA should be properly stored at -20oC or -80oC.

6. Viva questions • • • • •

What is plasmid? What is the function of of plasmid? What is the principle of this method? What is the function of RNase in this test? Which chemical is used for precipitation of nucleic acid?

13.8. RNA EXTRACTION BY KIT (EXPERIMENT 8) 1. Introduction Total RNA Isolation Kit provides quick and simple method of isolating total

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RNA from plant cells and tissues. It based on well-established spin column technology for RNA isolation.

2. Principle The Lysis Buffer creates an appropriate binding condition, which favors adsorption of RNA to the silica membrane. The washing steps remove salts, metabolites and macromolecular cellular components. Pure RNA is eluted under low ionic strength conditions with RNase free water.

3. Requirements • • • • •

Lysis buffer β-mercaptoethanol Absolute ethanol Wash buffer II Elution buffer

4. Procedure • • • • • •

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About 100 mg of fresh plant tissue was taken and placed in a prechilled autoclaved mortar and pestle. Small volumes of liquid nitrogen were added and sample was grinded to a fine powder. 450 µl of Lysis buffer- plants containing 4.5 µl of β-mercaptoethanol was added to the ground tissue. The sample was mixed thoroughly with the help of pestle to make a homogeneous lysate. The lysate was allowed to thaw with intermittent grinding and then was transferred in a fresh 1.5 ml vial. The lysate was centrifuged at 10,000 rpm at room temperature for 3 minutes and supernatant was carefully transferred to a new 1.5 ml vial. Pellet was discarded. To reduce the viscosity of the lysate, it was passed through the filtration column placed in a new 2 ml collection tube and was centrifuged for 1 minute at 10,000 rpm at room temperature. The column was discarded and the flow through was transferred to a new 1.5 ml vial. 300 µl of absolute ethanol was added to the flow through and was

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mixed immediately by repeated pipetting. The lysate including any precipitate that may have formed was transferred to the GeneiPureTM RNA column placed in a 2 ml collection tube. The lid was closed gently and was centrifuged for 1 minute at 10,000 rpm for 15 seconds to wash the spin column membrane. The flow through was discarded. Now add 500 µl of diluted Wash buffer II – plants (200 µl of wash buffer II + 800 µl of absolute ethanol) to the GeneiPureTM RNA column. The tube was centrifuged for 15 seconds at 10,000 rpm at room temperature. The above step was repeated. Again 500 µl of diluted Wash buffer II – plants was added to the GeneiPureTM RNA column. The column was centrifuged for 2 minutes at 10,000 rpm at room temperature to clean the remaining salt bound to the silica column. After this step remaining flow through was discarded. After centrifugation, the column was carefully removed from the collection tube so that the column does not contact the flow through. The column was then placed in a new 2 ml collection tube. The lid was closed gently and the empty column was centrifuged for an additional 1 minute at 10,000 rpm to eliminate any possible carryover of the flow through. The GeneiPureTM RNA column was placed into a nuclease free 1.5 ml vial. To elute highly pure RNA, 50 µl of prewarmed (65–70oC). Elution buffer was added at the center of the membrane. The column was incubated at room temperature for 1–2 minutes. The tube was then centrifuged at 10,000 rpm for 1 minute at room temperature. The eluted RNA was stored at -70oC as small aliquots.

5. Precautions • • •

Sterile distilled water should be used for experiment. Work should be done carefully when you are handle centrifuge machine. Wear Gloves during experiment.

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13.9. IDENTIFICATION OF BACTERIAL SPECIES BASED ON 16S RDNA SEQUENCES (EXPERIMENT 9) 1. Introduction One of the most unambiguous methods of the identification of bacterial strains on the species level is the 16S rRNA gene 16S rDNA sequence analysis. The major steps of this method are (DNA extraction, amplification of 16S rRNA gene with consensus PCR, determination of the amplicon’s nucleotide sequence sequencing and sequence comparisons using available databases). DNA extraction from bacterial strains. The procedure of DNA extraction can be separated into two main parts. The first step covers cell disruption, whereas in the second part DNA is purified from other molecules and cell debris.

2. Principle The cells disrupted can be achieved by chemical, enzymatic or physical methods or with the combination of these. Physical cell disruption can be performed, e.g., with a blade homogenizer, mixer mill (which shakes the cells together with glass beads), or grinding cells in liquid nitrogen using a mortar. The incubation at high temperature (98°C for 5 minutes) is also possible. This method utilizes the effect of detergents (SDS) with proteases, in the case of Gram-positive bacteria, lysozyme, or in the case of yeasts, lyticase. Several techniques are available for DNA purification. One of the easiest ways is desalting, when, following a quick spinning to get free from cell debris, DNA, proteins and other molecules are precipitated under high salt concentration. The precipitate is dissolved in water and then DNA is recovered with ethanol precipitation. The efficiency of this method is variable. Extraction with organic solvents is performed with the addition of phenol, chloroform, isoamyl alcohol or their mixture to cell lysate in a ratio of 1:1, which is followed by centrifugation, and the recovery of DNA is performed again with ethanol precipitation. This method is very efficient for DNA removal and also for the exclusion of organic cell components and contaminants, but its drawbacks are that the procedure is time-consuming, cannot be automated and hazardous waste is produced. In the case of CsCl gradient centrifugation, cell lysate is precipitated with ethanol and which is followed by centrifugation through a CsCl gradient using ethidium bromide staning, and subsequently the appropriate DNA-containing layer is separated. DNA can be purified from (ethidium bromide with

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isopropanol, and finally ethanol precipitation) is applied again. This method yields very pure DNA as a result, which is protected from fragmentation. Anion exchange methods are based on solid phase anion chromatography. The negatively charged phosphate-containing part of the DNA molecule can bind to a positive substrate at low salt concentration. The RNA, proteins and metabolites can be washed from the substrate at medium salt concentration, while at high salt concentration, DNA can be eluted. Finally, DNA is recovered with ethanol precipitation. This method produces long (150 kbp) and very pure DNA. The most important requirement of DNA extraction is the production of proper-quality DNA. Low quality DNA extracts contain other cell components (e.g., proteins, RNA) or contaminants that were introduced to the sample during the extraction process such salts, phenols, ethanol or detergent. All these substances can hinder subsequent applications. DNA purity can be checked by spectrophotometric analysis, and described with the quotient of absorption measured at (260 and 280 nm). This value is 1.6–1.8 in the case of DNA of high purity. Different DNA characterization methods need different DNA quantities. In the case of the most frequently used PCR-based techniques, the following considerations are useful. The quantity of sample used for DNA extraction depends on the DNA content of individual cells. Recommended cell numbers used for DNA isolation are for bacteria (109, for yeasts: 107, and for animal cell cultures: 106 cells). The result of DNA extraction (purity and size) can be checked with agarose gel electrophoresis. To avoid degradation, DNA should be kept in a freezer (at –20°C).

3. Requirements • • • • • • • • •

Inoculating loop Micropipettes sterile pipette tips micro centrifuge tubes Microcentrifuge tube rack micro centrifuge Vortex mixer 0.5 M NaOH solution TRIS [tris(hydroxymethyl) aminomethane] buffer (pH 8.0) (mixer mill (bead beater) Thermocycler or water bath dH2O [DEPC(diethyl pyrocarbonate)-treated distilled water] sterile glass beads

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DNA isolation kit laboratory scales Measuring cylinder 250 ml flask Electrophoresis system agarose 10×TBE solution DNA stain Loading buffer DNA ladder (e.g., Lambda DNA EcoRI/HindIII, Marker 3)

4. Procedure • • •

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The DNA isolation procedures are performed in parallel, that differ in the cell lysis step: chemical (steps 2–3.), physical (steps 5–8). The combination of chemical, physical and enzymatic lysis methods using a commercial DNA isolation kit (step 9). In the case of the simplest chemical cell lysis procedure (steps 1–3.), measure 25 μl 0.5 M NaOH solution into a 1.5 ml microcentrifuge tube that is labeled with the name of the bacterium strain. Suspend a loopful of bacteria in the solution, vortex thoroughly and incubate for 15 minutes at room temperature. Add 25 μl 1 M TRIS buffer and 300 μl dH2O. Check the DNA quality with agarose gel electrophoresis (steps 10– 13.). In the case of an easy physical cell lysis procedure, measure 300 μl sterile glass beads and 100 μl dH2O into a 600 μl microcentrifuge tube that is labeled with the name of the bacterium strain. Suspend a loopful of bacteria and shake the tubes for 1 minute at 30 Hz in a mixer mill. Spin the tubes quickly, and incubate for 5 minutes at 98°C (in a thermocycler or in a water bath). Vortex for 5 seconds, centrifuge the tubes for 5 minutes at 10,000 rpm and transfer the supernatant (approx. 70 μl) to a new, labeled microcentrifuge tube. Check the DNA quality with agarose gel electrophoresis (steps 10– 13.). The procedure of DNA extraction using DNA isolation kits will be explained during the practical session (in general, follow the instructions given by the manufacturer). Check the DNA quality with agarose gel electro-phoresis (steps

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10–13.). The first step of agarose gel electrophoresis is gel casting. In the case of 1 % agarose gel, add 8 ml 10× TBE solution to 0.8 gm agarose and fill up to a final volume of 80 ml with distilled water. Boil until the agarose is completely dissolved, cool to approx. 50°C, and add 2.5 μl DNA stain to the solution and mix. Insert combs into the gel casting system, and pour the agarose solution into it. Fill the buffer tank of the electrophoresis ap-paratus with 1× TBE solution. When the gel solidifies (requires 30–40 minutes), remove combs, and place the gel into the buffer tank. Mix 5 μl DNA sample with 3 μl loading buffer, and load this mixture into the wells of the gel. To achieve a semi-quantitative measurement of DNA, load a 2-μl DNA ladder next to the samples. Run electrophoresis for 20 minutes at 100 V, and detect the presence and quantity of DNA under UV light. Compare the quality and quantity of the three isolated DNA samples (e.g., fragmentation of DNA, signal intensity, approximate size compared to DNA ladder). Store isolated DNA at -20°C for further analysis.

5. Precautions • •

Sterile tubes should be used Aseptic condition should be maintained.

6. Viva questions • • • • • • • •

What is DNA? What is RNA? What are the function of DNA and RNA in bacteria? Why we used TBE buffer? What is the function of SDS in DNA extraction method? What is the significance role of this test? Why we used DNA ladder and marker? What is the meaning of cell lysis?

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What is the function of sequences to identify bacteria? Why not use 18S rDNA to identify bacteria instead of 16S rDNA?

13.10. ISOLATION OF MILK PROTEIN (EXPERIMENT 10) 1. Objective Isolation or milk protein (casein) from the milk sample.

2. Principle Proteins are present inside as well as outside the cells of all living organisms. They do may function inside the cells occurring as enzymes, hormones, etc. Most of them contain 20 amino acids. Proteins are the building block of all cells, cellular components and give structural topography. Caseins are the main group of milk protein. Further several proteins can be resolved from this group such as, b, g and k on the basis of electrophoretic mobility under an electric field. It is electively precipitated at pH 4.6. At this pH, caseins have move under an electric field. It explains that the number of positive charges is equal to the number of negative charges at this pH. This is called isoelectric point. Besides, minimum solubility of any zwitter-ionic molecule (those having both a negative and positive charge generating residues within a molecule) is found at its isoelectric point. Such molecules may be separated by filtration or net charge zero because they do not decantation. There are many methods for the estimation of proteins in the sample (milk) e.g., Lowery’s methods and Bradford methods. In this method, isoelectric precipitation principle is used for the isolation of casein from milk

3. Requirements • • • •

Beaker (100 ml), pH meter, cheese cloth Water bath, oven, refrigerator, centrifuge, thermometer, balance, dropper, glass rod. 1N HCI (100 ml); 0.4 M HCl (90.7 ml of stock solution and diluted to 2 liters) 0.4 M NaOH Skimmed milk sample (centrifuge 50 ml milk at 2,000 rpm for 10 minutes, remove fat layer and use the solution for the test).

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4. Procedure • • • • • • • • •

Take 50 ml skimmed milk sample and dilute it by adding 50 ml water. Keep the mixture in a water bath at 30°C. By using a dropper add 0.4 M HCl in the mixture drop by drop stirring continuously. (IV) Cheek the pH continuously by using a pH meter until pH 4.6 is obtained. Filter the content through cheese cloths when attaining the pH 4.6 Collect the precipitated casein curd and measure the weight of it. Take the known amount of wet sample and dry it at an oven at 100°C. Cheek the weight of dried sample continuously till it attains constant weight. Then store the remaining sample in the freezer.

5. Results • • • • • • •

Record the results as given below: Volume of the milk used = Temperature of the milk = Initial pH of the milk = (Amount of 0.4 M HCL was used (ml), till the pH rose to 4.6 (used volume) Wet weight of the sample = gram after drying at 100°C Dry weight of the sample = gram

13.11. PROTEIN ESTIMATION (EXPERIMENT 11) Determination proteins in an aqueous sample. There is a number of protein which have several physiological roles. There may be measured quantitatively by biuret method or Lowery’s method.

A. Biuret Test for Quantitative Estimation of Protein The proteins are made of amino acid Containing peptide bonds. In aqueous phase, two or more peptide bonds react with copper sulfate (blue solution of biuret) and result in purple-violet colored product. The number of peptides

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bonds presented in a sample governs the color intensity or the sample. Color intensity of this product is measured at 540 nm by a colorimeter. The absorbance value is directly proportional to protein concentration in the sample. Biuret reagent is a solution consisting of copper sulfate in alkaline solution of sodium potassium tartrate. Cu++ reacts with proteins coordinates with the peptide bond and reduced to Cu ++ ions resulting in purple-violet color to the solution.

2. Requirements • • • • •

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Test tubes (10) kept in a test tube rack, glass pipettes (1 ml, 5 ml) Colorimeter Unknown protein solution Reagents as below: Biuret reagent: Weigh 1.5 g CUSOSH, O and 4.25 g of sodium/ potassium tartrate and 4 NaOH and dissolve in 1 liter of distilled Store solution in a reagent bottle at room temperature It is stable for a week. Standard BSA (bovine serum albumin): Weigh 1 g of BSA and dissolve in a little amount of distilled water in a volumetric flask and make the volume to 100 ml. The frozen solution should be liquefied before use. Solution of an unknown protein: The BSA solution should be made in such a way that each student must get 3 ml Each student should be given solutions of different concentrations calculate the amount. Prepare BSA solution of different concentrations, e.g., 4.6, 8, 10 mg/ ml and give the concentration to different students and maintain the record accordingly

Procedure: • • • • •

Take test tubes and mark them with marking pen 1, 2,3…, and 8. Add in each tube BSA, solution of test protein and distilled water as below. To each tube add 4 ml of Biuret reagent and mix the contents by shaking the tubes. Mix the contents and incubate in a water bath set at 37 C for 10 minute. Cool the test tube by keep in the test tube stand for 20 minute at room temperature to develop the color.

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Measure optical density at 540 nm using a colorimeter.

3. Results Standard solution of BSA has 10 pmlie 0.1 ml will contain 1 mg Tube 1 containing 0.2 ml of BSA will have 2, and so on Calculate the protein content from a standard curve prepared using m g/ml of albunin, casein, etc. The protein standard is also prepared as described above. Serially dilute the protein solution by adding gradually increasing the amount of distilled water decreasing the concentration of protein solution. Prepare a ration protein:water in the order: 10:0, 9:1, 8:2. 7:3, 6:4, 5:5, 4:6, 3:7. 2:8, 1:9, 10:0. Measure the optimal density in a colorimeter. Plot a graph using concentration versus optical density.

B. Lowry’s Method for Quantitative Protein 1. Principle Lowry et al (1951) gave a simple and less time-consuming colorimetric method for quantative estimation of proteins. The principle of this method is based on the facts that the folin-ciocalteau reagent reacts with aromatic residues of protein and yields to blue color which in turn is read in a colorimeter. The different proteins contain different aromatic residues. Blue color develops because the alkaline copper reacts with protein. Tyrosine and tryptophan present in protein reduces phosphomolybdate (present in folin ciocalteu reagent).

2. Requirements • • • • • •

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Protein sample, albumin or case in as standard solution Pipettes (1 ml, 5 m), test tubes, water bath, colorimeter 1N NaOH solution, alkaline sodium carbonate solution (2 g Na, CO, dissolved in 100 md of rd due to lib 0. In NaOH solution) Reagents as below Copper sulfate, sodium, potassium tartrate solution in 1% of sodiumpotassium tartrate solution. Prepare fresh Alkaline copper reagent: Mix 50 ml of alkaline sodium carbonate solution and 1 ml of copper sulfate sodium potassium tartarate solution only on the day of use. Folin ciocalteu reagent: It is a solution of sodium tungstate and sodium molybdate in phosphonic and hydrochloric acid available commercially. Dilute the commercial reagent with an equal volume

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of distilled water only on the day of use.

3. Procedure • • • • •

Take a test tube, transfer 1 ml of 1 NaOH solution and heat up to 100° Suspend 1 ml of protein sample into the above solution for 5 minute. Add 5 ml of alkaline copper reagent, mix properly and leave this mixture at room temperature for 10 minutes. Add 0.5 ml of folin-e-calcium reagent rapidly with immediate mixing. Leave it for 30 minutes; measure the absorbance of solution at 750 nm.

3. Results Prepare a standard curve using casein or bovine serum and measure absorbance at 750 nm taking 1–10 ml solution with appropriate serial dilution. Prepare a graph between protein concentration and absorbance. Calculate the amount of protein present in the unknown sample following the standard curve of known protein. Report the result as mg protein/ml of solution.

13.12. ASSAY OF ACID PHOSPHATASE (EXPERIMENT 12) 1. Objective Determination of catalytic power of acid phosphatase.

2. Principle Enzymes are the major class of proteins, called biological catalysts or biocatalysts because they speed up the rate of reaction occurring inside the cells of all organisms In our body, numerous biological reactions occur which 1878 by Friedrich W. Kuhne to designate the biological catalysts that had earlier been called ferments. Acid phosphatase is located in lysosomes having acidic cytoplasm is nearly neutral. When an enzyme is active only in acidic pH. It is called acid-phosphatase. The enzyme catalyzes the release of inorganic phosphate from the compound of organophosphate, i.e., nucleotides, nucleic acid, phosphorylated proteins, etc. Enzyme activity is assayed by measuring

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the conversion of the substrate into products which is formed within a given lime. For determining the activity of acid phosphatase an artificial substrate, p-nitrophenyl phosphate, is used which is broken without the amount of enzymes, The term enzyme was coned is pH falling between 5 and 6., The pH of by acid phosphatase as below: Enzyme p-nilrophenyl phosphate + water p-nitrophenol + inorganic phosphate (P) (yellow) p-nitrophenyl phosphate (PNPP) is a colorless substrate but after the reaction it has yellow particles due to liberation of p-nitrophenol which can be easily measured colorimeter The color intensity can be correlated with enzyme activity by using a standard graph. Enzyme fraction is procured mm any source as aqueous solution. It is mixed with PNPP in a suitable buffer at 5.7 pH and allowed to react. At the end of the reaction an aliquot of potassium hydroxide is added which stops the reaction and inactivates the enzyme. Besides, this alkali intensifies the yellow color through favoring the tonance of PNP. This facilitates to detect the color change in spite of the presence of small amount of PNP.

3. Requirements • • • • •

Acetate buffer (1 M. pH 5.7). p-nitrophenyl phosphate (0.05 M) solution, p-nitrophenol (200 mM solution) solution. Potassium hydroxide (0.5 M solution, 500 ml). Sodium acetate, acetic acid, sodium chloride (0.9%, saline solution) Sprouted seed of mung dal (legume), beaker. Test tubes, mortar and pestle, glass pipettes (1ml, 5 ml), cheese clothes.

4. Procedure • • •

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(Take mung seeds and soak overnight to allow germination under dark condition. Weigh 10g of sprouted seeds, wash with distilled water (5 times) in a beaker). Place pestle and mortar, transfer small amount of sprouted seeds and pour a few volume of saline solution Care should be taken that the total amount of ground material should be about 50 ml. Take cheese cloth, make 5 layers of it and squeeze the ground material. Collect the filtrate in a test tube. This filtrate consists of the required enzyme. Since room temperature fluctuates during different seasons,

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the enzyme fraction should be place in a freeze till further use. Assay of enzyme by pouring the solutions in two different tests tubes in the following order: 0.4 ml Acetate buffer PNPP solution = 02 ml Distilled water 0.2 ml enzyme fraction (0.2 ml) • (Immediately after adding enzyme mix the contents and note the tire cover the test and place in the dark for 20–25 minute incubation at 25 t 1C)) • After 20 minutes of incubation, add 3 ml of KOH solution with the help of sterile auto pipette or sucker on the mouth of a pipette (mouth pipetting of KOH is very dangerous) • Mix the contents properly and gentle shaking of tube. • Take 1 to 6 & tubes and mark it properly.

5. Results Record the absorbance serially from 1 to 6. The solution of PNP is 200 mM it means 1 ml solution contains 02 m mol of PNP. Record absorbance after recording the readings.

13.13. IDENTIFICATION N-TERMINAL AMINO ACID OF A PROTEIN (EXPERIMENT 13) 1. Objective Identification of N-terminal amino acid of a protein using dansyl chloride method.

2. Principle Crystals of amino acids are white and its solution appearances colorless. Acids depends on the amount of carbon properties, etc. Specifically, dansyl chloride reacts with the free ammino group of the N-terminal acid residues of a protein. Upon acid hydrolysis it results it results in formation of dansylated amino acid and the other free amino acid are separated. They can be identified using ultraviolet light. Under UV light dansyl amino acids appears greenish-yellow in color.

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3. Requirements • • •

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Dansyl chloride solution (2.5 mg/ml in acetone) (prepare fresh) and keep at 4°C refrigerator. 0.2 M aqueous solution of sodium bicarbonate (keep at 4°C, when not in use) Ignition tubes/test tubes referred to as ‘dansyl tubes’ (5 cm x 2 mm) plates [polyamide thin layer plates both sides)] on both sides. Number the plates with a pencil in the top corner. Mark the origin for loading with the lower hand corner of the numbered side of the plate (the origin for loading on the reverse side of the plate should be immediately behind the loading position for the front of the plate, i.e., 0.3 0.5 mm from each edge in the lower right hand corner). Prepare two chromatography solvents as below: Solvent 1: Formic acid: water (4.5: 100, (v/v) Solvent 2: Toluene: acetic acid (8: Ultraviolet source (254 Lum or 265 um), vacuum pump, dessicator

4. Procedure • •

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For Amino Acids Prepare the stock solution (1 mg/ml) of standard dansyl amino acid in 0.2 M NaHCC (standard dansyl amnio acid are-Pro, Leu, Phe. Thr. Glu, Arg Store the stock solution at 4°C when not in use). Transfer 10 ul of each amino acid solution in a dansyl tube and mix gently. Add 30 ul of dansyl chloride solution (5 ul for each amino acid). Add 10ul of unknown amino acid and 5 ul of dansyl chloride in a separate dansyl tube. Seal the tube with parafilm and incubate at 37°C for 1 hour at room temperature for 3 hour). Dry the samples in vacuum because the small volume of liquid present will take about 5 minutes. Dissolve the dried samples in 10 ul of acetone using a capillary tube. Load 1 µl aliquots of the standard mixture at the origin (the diameter of the spot should not be allowed to exceed 1–2 mm). Load 1 ul of unknown amino acid on the reverse side. Place the plate in the first chromatography solvent when the loaded

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sample is fully dry Allow the plate to develop and the solvent to reaches the top. Dry both sides of the plate. Develop the dansyl plate in the first solvent. Dry the plate until the solvent front reaches the top. Observe the plate under UV light

2. For Proteins • • • • •

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Transfer protein in 10ul of 0.2 M NaHCO, in 1 ml stoppered tube. Pour 10 l of dansyl chloride solution in to it. Stopper the tube and incubate at 37 C for 1 hour (or at room temperature for 3 hours) Dry the samples in vacuum Transfer 20 µl of 6 N HCI to ml tube and close the tube tightly with stopper. Hydrolyse it keeping in an oven maintained at 105 t 1 for 24 hours. Again the dry samples in vacuum Dissolve the dried mixture using fine capillary tube. Diameter of the spot should not be allowed to exceed 1–2 mm Load 1 µl of protein hydrolysate containing the unknown dansyl amino acids on the reverse. Place the plate in the first chromatography solvent when the loaded completely dry. Allow the develop and reach the solvent to the top. Dry both sides of the plate Develop the dansyl plate in the second solvent at right angle to the direction of development as found in the first solvent. Wait to reach the solvent to the top, and dry the plate. Observe the plant under UV light.

5. Results You should identify three major bottom of the plate. Dansyl chloride is hydrolyzed into dansyl hydroxide. The hydrolyzed product appears as blue flour Dansyl amide (produced by side reaction with NiS) shows blue-green fluorescence and about one third way up plate. These two spots are useful internal marker which appears clear dansyle plates. The third spot fluoresces green and will correspond to the dansyl derivative of s amino acid. Solvent 2 separates the dansyl derivatives of hydrophobic amino acids and some natural amino acids. Derivatives of charged and other neutral amino acids remain at the

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lower end of the chromatogram. Chromatogram is compared with the standard provided in laboratory.

13.14. SDS-PAGE ANALYSIS OF PROTEINS (EXPERIMENT 14) 1. Objective Analysis of protein by gel electrophoresis under denaturing conditions (SDSPAGE)

2. Introduction The proteins, DNA and RNA (biomolecules) have electric charges which depend on molecule and the conditions of the medium (pH of buffer in which dissolved). Charged molecules can be separated by electrophoresis in gels. Polyacrylamide gel is used for protein isolation differences in amino acid composition proteins have a unique mass and charge. Hence the proteins have the negative charge and net positive charge or isoelectric point (no clarification at a given buffer). SDS-PAGE is a high resolution method used universally for analyzing the mixture of proteins to their respective size. SDS solubilized in soluble proteins makes possible the analysis of the other insoluble mixtures Separation of the proteins does not occur due to similar charge: mass (m). Therefore, such proteins are treated first with an ionic detergent called sodium dodecyl supfate (SDS) before the start and during the course of electrophoresis (PAGE) Therefore, such electrophoresis is called SDS PAGE Identical proteins are denatured by SDS resulting in their sub-units The polypeptide chains get opened and extended On the basis of the mass but not the charge, The molecule is separated Electrophoretic separation is normally used for these reasons, i.e., (i) gel acts as molecular sieves hence separates the molecules on the basis of their size, and (ii) gel suppresses conventional current produced by small temperature gradient which improves the resolution Polyacrylamide gel (supporting used for this purpose, temperature, 1onic strength and transparent). Polyacrylamide gel is better for size fraction of proteins.

SDS-PAGE by Laemmli Procedure Laemmli invented the gel disc electrophoresis which is a superior system to resolve proteins in a mixture. According to the procedure, proteins are

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electrophoresed by a discontinuous polyacryamide gel. A stacking gel (/3 of total gel) which includes be sample loading wells consists of 4% acrylamide prepared in Tris HCl buffer (pH6.8). The remaining 5 of the gel length is called separating gel. The separating gel consists of 7.5- 15.0% acrylamide. This range depends on the molecular size of proteins prepared in Tris HCL buffer pH 8.8). In lysis of a complex mixture of proteins, the resolution is improved by the initial movement through stacking gel. The final bands in the separating gel are sharper and focused in a better way. After electrophoresis mixture of proteins is separated as discrete bands.

3. Principle The proteins are denatured and have a negative charge with a uniform charge to mass ratio (z/m) n treated with SDS (anionic detergent) Proteins migrate towards anode at alkaline pH throughout gel during electrophoresis The smaller polypeptides moves faster followed by the larger peptides Therefore, the intrinsic charge on proteins is masked in SDS-PAGE. Hence, the separation is based on the size. Mercaptoethanol reduces interpolypeptide disulfide bridge and separates the sub -units of a polymeric protein. Molecular weight of the separated protein is analyzed by the molecular weight of the standard protein and its mobility.

4. Requirements • • • • • • • •

• •

Ammonium per sulfate (10%), coomassie brilliant blue (0.3%). Destaining mixture, gel staining dish. Electrophoresis apparatus with supply Running buffer, SDS (10%), 1.5 M Tris HCI (pH 8.8), 0.5 M Tr is HCI (pH 6.8), Laemmli. Stock acrylamide-bis-acrylamide solution. Reagents: The following reagents must be prepared in advance Stock acrylamind (bis-acrylamide solution: 29.2 g acrylamide, 0.8 g bis acrylamind: final volume raised to 100 ml) 1.5 M tris-HCI, pH 8.8: Dissolve 18.15 g of Tris in 50 ml of distilled water, adjust the n to 8.8 with HCI, make the final volume to 100 ml). 0.5 M Tris- HCI, pH 6.8: Dissolve 6 g Tris in 60 ml distilled water, adjust pH to 6.8 with HCL, make the final volume to 100 ml. 10% SDS: Dissolve 1g SDS in 5 ml distilled and raise the final volume equal to 10 ml.

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Gel running buffer: Dissolve 14.4 g glycine, 1 g SDS in 1 liter distilled water, adjust pH to 8.3 by adding solid Tris, make the final volume equal to 1 liter. Ammonium per sulfate (APS) (10%): Dissolve 500 mg of solid APS in 5 ml of distilled water. Always use freshly prepared APS solution only. Coomassie Brilliant Blue R 250: Dissolve 600 mg of Coomassie brillia (250) in 80 ml methanol, add 20 ml glacial acetic acid and make the final volume equal to 0 (CBBR – 200 ml) with distilled water. De-staining solution: Mix 400 ml methanol, 100 ml glacial acetic acid and 500 ml distilled to get 1 liter of this solution. Laemmli buffer: 62.5 mM Tris-HCl, pH 6.8 (use diluted 0.5M Tris HCl pH 6.8), 10 glycerol, 5% mercaptoethanol, 2% SDS. According to the manufacturer’s instructions, gel electrophoresis apparatus should be assembled protein sample should be prepared in Laemmli buffer or in the buffer supplied with the kit for the experiment.

5. Procedure •

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Make 7.5% uniform concentration of SDS resolving gel. The capacity of the apparatus golems the volume of the final gel solution. Accordingly, read carefully the instruction manual supplied with the electrophoresis unit before deciding the volume of the gel. Commercially available SDS-PAGE kit is also used according to instruction According to manufacturer’s instruction, assemble the gel electrophoresis apparatus. Make gel solution of the separating and stacking gels (find out the volume of solution needed by the apparatus). Prepare fresh solution just before. APS and TEMED are added to the rest of the solution just before pouring the get solution into the 48.0 ml 768 ml 96.0 ml glass sandwich. Fill up the glass sandwich with pipette up to mark with the separating gel solution prepared (the body must not make contact with chemicals. The acrylamide solution in unpolymerized state is neurotoxic). To make the gel surface straight after polymerization, overlay the acrylamide solution with water (the gel should be allowed to

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polymerize for 30 minutes. When the polyacrylamide layer becomes distinct below the water layer, polymerization is complete). Remove the distilled water by using the filter paper carefully. Thereafter, add the requisite amount of 4% stacking gel solution (freshly mixed). Then insert the comb for polymerization. With the polymerized gel place the glass sandwich into the electrophoretic chamber and add running buffer to level in both cathodic and anodic chambers. To prevent the sample wells from deforming, remove the comb under buffer With the help of micropipette, load the denatured protein solution into the well (protein sample should be denatured in Laemmli buffer by boiling for 5 minutes). Add standard molecular weight marker proteins in one lane. For detection by CBB dye, generally 20 to 50 µg protein is sufficient. Tightly connecting the electrodes of the apparatus with the power supply. Run the gel at constant current of 20 mA Track the mobility of sample in the matrix with the dye (generally bromophenol blue is added to the Laemmli buffer). After completion, switch off the button and disconnect the apparatus. Transfer the gel to the staining tray containing the gel staining dye. Under shaking conditions on a rocking shaker, stain the gel for at least 2 hours or overnight. At this stage, the whole gel turns blue. Carefully transfer the gel de-staining solution and shake on a rocker shaker for 30 minutes. Add fresh destaining solution. Repeat these steps until the bands are clearly visible in the gel. At this stage, take photograph of the gel. When the gel is clearly visible, analyze the photographated gel.

6. Results • • •

Several distinct blue colored bands can be seen in the gel. Each band represents to a single or multiple bands in the lane. Depending on the amount of the polypeptide present in the protein solution loaded in the gel, the intensity of these bands varies.

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13.15. RESTRICTION DIGESTION OF DNA (EXPERIMENT 15) 1. Objective Digestion of bacteriophage lambda DNA using a restriction enzyme 2.

2. Introduction Restriction enzymes have been described in Chapter 4 of this textbook. They are endonucleases which recognize and cleave the specific DNA sequences called restriction sites for example, EcoRI (isolated from Escherichia coli) that recognizes and cleaves the sequence 5 GAATIC-3 10 generate cohesive or sticky ends. Similarly, Hindlll isolated from Huemophites influenzae to recognizes and cleaves the sequences 5-AAGCTT-3 to generate cohesive or sticky ends. Enzyme activity is represented as IU (International Unit), One unit of a restriction enzyme is the amount of enzyme required to completely digest one microgram of lambda DNA [in a reaction volume of 50 µl) in one hour under optimal conditions of salt, pH and temperature (about 37ºC or most restriction enzymes)

3. Principle Phage lambda (λ) DNA is a liner double-stranded DNA containing of 48,502 base pairs (bp) Its DNA becomes circularized after release (inside the cell of E coli) at a cohesive site called COS site contains five recognition sites for EcoRI and seven recognition sites for Hindlll. The complete digestion of lambda DNA with EcoRI results in 21, 226, 7421, 5804, 4878 and 3530 bp long six DNA fragments. Similarly, a complete digestion of lambda DNA with HindIII results in eight DNA fragments viz., 23, 130, 9416, 6557, 4361. 2322. 2027, 564. 125 bp long fragments.

4. Requirements • • • •

Lambda DNA, restriction enzyme such as EcoRI or HindIII Assay buffer for restriction enzyme, sterilized water. Tips, Eppendorf tubes, micropipettes. Agarose gel electrophoresis apparatus 5x TBE or 5X TAE

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5. Procedure Always keep restriction enzyme (EcoRI or HindIII substrate (A DNA) and assay buffer in an ice bucket. Take 2–5 g of the lambda DNA as substrate in eppendorf tube and dissolved in an appropriate volume of water Add 2µL of about 10X assay huifer (available with the restriction enzyme) to the DNA in the eppendorf tube, followed by respective enzyme (S-12 units of EcoRI or 10–25 units of Hindll depending upon the amount of DNA used in the reaction step (ii) Add sterilized water to make the final volume of reaction mixture to 20 µL. Centrifuge gently or mix by tapping with fingers. Incubate the reaction mixture for 1 hour at 37°C in a water bath or incubator. As described in the plasmid isolation experiment, in the mean time prepare 1% agarose gel for loading and electrophoresis. (v) After an hour stop the reaction by addition of 3.33 l of 6X gei losding buffer to the eppendorf tube (as described in plasmid isolation experiment). Label the vial as ‘A’ and put on ice. As described in step (ii) take the same amount of 2 DNA in another fresh 1.5 ml eppendorf tube and mark as B. •

•

On a 1% agarose gel, load the samples A and B in separate wells. Note the order of the samples loaded in each well. As described in the plasmid isolation experiment, start electrophoresis. Run it until the bromophenol blue dye has reached 4 of the gel (it takes about 1 hour). Observe under UV transilluminator

6. Results A single band is seen in the lane in which sample ‘B’ (A DNA) has been loaded. While the second lane in which sample ‘A’ has been loaded shows multiple bands. This reveals the cleavage of sample ‘B’ by the respective restriction enzymes. The exact number and size of the bands obtained depend enzymes used for digesting the lambda DNA (sample ‘B’). As explained earlier, 6 DNA fragments observed if EcoRI is used and 8 DNA fragments are observed after using HindIII)

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13.16. DNA SEQUENCING (EXPERIMENT 16) 1. Objectives To perform DNA sequencing and determine the DNA sequence by reading an autoradiogram obtained from a sequencing gel.

2. Introduction There are several methods for DNA sequencing but the most popular one is the dideoxynutcleotide triphosphate (ddNTP) method or chain termination method proposed by F. Sanger’s group in 1977 The basic steps are followed in this method to get an autoradiogram. Step 1: Two complementary strands are separated yielding two single strands by denaturine the DNA fragment to be sequenced with alkali. Each of the two strands is separately sequenced. Step II: The sequencing reaction mixture includes a DNA single strand (which is to be sequenced) suitable primer radiolabeled. dnTPS and DNA polymerase. This mixture is distributed into four tubes labeled A. T, G and C. Small amount of ddATP ddTTP, ddGTP and ddCTP are added in A. T. G C tubes, respectively. Step III: Then, the reaction mixture from each tube is run on a polyacrylamide gel in four separate lanes labeled A, T, G and C. In each lane, the DNA strands are separated (according to their size wherein the DNA fragments differing even in one nucleotide in length can be resolved. Step IV: By aligning the gel tightly with an X-ray film followed by developing the film in suitable developer, an autoradiogram is prepared. The DNA bands appear as dark bands on the autoradiogram. Step V: Reading of the autoradiogram.

3. Principle Principle of this methods. DNA polymerase makes copies of the DNA single strand which is to be sequenced that extends a primer annealed to the 3’ end of the DNA strand. The extension terminates and a shorter fragment is generated when occasionally a dideoxy derivative is added in each of the four tubes. For example, in a tube where DDCTP is present, fragments of different lengths ending in residue C are obtained which correspond to various positions of G in the DNA strand which is being sequenced (C is complementary to G). In the same way in the ddNTP-labeled tubes, fragments of various lengths are

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obtained which correspond to the position of the complementary base in the DNA strand being sequenced. Using the radioactive primers that the position of a fragment can be viewed using an autoradiogram (X-ray) film.

Procedure (i) Procure an autoradiogram; its bands are read as A. T, G or C depending on the lane they appear and in the increasing order of their distance from the lower (anodic) end. Assume that the single strand of DNA to be sequenced consists of the following sequence: -5’TAATGATCGC3’(ii) Synthesis of the new various points due to the incorporation of ddNTPs. The DNA polymerase reads the sequence in 3’ 5’ direction. DNA strand -5 TAATGATCGC3The same sequence inverted end -3 CGCTAGTAAT Direction strand synthesis in each of the four tubes.

4. Results The sequence of the original DNA strand is -5”TAATGATCGC3 For detail go through the text.

13.17. PROTOPLAST PREPARATION AND FUSION (EXPERIMENT 17) 1. Objective Preparation of protoplasts from plant tissue and demonstration of somatic fusion.

2. Introduction Protoplast is a plant cell devoid of cell wall. These are useful for the study of physiological problems of the cells and isolation of organelles and study of the exogenously applied material on cell activity. Protoplasts are prepared from the plant tissues, callus and cultures of suspensions. When the plant tissue is cut into small sized pieces and treated with pectinase enzyme. The celis are separated from the connective tissue by the enzymes. Cellulase and hemicellulase enzymes breakdown the cell wall leaving only the cell membrane. Consequently

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protoplasts are liberated where cells lose their original shape and urns into round shape. The osmotic potential of the cells is maintained by using mannitol or sorbitol in the digesting medium. This prevents the bursting and shrivelng of the protoplasts. When the protoplasts from the same species are kept in close contact in proper buffer solution, they may fuse spontaneously. The frequency of fusion may be increased by using specific chemicals called fusagens (e.g., sodium nitrate. polyethylene glycol, Ca, high pil). The fusogens lower the Surface charge and thus permit the protoplasts to come into the close proximity for fusion, Protoplast fusion is a useful tool for plant agriculturists the use it in making crosses between sexually incompatible species. Through protoplast fusion one can transfer cytoplasmic traits between the species (interspecific) within the species (intraspecific) and withing the genera (intergeneric).

3. Principle When plant tissues are treated with cell wall lyzing enzymes and incubated properly, the plant tissues ure digested by the enzymes and protoplasts are released. In the presence of fusogen, these protoplasts can fuse with each other. When the protoplasts are obtained from colored plant tissue the protoplasts also appear colored and can easily be seen under the microscope. When protoplasts are obtained from two differently colored tissues, the fusion products can easily be seen and identifixl as homokaryon (fusion of protoplasts from same tissue) or heterokaryon (fusion of protoplasts obtained from different plant tissue).

4. Requirements • • • •

Plant tissues such as petals of flower, in vitro grown green tissue. young leaves, a microscope Pasteur pipettes. rubber bulbs, mannitol Macerozyme (a mixture of pectinase, cellulase and hemicellulase) (2% in 0.6 M mantol pH 5.8) Polyethylene glycal (PEG) (5 g/10 m)

5. Procedure A. Isolation of Protoplasts • Collect healthy leaves/petals of colored flowers/in vitro grown plant tissues. • Peel off epidermis and shred the leaf tissue (grown preferably under aseptic conditions) to small pieces. The colored petals of flowers (that have anthocyanin) can also be used.

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• •

Put 200 mg of tissue in 2 ml of enzyme solution taken in a Petri dish. Cover the Petri dish and incubate hours at 30°C at 50 rpm. Observe the isolated protoplasts under a simple microscope. B. Fusion of Protoplasts • Put one drop of suspension containing protoplasts and one drop of petal protoplasts on a slide. • Add 1 drop of PEG in the center of slide to allow the drops of protoplasts to come closer. The process of protoplast fusion is given in Observe the slides under the microscope after 10 minutes.

6. Results The isolated protoplasts appear as rounded structures of various sizes. The heterokaryon fusion products appears of different color.

14 Biostatistics

14.1. INTRODUCTION Statistics is the quantitative information of any data such as birth and death rate, health management, production of medicine, profit and loss of different industries. Another language, it is the collection, compilation, presentation, analysis and interpretation of both qualitative and quantitative information about data for example in one family planning, a number of born children can be collected from married women. The characteristics to be measured which depend on the objectives of the study. For example: study the knowledge of child health care, child mortality, child fertility and some other characteristics to be recorded are age of mother, education of mother, number of born children, duration of marriage, breastfeeding of period, number of dead children and proper vaccination is done or not in a month.

14.2. BIOSTATISTICS CONCEPT The term is used when the tool applied to the analysis of data that is come from biological organisms.

Importance of Statistics in Biotechnology • • •

It is apply for the aggregate of facts. Statistics are affected by number of causes by multiplicity. It should be capable of being related to each other, so they effect on relationship can be induced.

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This is applying for the standard of accuracy should be maintained for the analysis of data. • Statistics is help for the presenting of small to large quantity of data in a simple or very difficult form. • This tool is applied for the method of comparison of data. • There are different tools which used in number of fields viz. railways, bank, army, buildings, plants and animals, etc. • It tries to give material for the administrators so as to serve as a guide to shaping or planning in future policies. Problems Restricted in Statistics • It can be used to analyze only collective matters no single events. • It is applying for only the quantitative data not for the quality data. • It is associated for only the uncertain samples like biased. • This is done by only the true samples average run. Uses of Biostatistics Public Health and Medicine Community • The useful of vaccines and sera to be used in the field of percentage of attacks or deaths among the vaccinated subject is compared with the unvaccinated ones. • To measures the affected peoples in publically health related. • Differentiated epidemiological studies for the role of causative factors are applied for the evaluation. Medicine • Identify the symptoms and sign of a syndrome or a disease. • Typhoid and cough is found by chance and fever is almost found among the every cases. • The association is done between the attributes such as cancer and smoking. • To find out the effect of drug on the patient or efficacy of drugs among the people who are taking for the analyzes. • To check the data or drug to know the percentage of cured and died people after applying drug to the patients with control groups for the comparisons. Anatomy and Physiology • Check the correlation between the two factors or variables, e.g., X and Y (such as height or weight and color or height) for the observation of normal in a population.

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•

To check out the means or average proportions of normal or two places in different mode. • To compare the healthy or unhealthy population in data. Pharmacology • To know the potency of the new drug with respect to the standard drug (standard drug is take for the comparisons of tested drugs. • Take different drug or two successive dosages of the same drug for the comparisons which one is more affective for the prevention of diseases. • The drug is given to the animals or humans to view the changes occur due to the drug effect or due to the off target effect (means by chance) to know the action of the drug.

14.3 FREQUENCY DISTRIBUTION There are mathematical function which showing numbers of instances in which variables takes each of possible values in experiment. The statistical table which shows the values of variable arranged in order of magnitude wither in individually or in a group and also the frequencies side by side are known as frequency distribution (Tables 14.2 and 14.3). Table 14.2: Simple Frequency Distribution Number of subject test

Marks obtained

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23

Cell biology

45

Molecular biology

34

Microbiology

56

Environmental biology

45

Molecular virology

65

Biodiversity

56

Plant genetic resources

78

Animal biotechnology

75

Plant biotechnology

34

Recombinant DNA technology

56

Animal biosciences

55

Applied Microbiology

77

Immunology

56

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Pharmaceutical chemistry

68

Chemistry

79

Intellectual property rights

67

Entrepreneurship

56

Table 14.3: Group Frequency Distribution

• 1. 2. 3. 4. 5. • 1. 2.

Age in years

Frequency (No. of persons)

10–15

10

15–20

15

20–25

20

25–30

25

30–35

30

35–40

35

40–45

40

45–50

34

50–55

43

55–60

37

60–65

34

65–70

24

70–75

9

75–80

8

80–85

6

85–90

5

90–95

4

95–100

7

Remember for the forming frequency distribution The number of class should neither to be small nor to be large in distribution pattern. The normally classes should be made of equal width are to be expected. The classes should be exclusive pattern, i.e., non-overlapping. The class must be exhaustive i.e, each raw data must be included in the classes. The class should be clearly defined. Remember for the grouped frequency distribution Cumulative frequency Class frequency

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299

Total frequency Percentage frequency Frequency density Class marks Class width Class boundaries Class interval Class limits

Class or Class Interval The large number of observations varying in a wide range is available. There are classified in several groups according to the size of values. Each of these groups defined by an interval is known as class or interval. The class intervals are classified two types such as: •

Continuous: The class interval does not contain the upper boundary of the class will be known as class interval of continuous. A class interval of the form 10–20 in continuous class will contain values from 10 to less 20. An example such as Class interval

Range

0–10

From zero less than 10

10–20

From 10 less than20

20–30

From 20 less than 30

30–40

From 30 less than 40

40–50

From 40 less than 50

50–60

From 50 less than 60

60–70

From 60 less than 70

70–80

From 70 less than 80

80–90

From 80 less than 90

90–100

From 90 less than 100

•

Discontinuous: The class interval where each value includes the end values will be called discontinuous class interval. The class interval of the form 0–9 in discontinuous class will contain values from 0–9 both inclusive. An example such as: Class

Range

0–9

From 0 to9

10–19

From 10 to 19

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From 20 to 29

30–39

From 30 to 39

40–49

From 40 to 49

50–59

From 50 to 59

60–69

From 60 to 69

70–79

From 70 to 79

80–89

From 80 to 89

90–99

From 90 to 99

Continuous classes intervals are formed generally with continuous type values or non-integral values e.g., Kg and Rupees, etc. Discontinuous class intervals are formed generally with discrete or integral values e.g., marks.

Open End Class The one end of class is not specified is known as open end class. A frequency distribution may have either one or two end classes. Income (Rupees)

Frequency

0–50

90

50–100

100

100–150

80

150–200

50

200–250

40

250–300

150

300–350

50

350–400

30

400–450

40

450–500

50

500–550

40

550–600

30

600–650

20

650–700

10

Class Limits The construction of groups frequency distribution the class interval must be defined by pairs of numbers such that the upper end of one class does not coincide with the lower end of the immediate following class. The two numbers are used to specify the limits of classes’ interval for the purposes of tallying the original observations into the various classes are called

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as class limits: • •

The small of the values or pair is known as lower class limits. The larger of the values or pair is known as upper class limit.

Class Boundaries The measurements of the continuous variables all data are recorded nearest to a certain unit or integer value. The most extreme values which would ever be included in a class interval are known as class boundaries, In fact, this is real or actual limits of a class interval. • •

The extreme point is lower is known as lower class boundary. The extreme point is higher is known as higher class boundary.

Calculation If x is the gap between the upper class limit of any class or class interval and the lower class limit of the next class or class interval. Upper class boundary

= upper class limit + 1 x 2

Lower class boundary

= lower class limit – 1 x 2

Note Class limits are used only for the construction of the grouped frequency distribution but in all statistical calculations and diagrams involving end points of classes (e.g., medium, mode, histogram and ogives, etc.) Class boundaries are used. Class Mark • It is mid value of a class interval exactly at the middle of the class or class interval. • It lies half way between the class limits or between the class boundaries. Class mark = Lower class limit + upper class limit • It is used for the representative class interval for the calculation of means and standard deviation and mean deviation, etc. Class Width It is length or range of class interval or difference between the upper or lower class boundaries.

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Width of the class = upper class of the boundaries – lower class of the boundaries.

Class Frequency or Total Class of the Frequency

The number of observation falling within a class is known as class frequency or simple frequency. The sum of all the classes frequencies are called total frequencies.

Percentage Frequency The percentage of the class interval is expressed as percentage of the total frequency distribution. Percentage frequency of a class = Frequency of the class × 100 Total frequency

Relative Frequency The representation ratios are of the total frequency values. It is not expressed in percentage. It is used to compare two or more frequency distributions or two or more items in the same frequency distribution. Relative frequency of the class = Frequency of the class

Total frequency

Frequency Density The frequency density of a class interval is its frequency per unit width. It shows the concentration of frequency in a class. It is used in drawing histogram when the classes are of unequal width. Frequency density = Class frequency Width of the class

0

0

0

100

55–59

65–69

Total

0

50–54

60–64

6

0

40–44

45–49

12

9

30–34

21

25–29

35–39

18

34

15–19

20–24

2

1

-

70

65

60

55

50

45

35

30

25

20

15

Lower 3

Class Frequency Class Limits

Class Interval

-

84

79

74

69

64

59

44

34

29

24

19

Upper 4

-

64.5

59.5

54.5

49.5

44.5

39.5

34.5

29.5

24.5

19.5

14.5

Lower 5

-

69.5

64.5

59.5

54.5

49.5

44.5

39.5

34.5

29.5

24.5

19.5

Upper 6

Class Boundaries

-

77

72

67

62

57

52

39.5

32

27

22

17

7

Class Marks

-

5

5

5

5

5

5

5

5

5

5

5

8

Class Width

-

0.01

0.04

0.06

0.08

1.0

1.2

1.8

2.4

4.2

6.8

3.6

9

Frequency Density

1.15

0.01

0.02

0.03

0.04

0.05

0.06

0.09

0.12

0.21

0.34

0.18

10

Relative Frequency

Table 14.4: Class Interval, Class Frequency, Class limits, Class boundaries, Class marks, Class width, Frequency density, Relative frequency

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Cumulative Frequency Distribution: The corresponding to a class is the sum of all the frequency up to and including that class. It is obtained by adding to the frequency of that class and all the frequencies of the previous classes. It is two types 1. Less than cumulative frequencies: The number of observations up to a given value is called less than cumulative frequency. 2. More than cumulative frequencies: The number of observations greater than a value is called as more than cumulative frequencies. Table 14.5: Cumulative Frequencies Class Interval

Uses • • •

Frequency

Cumulative Frequency Less than

More than

30–40

8

8

100

40–50

12

20

92

50–60

20

40

80

60–70

25

65

60

70–80

18

83

35

80–90

17

100

17

90–100

5

105

12

Total

105

To analysis the number of observations less than or more than any given value. To find out the number of observations falling between any two specified values of the variable. To measure out pentiles, quartiles and median. (Dr. Pranav Kumar Banerjee, a textbook of biometry).

Theoretical Distribution Normal distribution was first discovered in 1733 by mathematician De Movire. He obtained this continuous distribution as a limiting case of binomial distribution. So the normal distribution is also called as Gaussian distribution named after Karl Friedrich Gauss, who used this normal curve to describe the theory of accidental errors of measurements involved in calculation of orbits of

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heavenly bodies. It is a continuous probability distribution which is bell shaped unimodal and symmetrical. The normal distribution of a variable when represented graphically takes the shape of a symmetrical curve is called as Normal curve. Properties of the normal distribution and normal probability curve: • It has two parameters viz. mean (µ) and standard deviation (σ) It is bell shaped and symmetrical about the line x = µ. • It has only one mode occurring at µ i.e., it is unimodal. • The mean, median and mode value will coincide at the center (x = µ) because of the symmetrical and single peaked. • Mean = Median = Mode = µ • The normal curve has asymptotic tails, i.e., progressively nearing the abscissa or x-axis. • The range is unlimited in both directions but as the distance from µ increases, the curve approaches the horizontal axis more and more closely and never touches the horizontal axis (X). • The two points of inflections, the point where the change in curvature occurs at a distance σ on either side mean. • The points of inflections of the normal curve are at x = µ ± σ. • The curve changes from concave to convex and vice versa. • The quartiles of the first (Q1) and the third (Q3) are equidistant from µ. Q 3 – µ = µ – Q1 Quartile deviation = Q3 – Q1/2 = 0.67458 Q1 = µ – 0.6745 Q3 = µ + 0.6745 • •

Thus the range of the normal distribution from -0.6745 to 0.6745. The normal distribution is bilaterally symmetrical so, it is free from skewness so its coefficient of skewness amounts to zero. Skewness = 0 Kurtosis = 0 • • •

The ordinates maximum (y) lies at the mean, i.e., at x = µ. Its value is y = 1/ σ √ 2x. When S.D increases the maximum ordinate decreases. Since the mean = median = µ, the coordinates at x = µ (or Z = 0)

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• •

•

•

•

divides the whole area into two equal parts. The area right to the ordinate and left to the ordinates at x = µ (Z = 0) is 0.5. The mathematical equation is completely determined if the mean (µ) and standard deviation (σ) of the variable is completely known to fix the curve. The curve will always remain in symmetrical about the maximum ordinate. But the change in the value of mean (µ) and the standard deviation (σ) or in both will alter the shape of the curve. The maximum ordinate is at the mean (µ) and at various standard deviation (σ) the distances are in a fixed proportion to the ordinate at the mean when the total mean are under the normal curve is equal to unity. There are following table which gives are under the normal probability curve for some important values of Z. Distance from the mean ordinate in Area under the curve terms of ± σ Z = ± 0.6745

50% = 0.50

Z = ±1.0

68.27% = 0.6827

Z = ±1.96

95% = 0.95

Z = ±2.00

95.45% = 0.9545

Z = ±2.58

99% = 0.99

Z = ±3.0

99.73% = 0.9973

Uses of normal distribution • It is apply in sampling theory. • It can be used to approximately the binomial and Poisson distribution. • It has considerable application in statistical quality control. • It is mostly used in statistical hypothesis. • It is used in to test the significance value. • It has different mathematical properties which make it popular and comparatively easy to manipulate foe the use in social natural sciences. Best fitting normal distribution It is the normal distribution which fits best with an observed the distribution and has the same mean (X), the same standard deviation (σ) and the same sample size (n) as the latter. The mean value, standard deviation is computed.

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So the Xm is transformed into a Z score. Z = Xm – X/ σ Example 1: The best fitting of normal distribution for the following blood glucose concentration of 80 patients of Mmidnapure district hospital. Calculate the mean and the standard deviation of the given data. Class Interval

100–109

110–119

120–129

130–139

140–149

150–159

160–169

Frequency

6

11

10

17

16

13

7

Solution: Let assume the mean 134.5 Class Interval

Mid value (Xm)

Frequency (f)

Assumed mean A = 134.5 Xm – A

Xm – A/ C = d

fd

fd2

100–109

104.5

6

104.5–134.5 = -30

-3

-18

54

110–119

114.5

11

114.5–134.5 = -20

-2

-22

44

120–129

124.5

10

124.5–134.5 = -10

-1

-10

10

130–139

134.5

17

134.5–134.5 = 0

0

0

0

140–149

144.5

16

144.5–134.5 = +10

+1

+16

16

150–159

154.5

13

154.5–134.5 = +20

+2

+26

52

160–169

164.5

7

164.5–134.5 = +30

+3

+21

63

∑ fd = 63– 50 = 13

239

∑ f = 80

Mean = A + ∑ fd/N × i X = 134.5 + 13/80 × 10 = 134.5 + 1.625 = 136.125 = 136.1 Standard Deviation (SD) = √ ∑ fd2/N – (∑ fd/N)2 × i = √ 239/80 – (13/80)2 × 10 = √ 2.988 – 0.02656 × 10 = 10 √2.988 – 0.0266 = 10√ 2.96 = 10 × 1.72 =17.2 • The deviation of each (Xm) from (X) is then transformed into Z score which entered in the following table. Class Interval

Frequency (f)

Mid Value (x)

Xm – X

Z = Xm – X/ S.D

y

Y

100–109

6

104.5

104.5 – 136.1= -31.6

-31.6/17.2 = -1.84

0.0734

3.4

110–119

11

114.5

114.5 – 136.1 = -21.6

-21.6/17.2 = -1.26

0.1804

8.4

120–129

10

124.5

124.5 – 136.1 = -11.6

-11.6/17.2 = -0.67

0.3187

14.8

130–139

17

134.5

134.5 – 136.1 = -1.6

-1.6/17.2 = -0.09

0.3973

18.5

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140–149

16

144.5

144.5 – 136.1 = +8.4

8.4/17.2 = 0.49

0.3538

16.5

150–159

13

154.5

154.5– 136.1 = 18.4

18.4/17.2 = 1.07

0.2251

10.5

160–169

7

164.5

164.5– 136.1 = 28.4

28.4/17.2 = 1.65

0.1023

4.8

∑ f = 80

Neglecting the algebraic sign of the Z score, the height y of the ordinate at each Z score is then recorded from units normal curve table. The Y ordinate height of the best fitting normal distribution is computed for each Z score by multiplying its y score with in/ SD (I = class interval, n = total frequency, SD = standard deviation) for example Y = y × in/ SD = 0.1804 × 10×80/17.2 = 0.1804×46.51 =8.39 = 8.4

14.4. VARIABLES These characteristics are different from individual to individual. So, it is known as variable. There are two types of variable. The information of the whole group is examining the part of the each whole group individual to individual. • Qualitative variable • Quantitative variable

14.5. QUALITATIVE VARIABLE The variable is measured normally by qualitative properties such as type of disease caused in human, rank of doctors, occupation of persons, and color of girl and qualification of girls. It is also called as attribute.

14.6. QUANTITATIVE VARIABLE The variable is measured by numerical basis such as weight of boys, height of girls, blood pressure of patients, and number of marks obtains in class, age of small baby girl and number of patient in a hospital. It is again divided into two parts: discrete variable and continuous variable.

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14.7. DISCRETE VARIABLE It is measured by only integral value such as number of patient admitted in a day in a hospital.

14.8. CONTINUOUS VARIABLE It is measured by integral or fractional values such as age of boys and height of boys. According to recorded variable is known as random variable and nonrandom variable

14.9. RANDOM VARIABLE The values are selected randomly from different units selected by random process such as 500 students in a school and n = 100 students are selected by any random process for age investigation.

14.10. NON-RANDOM VARIABLE The values are not selected randomly; select all students’ population in a school for age investigation. The data can be collected from selected all populations or some units. There are two types of data collection method: (i) sample survey method; and (ii) census method. Discrete variables: Serial Numbers

Number of Tiger in One species

1

4

2

5

3

6

4

2

5

5

6

5

7

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Continuous Variable: Serial Numbers

Weight of the Tiger

1

86.023 kg

2

105.976 kg

3

104.034 kg

4

160.89 kg

5

145.087 kg

6

128.568 kg

7

100.572 kg

14.11. SAMPLE SURVEY METHOD In this method, we are collected data from some selected units under investigation example quality of data can be ensured.

14.12. CENSUS METHOD In this method, we are collected data from all population unit under investigation example money and time.

14.13. GRAPHICAL REPRESENTATION OF DATA There are two method of data collection has been discussed. • •

Primary data; and Secondary data.

14.14. PRIMARY DATA The data are collected by personal interview or by mailed. These data are called as raw data. Example The amount of fasting blood sugar of some persons (male and female) recorded in first 10 minutes by a working day.

14.14.1. Primary Data Collection

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Schedules sent through investigators Direct personal observation Questionnaires sent by email Indirect oral presentation

14.14.2. Questionnaire This is the perform containing a sequence of questions for the statistical enquiry. This is used for collection of primary data from individual persons through their responses.

14.14.3. Primary Data Advantages • • • • • •

It provides details information but information may be suppressed in secondary data. It is free from errors. It contains information regarding methods of procuring data where as primary data often included. It is cost effective. It is time less consuming process or suitability. It gives accuracy results for the data analysis.

14.14.4. Population There are group of peoples or study the elements for measurements having some common fundamentals characteristics. • •

Finite: Population is fixed value, e.g., number of days in a year. Infinite: Population consists of an endless succession of values e.g., number of plants in ocean.

14.15. SECONDARY DATA The data are collected from official recorded, from published work or by second person. Example census reports are known as secondary data. Statistical Error: There are errors during the collection of data or error shows the extent to which the observed value of a quantity exceeds the true value. Error = Observed value-True value Types: It is classified as:

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Biased errors: due to personal prejudices or bias of investigator Unbiased errors: statistical enquiry due to chance causes. Array Presenting data in ascending order of magnitude is known as array. Tally A value occurs more than four times the fifth occurrence is denoted by a cross (\) tally mark and running diagonally across the four tally marks. Examples: Form a frequency table for the different variables. 1, 4, 7, 8, 5, 8, 4, 9, 23, 56, 23, 45, 67, 78, 25, 4, 8. 9, 4, 1, 23, 78, 56, 9, 45, 78, 25, 67, 25, 78, 45, 23. Solution: Variables

Tally

Frequency

1

II

6

4

IIII

7

7

I

4

8

III

6

9

III

7

11

III

3

23

IIII

5

55

IIIII

3

56

II

5

45

III

7

67

II

7

78

IIII

3

25

IIII

5

78

IIII

6

14.16. BIOSTATISTICS Statistics of marriage, birth death rate, migration, family planning, level of education, health care of pregnant women and many more problems which are affecting the welfare of mankind.

14.17. PRESENTATION OF DATA The data are collected through survey is called raw data. The raw data are not always suitable for proper statistical analysis. So, we have to classified,

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tabulated and presented by graphs and diagrams.

14.18. DIFFERENT GRAPHS AND DIAGRAM There are many important diagrams and graphs used in biostatistics. The representation of data through chart and diagrams is known as graphical representation of data.

Advantages of graphical representations • • • • • • •

It is easily understood. The data presented in more attractive form. It shows the tendency and trends of values of the variable. It is useful to detect mistakes. It shows easily the relationship between two data sets. It has universal applicable. It is helpful for the assimilation of data quickly.

Limitations of graphical representation data • • •

It does not show all the facts. It can reveal only the approximate position. It can take lot of time so its time consuming pattern.

Different types of diagrammatic representations There are different types of graphs in the form of diagrams and charts such as: • line diagram • bar diagram • pie diagrams • These three are used for qualitative presentation of data • stem- and leaf plot • histogram • ogive (cumulative frequency polygon). • scatter diagram • frequency polygone These five are used for quantitative presentation of data.

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Statistics is an important part for all students from graduation to PhD level. Here we are going to discuss stats via using some important software’s which make them very easy as well as less time-consuming. Excel is basic stat software which should be known to each and every student of any discipline. After excel, some other advanced stats tool are also discussed here in this chapter such as Origin Pro software. The data in the form of raw scores is known as ungrouped data and when it is organized into frequency distribution then it is referred to as grouped data. Separate modes and methods are used to represent these two types of data ungrouped and grouped.

14.19. LINE DIAGRAM The data is usually represented in line. The plotted data in Y-axis is added to the X-axis by a line. The resultant diagram is called Line diagram. Example 1: The following data are representing the number of concentration Fluoride (0, 25, 50, 75, and 100) mgkg-1 NaF effect on Root length. Represent the data by a line diagram work on Excel software. Solution: Firstly open excel software and enter data in excel sheet (Figure 14.1). After getting design line diagram choose layout and select error bars with standard error.

Select the data and click on cell

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Go to insert page and select data

Click on line diagram and choose graph.

In line diagram many more style is there, so which you want to like in design select and click “OK”

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After you get graph then go to design page and select any design which you want, so your graph will attractive presentation of data.

Select any design which you want to like in graph.

Now you get graph according your choice.

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Now, go to layout page.

Select “error bars with standard error,” so you can present your data without any error.

Go to grindlines, select “horizontal lines,” and select “None”

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Now you find graph without any lines in graph.

Go to data tables and select below, so your data will show on graph.

Go to data labels and select “above” then data show on line symbol.

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Go to legend and “select show legend on top”

Go to Axis titles select “Title below Axis”

Figure 14.1: Screenshot represents the data input in excel sheet and form Line diagram.

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14.20. BAR DIAGRAM The diagram is similar to line diagram but the value is shown by a rectangle instead of a line. Example 1. The following data are representing the number of concentration Fluoride (0, 25, 50, 75, and 100) mgkg-1 NaF effect on Root length. Represent the data by a Bar diagram work on Excel software. Solution: Same as line diagram (Figure 14.2).

Figure 14.2: Screenshot represents the data input in excel sheet and form Bar diagram.

14.21. PIE DIAGRAM This diagram is used to present the values of different levels of qualitative data, where data are move to angle and the angles are drawn within a circle. The total angle of a circle is 360o. Diagram is made same as above given data (Figure 14.3). Enter data, select data, go to insert page, choose pie diagram “3-D diagram.”Same as above

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Figure 14.3: Screenshot represents the data input in excel sheet and form Pie diagram.

14.22. STEM AND LEAF PLATE The stem is the number displayed to the left of the vertical bar (│) and the eachleaf is digit displayed t the right of it. Each leaf represent a separate data value.

14.23 HISTOGRAM It is the accurate representation of the distribution of numerical data. It is an estimate of the probability distribution of a continuous variable and was first introduced by Karl Pearson.

14.24 OGIVES It is the graph showing the curve of a cumulative distribution function. The points plotted are the upper class limit and the corresponding cumulative frequency.

14.25. SCATTER DIAGRAM In this diagram the values are assumed to be correlated for example the given concentration of F is affected the root length (If we are increase F concentration than length are reduced, so in between there is correlation).

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Enter the data on excel cell, after select the data, for graph formation. When you will get graph than right click on dots. 2. Select the Trendline option in layout option. 3. After Trendline option select the last two options display equation on chart and display R- squared value on chart. 4. Note down the displayed equation and calculate the x value from the given equation (in y = mx + c) 5. Y= -0.065x + 15.33 6. R2 = 0.821 (Figure 204.4). Go to insert page, select scatter, choose scatter which you want to like in graph. 1.

Go to design page, select any design, and find graph.

Right click on trendline, after that new window open then select liner, display equation on chart and display R square value.

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Figure 14.4: Screenshot represents the data input in excel sheet and form scatter diagram Methods of Presentation of Statistical Data • Textual presentation: data presented in descriptive form. • Tabular presentation: data presentation in table form. It is two types: • Simple form • Complex form Example 1: Simple form: Number of students in HIMT College Greater Noida, Dr. M.P.S College, Agra, IIMT Aligarh. Name of the Colleges

No. of Students

HIMT College Greater Noida

4567

Dr M.P.S College, Agra

6000

IIMT Aligarh

5500

Example 2: complex form: Number of students in different course in different colleges. Name of the colleges

No. of BA students

No. of BSc students

No. of MA students

No. of MSc students

HIMT College Greater Noida

123

342

145

140

Dr M.P.S College, Agra,

234

230

202

130

IIMT Aligarh

231

123

250

145

(a) Graphical presentation Statistical Tables: It is systematic arrangement of quantitative data under appropriate heads in rows and columns. Tables parts description • TITLE • STUB • CAPTION

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• BOX HEAD • BODY • SOURCE • FOOT NOTE Features of good table 1. Title must have clear and concise which gives precise idea about the table contents. 2. The items arrangement in the table should be arrange logically. 3. Special notes at the end of the table for experiment should be bear to resolve or solve the confusing entry. 4. All the necessary details should be containing in the table. 5. Column or sub column should be distribution like single or double ruling, etc. 6. Figure should be kept close as possible to the table for the compatible comparison. 7. Table should be well proportional or justify in breadth or length. 8. Measurements of units or abbreviations should be shown clearly on top of the column or below in the “Note” text line. 9. Pattern of table given in Table 14.1. Table 14.1: Geo-Statistics of Individual Layers of Groundwater Quality Parameters (*WHO/Indian Standard for Drinking Purpose) Banasthali, Tonk, Rajasthan, India No.

Layer

Min.

Max

Mean

SD

Desirable Limit*

Units

1

Ca+2+Mg+2

0.00

7.63

2.76

1.05

15–40

meq/L

Class

Value

2

Mn

0.32

4.85

1.99

1.11

5.00

µg/L

High

0.90 to 0.70

3

Fe

4.18

9.79

6.55

0.92

100.0

µg/L

Medium High

0.70 to 0.50

4

Cu

0.48

5.32

2.31

1.19

50.00

µg/L

Medium

0.50 to 0.35

5

Zn

0.38

2.12

1.01

0.20

5.00

µg/L

Low

Below 0.35

High

0.50 to 1.50

6

Cl

2.66

24.17

9.02

4.04

1.00

meq/L

7

CO3

0.00

3.33

1.10

0.57

 

meq/L

8

HCO3

1.83

19.60

7.12

3.11

 

meq/L

9

pH

7.30

9.39

8.49

0.32

6.50–8.50

 

10

EC

2.73

29.05

9.68

3.70

G.M > HM. For example: We take two positive items 4 and 9 Mean (AM.) = 4+9/2 = 6.5 G.M = √4×9 =6 H.M. = 2/1/4+1/9 = = 2/13/36 = 2×36/13 = 5.5 So, A.M > G.M > H.M = = 6.5 > 6 > 5.5. Questions: Which type of average would be suitable? i. Average sales for various years ? ii. Sale of shirts with collar size in Cm 36, 37, 35, 36, 33, 36 ? iii. Size of agriculture holdings ? iv. Per capita income in several countries ? v. Runs scored by a player in different matches ? vi. Comparison of intelligence of students ? vii. Marks of candidates obtained in an examination ? viii. Size of the shoes sold at a shop ? Answers: (i) Mean (ii) Mode (iii) Mode (iv) Mean (v) Mean (vi) Median (vii) Median (viii) Mode.

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14.32. STANDARD DEVIATION The standard deviation concept was introduced by Karl Pearson in 1893. Standard deviation is used to measure the amount of variation in the set of the data. At the time of data collection or in the case of experiments results we used to collect or calculate multiple sets of data to minimize the chance of experimental error. [σ = √ ∑ (X – X)2/N] It is the square root of the arithmetic mean squares of deviation from arithmetic mean. In short of S.D may be defined as the “Root Mean Square of deviation fro Mean.” It is denoted by sigma σ If x1, x2 x3……xn be set of observations and x their arithmetic mean.

Deviation from mean = (x1 – x), (x2 – x), (x3- x)…….(xn – x).

Square deviation from mean = (x1 – x)2, (x2 – x)2, (x3- x)2…….((xn….x)2. Mean Square deviation from mean i.e.,

= (x1 – x)2, (x2 – x)2, (x3- x)2…….((xn….x)2/ n = ∑ (x – x)2/n Root mean square deviation from mean, i.e., standard deviation (σ) = √ 1/n ∑ (x – x) 2 Coefficient of Standard Deviation: It is ratio of the standard deviation to its arithmetic mean i.e., Coefficient of standard deviation = σ/X Standard deviation calculates: Simple series: It can calculate the mean It can find out the differences of each observation from the mean. The square of the differences of observation from the mean is fixed. Add the square values to get the sum of the squares. Divide by the numbers of observations. S.D (σ) = √∑ (X – X)2/n = √d2/n where d = (X – X) X = Value of the variable X = Arithmetic mean

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n = Total number of observations. Example 1. Find the standard deviation. 11

12

13

14

15

16

17

18

19

20

21

Solution: Here there is arithmetic mean X = 11+12+13+14+15+16+17+18+19+20+21/11 X = 176/11 =16 Lets calculate the standard deviation X

X–X=d

(X – X)2 = D2

11

11 – 16 = -5

25

12

12 – 16 = -4

16

13

13 – 16 = -3

9

14

14 – 16 = -2

4

15

15 – 16 = -1

1

16

16 – 16 = 0

0

17

17 – 16 = +1

1

18

18 – 16 = +2

4

19

19 – 16 = +3

9

20

20 – 16 = +4

16

21

21 – 16 = +5

25

N = 11

∑ D2 = 110 S.D (σ) = √d2/n = √110/11 = √10 = 3.16

14.33. SHORT CUT METHOD This method is used for the calculation of the standard deviation when the arithmetic mean of the data comes out to be a fraction. It is very difficult and tedious to find out the deviations from the mean. Here it is, S.D (σ) = √ ∑d2/n – (∑d/n)2 Where d = X – A A = Assumed mean n = Total number of observations Example 1: Find the standard deviation of the following items.

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43

65

57

Solution: Calculate the standard deviation.

(b) (i)

31

60

37

Value (X)

d = (X-A) (A= 57)

d2

48

48 – 57 = -9

81

43

43 – 57 = -14

196

65

65 – 57 = +8

64

57

57 – 57 = 0

0

31

31 – 57 = -26

676

60

60 – 57 = +3

9

37

37 – 57 = -20

400

48

48 – 57 = -9

81

59

59 – 57 = +2

4

78

78 -57 = +21

441

-78 +34 = -44

1952

48

59

351 78

Here assumed mean = 57 ∑ d2 = 1952 ∑ d = – 44 n = 10 S.D (σ) = √ ∑d2/n – (∑d/n)2 = √1952/10 – (44/10)2 = √195.2 – 19.36 = √175.84 = √13.26

Standard deviation (Grouped data) Discrete Series: Direct Method

S.D (σ) = √∑ f (X – X)2/ n X = Arithmetic mean f = Frequency n = Number of item

(ii) Short Cut Method The mean has a fractional value then the following formula is used S.D (σ) = √∑ fd2/n – (∑ fd/n)2 d=X–A A = assumed mean n = ∑ f Example 1: Find out the mean and standard deviation of the following data. Size of item

10

11

12

13

14

15

16

Frequency

2

7

11

15

10

4

1

Solution: Prepare the following table:

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Size of the item

Frequency

d = X – A (13)

fd

fd2

10

2

10 – 13 = -3

-6

18

11

7

11 – 13 = -2

-14

28

12

11

12 – 13 = -1

-11

11

13

15

13 – 13 = 0

0

0

14

10

14 – 13 = +1

10

10

15

4

15 – 13 = +2

8

16

16

1

16 – 13 = +3

3

9

∑ f = 50

∑ fd = +21 – 31 = ∑ fd2 = 92 -10

X = A + ∑ fd/n = 13 – 10/50 =13 – 0.2 = 12.8 X = 12.8 S.D = √∑ fd2/n – (∑ fd2/n)2 = √ 92/50 – (-10/50)2 = √1.84- (0.2)2 = √1.84 – 0.04 = √1.80 = 1.342

Standard deviation in continuous series (a) Direct Method S.D (σ) = √∑f (X – X)2 X = Mid value X = A.M f = Frequency ∑f = N frequency (b) Short Cut Method: d=X–A d = X – A/i = d/i (σ) = √∑ fd2/n – (∑ fd/n)2 × i d = X-A/i A = Assumed mean N = total frequency i = class width

Example 1: Calculate the mean, median, S.D variance and covariance of the following items. Heights in inches Number children

95–105

of 19

105–115

115–125

125–135

135–145

23

36

70

52

Solution: Let assumed the mean value is 130

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Class Interval

Mid value X

Number o f Children

Cf

X – A/ C = d

fd

fd2

95–105

100

19

19

100–130/10 = -3

-57

19 × 9 = 171

105–115

110

23

42

110–130/10 = -2

-46

23 × 4 = 92

115–125

120

36

78

120–130/10 = -1

-36

36 × 1 = 36

125- 135

130

70

148

130–130/10 = 0

0

70 × 0 = 0

135–145

140

52

200

140–130/10= +1

+52

52 × 1 = 52

-139 +152 = -87

= 351

N = 200

Mean = A + ∑ fd/ N × i = 130 + -87/200 × 10 = 130 – 4.35 = 125.65 S.D (σ) = √∑ fd2/N – (∑ fd/N)2 × i = √ 351/200 – (-87/200)2 ×10 = √1.75 – 0.189 × 10 = √ 1.56 × 10 = 1.2489 × 10 = 12.489 Median = L1 + N/2 – C/ fm × i Median class 125 – 135 = 125 + 200/2 – 78 /70 × 10 = 125 + 100 -78/70 × 10 = 125 +22/7 = 125 + 3.14 = 128.14 Variance = (σ2) = (12.489)2 = 155.97 = 156 C.V = 12.48/125.65 ×100 1248.00/125.65 – 124800/12565 = 9.93

Standard Error The sampling distribution of any statistics will have its own mean, standard deviation, etc. The sample estimates of statistics will differ from population parameter. The difference between the sample or particular sample and population variation is called as sampling error or standard error. Standard error can be calculated by S.E – (X) = S.D/√n

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S.E – X = Standard error S.D = Standard deviation n= Size of the sample If the sample have same standard deviation then S.E of (X1 –X2) = S.D √1/n1 +1/n2 (X1) (X2) = Population n1 and n2 = size of the sample

Factors affecting or controlling the Standard Error: • The sample size: Increase the size of the sample decease S.E. • The nature of statistics: e.g., means, variance, etc. • The standard deviation: the value of S.E varies directly with the size of S.D. Uses: • To measure of the extent sampling error in the mean. • To calculate the size and also determine whether the population is drawn from known population or not. Example 1: To find out the standard error of the mean and when the unbiased S.D of sample is 36 cases is 23.61. Solution: S.E of X = S.D/√n S.D = 23.61 n = 36 = 23.61/√36 23.61/6 = 3.935. Example 1: Compute the variance, SD, the coefficient of variance and the coefficient of dispersion of the following frequency distribution of interorbital width (m.m) of a sample of 100 pigeons. Class Interval

11–13

14–16

17–19

20–22

23–25

Frequency

8

20

40

25

7

Solution: Class Interval

Mid Value (x)

f

fx

d = (x – x)

d2

fd2

11–13

12

8

96

-609

37.1

296.7

14–16

15

20

300

-3.09

9.55

190.96

17–19

18

40

720

-.09

0.0081

0.324

20–22

21

25

525

2.91

8.47

211.7

23–25

24

7

168

5.91

34.92 = 35

244.49

∑ f = 100 ∑ fx = 1809

Mean (x) = ∑ fx/∑f

∑ fd = 944.174 2

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= 1809/100 =18.09 S.D = √∑ fd2/N = √ 944.174/100 = √9.44174 = 3.07 Variance = (S.D)2 = 9.4249 Coefficient of variance = S.D/A.M × 100 = 3.07/18.09 × 100 = 16.97 Example 2: Find out the mean and S.D from the following frequency distribution? Scores

20–22

23–25

26–28

29–31

32–34

35–37

38–40

Frequency

2

5

7

13

8

4

1

Solution: Score

Mid Value (x)

f

fx

d = (x – x)

d2

fd2

20–22

21

2

42

-3.7

75.69

151.38

23–25

24

5

120

-5.7

32.49

162.46

26–28

27

7

189

-2.7

7.29

51.03

29–31

30

13

390

0.3

0.09

1.17

32–34

33

8

264

3.3

10.89

87.12

35–37

36

4

144

6.3

39.69

158.76

38–40

39

1

39

9.3

86.49

86.49

∑ f = 40

∑ fx = 1188

∑ fd2 = 698.41

Mean = x = ∑ fx/ ∑ f = 1188/40 = 29.7 S.D = σ = √ ∑ fd2/N = √698.41/40 = √17.46 = 4.18

Example: Calculate the mean and median from the following frequency distribution. Scores

20–24

25–29

30–34

35–39

40–44

45–49

Frequency

7

9

12

6

4

2

Solution: Scores

Mid value (x)

Class Boundaries

f

fx

cf

20–24

22

19.5–24.5

7

154

7

25–29

27

24.5–29.5

9

243

16

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30–34

32

29.5–34.5

12

384

28

35–39

37

34.5–39.5

6

222

34

40–44

42

39.5–44.5

4

168

38

45–49

47

44.5–49.5

2

94

40

∑ f = N = 40

∑f x= 1265

Mean (x) = ∑ fx/ ∑ f = 1265/40 = 31.625 Median = L1 + N/2-C/fm × i = N/2 = 40/2 = 20 L1 = 29.5

fm = 12, C = 16, I = 5 Median = 29.5 + 20–16/12 × 5 29.5 +4/12 × 5 = 29.5 + 1.667 = 31.167 Example: Calculate the mean, median and S.D from the following distribution? Scores

10–19

20–29

30–39

4 0 – 49

50–59

60–69

70–79

80–89

90–99

Frequency

2

5

3

5

8

12

25

30

10

Solution: Scores

Mid value (x)

Class boundaries

f

fx

c.f

d = (x-x)

d2

fd2

10–19

14.5

9.5–19.5

2

29

2

-55.8

3113.64

6227.28

20–29

24.5

19.5–29.5

5

122.5

7

-45.8

2097.64

10488.2

30–39

34.5

29.5–39.5

3

103.5

10

-35.8

1281.64

3844.92

40–49

44.5

39.5–49.5

5

222.5

15

-25.8

665.64

3328.2

50–59

54.5

49.5–59.5

8

436

23

-15.8

249.64

1997.12

60–69

64.5

59.5–69.5

12

774

35

-5.8

33.64

403.68

70–79

74.5

69.5–79.5

25

1862.5

60

4.2

17.64

441

80–89

84.5

79.5–89.5

30

2535

90

14.2

201.64

6049.2

90–99

94.5

89.5–99.5

10

945

100

24.2

585.64

5856.4

n = 100

∑ fx = 7030

Lets assume mean 74.5 Mean (x) = ∑fx/N = 7030/100 = 70.30

∑ fd2 = 38636

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Median = L1 + N/2 – C/ fm × i = 69.5 + 50 – 35/25 × 10 = 69.5 + 150/25 = 69.5 + 6 = 75.5 S.D = √∑ fd2/ N = √ 38636/100 = √386.36 = 19.656 Merits and Demerits of the Standard Deviation Merits • It is less affected by fluctuations of sampling as compared to other measure of dispersion. • It is used in correlation. • It is defined rigidly. • It is based on all the observations. Demerits • It gives more weightage to extreme values • It is not simple to understand. • It is difficult to compute the other measures of dispersions. Uses • It is also help in finding the suitable size of sample. • It is also helpful for the calculation of the standard error. • It is used for the summaries of the deviations of a large distribution from mean.

14.34. CALCULATE STANDARD DEVIATION ON EXCEL Select new column for the SD and then click on cell type STDEV choose option and enter and select the data and click on enter button. There are many snapshot below which will help you for calculating standard deviation. Same as mean (Figure 14.8).

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Same as mean

Same as mean

Same as mean

Same as mean

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Same as mean

Figure 14.8: Screenshot represents the standard values of data. 14.34. VARIANCE The Square of standard deviation is known as variance and it is denoted by (σ2) Variance = (S.D)2 = (σ2) Coefficient of Variance: It is the product of the coefficient of standard deviation × 100 Coefficient of variance = S.D/AM × 100 it means Sigma/A.M × 100 Covariance: The measure of the statistical between two variables (X and Y) where the average product of the simultaneous deviation of the variables from their respective mean. (σ2) = ∑ (X – X)2/ n-1 or (σ2) = ∑ (X –X)2/n COVXY = ∑ (X-X) (Y-Y)/n Variance on excel Select new column for the variance and then click on cell and type VAR, selection option and enter (Figure 14.9).

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Same as standard deviation

Same as standard deviation

Same as standard deviation

Same as standard deviation

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Same as standard deviation

Figure 14.9: Screenshot represents the Variance of data.

Frequency Values of Variable The different observations are known as values of variable. The values of variable obtained by observations are known as observed values or observation.

14.35 CORRELATION (R) Select new column for the correlation and then click on cell type CORREL and choose option and then enter. There are two type of correlations +1 (perfect correlation) 0 (no correlation) to –1 (perfect negative correlation) (Figure 14.10). After calculating correlation we can determine the probability of observed correlation occurred by chance means we can conduct a significance test. Most often we are using this to determine while our hypothesis is a real one and not a chance occurrence. There are two hypothesis (1) Null hypothesis and (ii) Alternative hypothesis. 1. Null hypothesis The hypothesis is usually verified for possible rejection by test, even it is true known as null hypothesis. It is denoted by H0. The formula given as follows: H0: µ = µ0 2. Alternative hypothesis

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The statement against null hypothesis is known as alternative hypothesis. Thus, if null hypothesis is H0: µ = µ0 then alternative hypothesis may be HA: µ ≠ µ0 or HA: µ < µ0 or HA: µ > µ0. The commonly are used significance level 0.05 for agriculture and 0.01 for pharmaceutical industry. For example, if we get correlation value r = 0.972, which is more than 0 means our value accepts the alternative hypothesis and reject the null hypothesis. Choose the cell and text the “cor” and select the CORREL

Now same follow the instruction as same as in standard deviation

Same as standard deviation

Same as standard deviation

Same as standard deviation

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Same as standard deviation

Figure 14.10: Screenshot represents the correlation values of data.

14.36. REGRESSION It is used to denote estimation or prediction of the average value of one variable for a specified value of the other variable. One of the variables is known as independent or the explained variable and the other is called dependent or the explaining variable. (It is the measure of the average relationship between two or more variable in terms of the original units of the data; M.M. Blair). Regression Lines The bivariate data are plotted as points on graph paper, it will be found that the concentrations point follows a certain pattern showing the relationship between the variables. When the trends points are found to be linear, we determine the best fitting straight line by Least Square Method. Such straight lines which are used to obtain best estimated of one variable for given values of the other are called regression lines. If two variables are linearly related then that relation can be expressed as Y = bx +a. Where as “b” is the slope of the line relating Y to X and “a” is the “Y” intercept of that line. “A line of regression is the straight line gives the best fit in the least square sense to given sets of data.”

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Regression coefficient It is expression of how much one dependent variable (Y) may be expected to change per unit change in some other independent variable (X) is known as regression coefficient (b). Types of Regression • Simple regression: Dependent variable is a function of a single independent variable. • Multiple regression Dependent variable is a function of two or more variable. • Linear regression Dependent variable is linearly correlated with the predictor (independent variable). It forms the straight line. • Nonlinear regression Dependent variable has a nonlinear correlation with the independent variable. It forms sigmoid or hyperbolic curve. Properties Regression • The expression of the dependent variable is applied as a function of independent variable. • It is work out only when significant correlation between the dependent and independent variable. • Regression predicts. • It is worked out using statistics for regression coefficient.

Methods of Studying Regression There are two types of method such as: • •

Graphic Method Algebraic Method

Useful Properties of Regression • • • • • • •

It is a mathematical measure showing the average relationship between two variables. It is random variables (x) and y is fixed variable. Sometimes both variables are randomly collect. It causes and affects the relationship between the variables. It is used for prediction of one value in respect to the other given values. It is an absolute figure. It has wide application as it studies linear and nonlinear relationship

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between the variables. It is explain that the decrease in one variable is associated with the increase in the other variable.

14.37 NULL HYPOTHESIS In statistical hypothesis is asset up and whose validity is tested for possible rejection on the basis of sample observations is known as Null Hypothesis. It is denoted by H0 It is tested against the alternatives. “Null hypothesis is the hypothesis which is to be tested for possible rejection under the assumption it is true” (Prof R. A Fisher remarked).

14.38. ALTERNATIVE HYPOTHESIS • • • •

The negation of null hypothesis is called the alternative hypothesis. It is denoted by H1 and H∞. It is not tested, but its acceptance (rejection) depends on the rejection (acceptance) of the null hypothesis. The alternative hypothesis contradicts depends on the rejection of the null hypothesis.

Statistical Hypothesis The statement or assertion about the statistical population or the value of its parameters is called statistical hypothesis. It is two types of hypothesis • Simple • Composite

1. Simple hypothesis The hypothesis which specifies the population completely is called simple hypothesis.

2. Composite hypothesis The hypothesis which does not specify the population completely is called composite hypothesis.

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Rejection Region The set values of the statistics which lead to rejection of the null hypothesis is called rejection region of the test. The probability of the null hypothesis is rejected by the test is often referred to as “size” of the critical region. On the other hand which lead to the acceptance of null hypothesis which gives us a region is called as “Acceptance region.”

Statistics Test After the arrangement of the null hypothesis and alternative hypothesis the test of statistics is computed and it is based on the probability distribution. It is used to test whether the null hypothesis set up should be accepted or rejected.

Significance Level The probability is maximum with which a true null hypothesis is rejected is known as level of significance of the test. It is denoted by α. The level of significance is arbitrarily chosen in advance depending on the consequence of statistical decision for the farming decision rules. The level of significance generally 5% and 1% is taken other levels such as 2% or ½ (0.05) % are also used.

Degree of Freedoms The sample which is freely variable without affecting the mean or it is an integer used to determine whether a chi-square value is statistically significant. There are the number of data which are given in the form of a series of variables in a row or column or the number of frequencies that are put in cells in a contingency table which can be calculated independently is called the degrees of freedom and is denoted by df.

Calculation for Degree Freedom The data is given in the form of series of variables in a row or column then the degrees of freedom (df) = (the number of items in series) -1, i.e., df = n–1. Where “n” is the number of observations. The number of frequencies are put in cells in a contingency table the degree of freedom will be the product (number of rows less one) and the (number of column less one) i.e., df = (R-1) (C-1) where “R” is the number of rows.

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“C” = is the number of column.

Condition for using the Chi-Square test: • • • • •

• •

It should be on random basis of sampling. It should be absolute not relative terms. It is dependent degree of freedom. It should be independent to each other for making the sample of each observations. If X2 test applied in a fourfold table then it will not give a reliable result with one degree of freedom if the expected value in any cell is less than 5. In such cases to apply X2 test yates correction necessary. In this test the total number of observation should be large it mean n ≥ 50.

Types of chi-square test • • •

Homogeneity Chi-Square. Contingency Chi-Square. Goodness of fit.

Goodness of Fit (Pearsonian – x2): The chi-square test is also applied for the test of “goodness of fit” to determine whether the actual or (observed) numbers or frequencies are similar or in “good agreement” with the expected or (theoretical) number of frequencies. The test is called “goodness of fit” X2 = ∑ (│O-E│ -1/2)2/E where “E” Expected. “O” Observed ½ Yates correction. Example: The Model’s reported the results of the garden pea test each for goodness of fit for the following tested. Solution: Cross

Progeny

Hypothesis

Green × Yellow Pods

(F2)428: 152

3: 1

Violet red × White Flower

(F1) 47: 40

1: 1

Round yellow × Wrinkled green

(F1) 31: 26: 27: 26

1: 1: 1: 1

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Solution: • Null Hypothesis = 3: 1 • Alternative Hypothesis = 1: 1 • Calculation = Observed (O)

Expected (E)

(O-E)

(O-E)2

(O-E)2/E

428

¾ × 580 = 435

428 – 435 = -7

49

49/435 = 0. 113

152

¼ × 580 = 145

152 – 145 = 7

49

49/145 = 0. 338 x2 = 0. 451

Total = 580

The critical value of X2 at 0.05 and for 2–1 = 1 degree of freedom is 3.84. The decision; the calculated value of chi – square (x2) = 0.451 < critical value of x2 for df = 3.84 so the null hypothesis is accepted, i.e., there is no significant variation with the data So it is result of F2 monohybrid cross. Solution (b) • Null Hypothesis = 1: 1 • Alternative Hypothesis = 3: 1 • Calculation = Observed (O)

Expected (E)

(O-E)

(O-E)2

(O-E)2/E

47

87/2 = 43.5

47 – 43.5 = 3.5

12.25

12.25/43.5 = 0.281

40

87/2 = 43.5

40 – 43.5 = -3.5

12.25

Total 87

12.25/43.5 = 0.281 X2 = 0.562

Critical value: The control vale of chi square at 0.05 and for 2–1 = 1 degree of freedom is 3.84. Decision: the calculated value of the chi square x2 = 0.562 < critical value of x2 for 1 df = 3.84 so the null hypothesis is accepted, i.e., the variation is non significant. Solution (c) • Null Hypothesis = 1: 1 • Alternative Hypothesis = 3: 1 • Calculation = Observed (O)

Expected (E)

(O-E)

(O-E)2

(O-E)2/E

31

¼ × 110 = 27.5

31 – 27.5 = 3.5

12.25

12.25/27.5 = 0.445

26

¼ × 110 = 27.5

26– 27.5 = 1.5

2.25

2.25/27.5 = 0.082

27

¼ × 110 = 27.5

27– 27.5 = -0.5

0.25

0.25/27.5 = 0.009

26

¼ × 110 = 27.5

26 – 27.5 = -1.5

2.25

2.25/27.5 = 0.082

Total = 110

Critical value

x2 = 0.618

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The chi square value of chi-square at 0.05 and 4–1 = 3 so, df = is 7.82 Decision Calculated chi-square value (x2) = 0.618 < critical value of X2 of 3 df = 7.82 so the null hypothesis is accepted and the variance is non significant. Uses of chi square test • Test of homogeneity • Test of independent of attributes. • Test of Goodness of Fit.

14.39. CENTRAL TENDENCY The tendency for the given values of random variable to cluster round its mean, mode or median.

14.40 MEASURES OF VARIATION The variability is essential a normal character. The variability is a biological phenomenon. It is an important characteristic indicating the extent to which observations vary among themselves. There are three main types of variability • Biological variability • Experimental variability • Real variability

Measures of variability It is helpful to find out on how individuals observations are dispersed around the mean of a large series. The variability of a given set will be zero and only when observations are equal so it takes positive when observations are unequal. The measurements and variability are both of fundamental importance in the biological science. Dispersion

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Absolute measure of dispersion 1. It is expressed in the same statistical unit in which the original data are given. 2. It is used for the comparison of two sets of observations provided the variables are expressed in the same units and of the same average size. 3. The sets of data are given in dissimilar units again then the absolute measures of dispersion are not comparable. Relative measures of dispersions 1. It may also be used to compare the relative accuracy of data. 2. It is the ratio of a measure of absolute dispersion to an appropriate measure of central value and it is expressed in pure number. 3. It is independent of the units of measurement. Good measures of dispersions • It should be defined. • It should be easily calculated. • It should be based on all observations. • It should be algebraic treatment. • It should be based on all observations. • It should not be affected by extreme items. • It should have stability of samples.

Range It is the simplest measure of dispersion. It is the difference between the value of smallest item and the largest item included in the distribution. Range (R) = Largest value (L) – Smallest value (S) Coe-efficient of Range: The relative measure corresponding to range is coefficient of range. R=L–S L+S

Example: Find out the ranges of daily wages of 8 persons in a family given below.

Biostatistics Rupees

Solution:

10

11.50

12

21

6.75

18

13

371 20

Here the largest values Rs. 20 & smallest value Rs. 675 R = 20 – 6.57 = 13.25

Example 2: Find the range of the following: Class

10–19

0–29

30–39

40–49

50–79

80–99

No. of person

5

15

25

35

15

5

Solution: There are class discontinuous type we change class limits to class boundaries 7 then the lower class boundary of lowest class 9.5 (S) and the upper class boundary of the highest class = 99.5 (L) The range (R) = 99.5 – 9.5 = Rs. 90. Example: Find the range and the coefficient of range Days

Mon

Tue

Wed

Thus

Fri

Sat

Prices

20

21

23

16

25

22

Solution: R=L–S Here L = 25, S = 16 R = 25 – 16 = 9 (R) Co-efficient of range = L – S L+S = 25 -16 25+16 = 9/41 = 0.219

Merits and Demerits of Range Merits • It takes time to calculate. • It is also simple to calculate. • It is easy to understand. Demerits • It is not depend on all observations. • It is based on only the largest and smallest among the values. • It is highly affected by extreme values. • It cannot be calculated by from frequency distribution with open classes. Uses 1. Estimating the Fluctuations in Prices: It is useful for the prices variation in stocks and shares. 2. Weather Forecasts: It is preferably used in determining the

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difference in minimum and maximum temperature for predicting the variation of temperature in a day. Quality Control: It plays an important role in preparing control charts in the methods of statistical quality control.

3.

Mean Deviation The mean deviation is called the average deviation. It is the average difference between the items in a distribution and the median and mean that series. • It is about the mean. • It is about the median Co-efficient of Mean Deviation It is the ration of mean deviation to its arithmetic mean or median multiplied by 100. C.M.D = MD× 100 Mean/ Median

Calculation of Mean Deviation Ungrouped data: It can be calculated mean and median. Calculate the deviation from mean denoting by “D” and ignoring the sign positive (+) or negative (-) Where “n” is the total number of observation items. Divide sum of the ∑ D by the total number of items. Mean Deviation (MD) = ∑ │X – X│ = ∑ │D│ nn Mean Deviation about the mean MD = 1/ N ∑ │X – X│ f Mean Deviation about the median MD = 1/N ∑ f (x- median)

Example: Calculate the mean deviation for the following items. X

10

11

12

13

14

F

3

12

18

12

3

Solution: Median deviation = ∑ f D N = 36/48 = 0.75

Example: Find out the mean deviation of the following data 13, 84, 68, 24, 96, 139, 84, 27 and bout the median? Solution: Here there are many even number of observations viz 8, median is the average of the two middle most observations Let us arrange the data

Biostatistics 13

24

27

68

84

84

96

373

139

Median = 68+84/2 = 152/2 = 76 X

X – Median = D

13

76 – 13 = 63

24

76 – 24 = 52

27

76 – 27 = 49

68

76 – 68 = 8

84

84 – 76 = 8

84

84 – 76 = 8

96

96 – 76 = 20

139

139 – 76 = 63

N=8

∑ D = 271

Mean Deviation about the median = MD = 1/N ∑ │x – median│ = 1/8× 271 = 33.88 Grouped data: 1. Discrete series: It is calculated mean and median and the deviation from the mean and the median ignoring the sign and denote them by │D│ It is multiply these deviation by respective frequencies & obtain the total ∑ f│D│. Divide the total (∑ f │D│) by the number of observations giving the required value of mean deviation. MD = ∑ f │D/ ∑ f = 1/N ∑ f │D│ =X–X ∑f=N

2.

Continuous Series: It can be calculated the mean and the median series and also take the deviation of the items from the mean or median, ignoring signs and denote them by │D│. The mean deviation is taken from the mid value of each class. Multiply the deviation by frequencies and obtain the total. It is divide the total sum of the total number of observations.

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Merits and Demerits of Deviation Merits • • • •

It is relative simplicity. It is easy to compute. It is easy to understand. It is less affected by the value of extreme items as compared to standard deviations.

Demerits • It is rarely used in social sciences. • It is yielding best result while taken from median. • This method may not yield accurate results. • It is not suitable for further algebraic treatment. Example 1. Calculate the mean deviation from the mean from the following data. Marks

0–10

10–20

20–30

30–40

40–50

Total

No. of students

5

8

15

16

6

50

Solution:

Class Interval Mid Value (X)

Frequency (f)

fx

│D│= X -27

fD

0–10

5

5

25

5 -27 = 22

110

10–20

15

8

120

15 – 27 = 12

96

20–30

25

15

375

25 – 27 = 2

30

30–40

35

16

560

35 – 27 = 8

128

40–50

45

6

270

45 – 27 = 18

108

∑ f = 50

∑ fx = 1350

∑ fD = 472

X = ∑ fd/ N = 1350/ 50 = 27 MD = ∑ f │D│/ ∑ f = 472/50 = 9.44

Example: From the following frequency distribution calculate the value of quartile (Q1) median (Q2) and upper Quartile (Q3). Marks in 10–19 Mathematics

20–29

30–39

40–49

50–59

60–69

Total

Frequency

11

15

17

12

7

70

Solution:

8

Q1 = N/4 Q2 = N/2 Q3 = 3N/4

Biostatistics Class interval

Frequency

10–19

8

20–29

11

30–39

15

40–49

17

50–59

12

60–69

7

Class boundary Q1 Q2 Q3

375

Cumulative frequency

9.5

0

19.5

8

29.5

19

39.5

34

49.5

51

59.5

63

3 N/4 = 52.5

69.5

70

=N

N/4 = 17.5 2 N/4 = 35

Q1 = L1 + N/4 –F1/f1 × i L1 = Lower boundary of quartiles F1 = Cumulative frequency f1 = frequency of quartile class i = width of class interval Q1 = 19.5 + 17.5–8/11 × 10 = 19.5 + 95/11 = 19.5 +8.6 = 28.1 Q2 = 39.5 + 35–34/17 ×10 = 39.5+ 10/17 =39.5+0.58 = 40.08 =40 Q3 = 49.5 + 52.5 – 51/12 × 10 49.5 + 1.5 × 10/12 = 49.5 + 1.25 = 50.75 = 51 Q1 = 28 Q2 = 40 Q3 = 51

Example: Calculate the Quartile deviation and Coefficient of Quartile deviation from the following frequency distribution? Class Interval

10–15

15–20

20–25

25–30

30–40

40–50

50–60

60–70

Frequency

4

12

16

22

10

8

6

4 = 82

Solution: Class Interval

Frequency

10–15

4

Class Boundary 10

Cumulative frequency 0

15–20

12

15

4

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16

Q1

20

16

20.5 = N/4

25–30

22

Q2

25

32

41 = N/2

30–40

10

Q3

30

54

61.5 = 3N/4

40–50

8

40

64

50–60

6

50

72

60–70

4

60

78

70

82

=N

Calculate Quartile deviation and its coefficient We have to find Q1, Q2 and Q3 i.e., Cumulative frequency N/4 Cumulative frequency N/2 Cumulative frequency 3N/4 Total frequency N = 82 N/4 = 20.5 N/2 = 41 3N/4 = 61.5 Q1 = L1 + N/4 – F1/f × I Q1 = 20 + 20.5 – 16/16 ×5 Q1 = 20 + 4.5/16 × 5 = 20 + 22.5/16 = 20 + 1.4 = 21.4 Q2 = 4 + N/2 – F/f × i = 25 + 41–32/22 × 10 = 25 + 90/22 = 25 + 4 = 29.0 Q3 = 30 + 61.5 – 54/10 × 10 = 30 + 7.5 = 37.5 Q = (Quartile Deviation) = Q3 –Q1/2 =37.5 – 21.4/2 = 16.1/2 = 8.0 Coefficient of Quartile Deviation = Q/Q2 × 100 = 8/29×100 =27.58%.

Example: Find the quartile deviation of the following distribution? Class Interval

40–45

45–50

50–55

55–60

60–65

65–70

Frequency

10

22

28

20

12

8

Solution:

Class Interval

Frequency

Class boundary

Cumulative frequency

40–45

10

40

0

45 – 50

22

Q1

45

10

50 – 65

28

Q2

50

32

55 – 60

20

Q3

55

60

60 – 65

12

60

80

25 = N/4 75 = 3N/4

Biostatistics 65 – 70

8

65

92

70

100

377

=N

Q1 = L1 + N/4 – F/ f × i N/4 = 25 F = 10 L1 = 45 f = 28 i=5 = 45 + 25 – 10/22 ×5 = 45 + 15/22 × 5 = 45 + 3.4 = 48.4 Q3 = 55 + 75 -60/20 ×5 = 55 + 3.75 = 58.75 Quartile Deviation (Q) = Q3 – Q1/2 = 58.75 – 48.4/2 = 10.35/2 = 5.175

14.41 BINOMIAL DISTRIBUTION Binomial distribution was discovered by Swiss mathematician James Bernoulli (1654–1705). It is derived from the process is called Bernoulli trial. It is the discrete probability distribution which is obtained when the probability (P) of the happening of an event is same in all the trials and there are only two events in each trial. It is theoretical probability distribution because it can be worked out theoretically using the series of the terms of binomial equation.

Condition Under Binomial Distribution • • • • • •

The trials or events must be repeated under the conditions. The number of trials should be finite and also small. The events or trials must be independent it means happening of one event or trials must not affect the happening of other events. The variable should be discrete it means the values of X should be 1, 2, 3, 4, or 5, etc. It never be 1.5, 2.5, 3.7, 5.5, etc. It should be either success or failure so that it can dichotomy exist, i.e., the happening of an event has two possible outcomes.

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Properties of Binomial Distribution • •

It is presented the discrete probability distribution. It has two parameters “p” or “q” the probability of success or failure and “n” the number of trials. • The parameters “n” is always integer. • The mean (µ), SD (σ) variance (σ2) and the coefficient of dispersion (C.D) of a binomial distribution of cases of the class having the proportion “p” in the population are obtained from the sample size (n) and the proportions (p and q) of the cases in the two classes. • Mean (µ) = np • Standard deviation (σ) = √npq • Coefficient of dispersion (CD) = npq/np = q • Variance (σ2) = npq • Kurtosis and Skewness of binomial distribution depends on the proportion of p and q in the population. • Skewness (Y1) = (q – p)2/npq • Kurtosis (Y2) = 3 + 1 – 6 pq/npq • It is symmetrical of p = q = 0.5 • It is positively skewed if p < 0.5 • It is negatively skewed if p > 0.5 • The binomial coefficients are given by the Pascal’s triangle. Computational Binomial Probabilities

Multinomial distribution: It may be regarded as generalization of binomial distribution for accommodating any number of variables when they are more than two mutually exclusive outcomes of a trial the observations leads to multinomial distribution. Suppose that: E1, E2, E3 E4……EK are k mutually exclusive and exhaustive outcomes of an event trial with respective probabilities p1, p2, p3, pk will occur k1, k2, k3, k4…kn times respectively is C = N/ k1│ k2 │k3│Kn × P1k1 P2k2….Pkn Where k1 + k2 + k3 + kn = N C = the number of permutation of the events E1, E2….Ek (1) Single term of the expansion: It represents the number of ways in which the conditions of each term may be satisfied. The number of combination (C) of n different taken k at a time is

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expressed by nCk = n/ k n-k (2) Bernoulli expansion: It is having total number of classes, events with probability of Bernoulli expansion n = total number of events p = classes q = classes X = number of classes in p classes n-X = number of cases in q classes Probability of P (X) is expressed by Bernoulli expansion P (X) = n px qn-x/X │N – X│ (3) Binomial expansion: It is having a total number of events and trials with probability of occurrence of success or failure. n = total number of events/ trials p = probability of occurrence of success q = probability of occurrence of failure. p + q = 1 or q = 1- p The general expression of Binomial expansion (p +q)n = pn + npn-1 + n (n-1)/ 1×2 pn-2 q2 + n (n-1) (n-2)/ 1×2×3 p2–3 q3 +.+ n (n-1) (n-2) ×3×2/ 1×2×3×………× (n-1) × pg n-1 + qn Probabilities distribution of binomial expansion for r success in n trials is given by P (r) = nCr qn –r pr It has mean = np and variance = npq. Mean, Variance and Standard deviation of Binomial distribution The probability of distribution of binomial distribution for r success in n events and trials is given by P (r) = nCr qn –r pr So, mean (µ) = np Variance (σ2) = npq Standard deviation (σ) = √npq n =number of independent events p = probability of success q = probability of failures

Example 1: Calculate the probability of getting head three times when a coin is tossed 5 times. Solution: When coin is tossed there are only two outcomes either head or tail. Therefore it shows binomial distribution. If the probability of getting head then it is “p” and of tail it is “q.” The probability of getting head 3 times can be

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calculated in the following way. n =5, X = 3, P = ½ = 0.5, Q ½ = 0.5 P (X) = n px q n-x/ X (n-X) = 5 (0.5)3 (0.5)5–3/3 (5–3) = 5.4.3.2 × (0.5)3 × (0.5)2/ 3.2.2 = 5 × (0.5)5 = 10 × 0.5 ×0.5 ×0.5 × 0.5 × 0.5 = 5 × 0.03125 = 0.3125 Example 2: The two male and female children in a family of four children by applying binomial theorem. Solution: Total number of children = 2 + 2 = 4 Male = 2 Female = 2 Initial probabilities of male child (p) = 2/4 = ½ = 0.5 Initial probabilities of female child (q) = 2/4 = ½ = 0.5 According to the binomial equation: (p + q)4 = p4 + 4p3q + 6 p2 q2 + 4pq3 +q4 p = 6p2q2 = 6. (0.5)2 (0.5)2 = 6 × 0.5 × 0.5 × 0.5 × 0.5 = 6 × 0.0625 = 0.375 So, the probabilities of two boys and two girls in a family are 0.375. Example 3: In a two hundred families with three children a population of Arambagh subdivision is sampled at random. How many families do we expect to have (a) no girls (b) one girl (c) two girls? Assume the sex ration to be 1:1. Solution: Probabilities for girls and boys = ½ g for girls and b for boys. Now we expand the binomial (g and b), n = 3 (g +b)3 = g3 + 3g2b + 3gb2 + b3 No girls relate to b3 term (1) ½ × ½ × ½ = 1/8 = 1/8 ×200 = 25 (200 = families) (2) One girls relates to 3gb2 term 3 × ½ (1/2)2 = 3 ×1/2 × ¼ = 3/8 = 3/8 ×200 = 75 (3) Two girls relate to 3g2b term 3 × (1/2)2 ½ = 3× ¼ ×1/2 = 3/8 = 3/8 × 200 = 75 Example 4: A plant breeder has 45 different inbred strains of pea plants. How many different hybrids can be obtained from a total 45 plants?

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Solution: Hybrid has two genes N = 45 r = 2 According to the formula: nCr = n/r (n –r) = 45/ 2 (45 -2) = 45 × 44 ×43/ 2.1 ×4 ×43 = 45 ×44/2 45 × 22 = 990 Example 5: In a family with two children in serampore subdivision where both parents are heteroztgous for albinism. What proportion of these family would be expected to have (a) neither child with albinism (b) one child with albinism, (c) both children with albinism? Solution: Let the symbol “a” for albinism and “A” for normal Expand binomial expansion (A + a)2 = A2 + 2Aa + a2 The parents are heterozygous so therefore probabilities of normal ¾ and albino ¼ Two children in a family both are normal, i.e., A2 = (3/4)2 = 9/16 Among the two children, one is with albinism, i.e., 2Aa = 2Aa = 2 × ¾ × ¼ = 6/16 Both children with albinism, i.e., a2 = (1/4)2 = 1/16. Example 5: Consider the parents of a Sinha Roy family in which both of them heterozygous for a sever genetic syndrome, that is autosomal recessive. Of their six children five of them have this particular syndrome. How unlucky is this family? Solution: n=6 p=¾ q=¼ Probabilities for diseases (t) = 5 Normal (s) = 1 P = n × p i q 5 /s × t = 6 × (3/4)1 (1/5)5 = 6 ×5/ 5 ×3/4 ×1/1024/1 × 5 = 18/4096 = 0.00439 = 0.0044 Example 6: A couple of heterozygous for albinism (Aa). What is the probability that (a) 4 out of 6 children born to them are normal: (b) 4

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normal and 2 albibo out of 6 children? Solution: Let “a” = allele for albinism A = allele for normal skin color Generally heterozygous parents have ¾ normal children and ¼ albino children. Pa = ¾ q (a) = ¼ and n = 6 Probability of 4 children being normal PA = (3/4)4 = 3/4 × 3/4 × 3/4 × 3/4 = 81/256 = 0.316 n = 6, s = 4 and I = 2 (4 normal and 2 albino) P = n × (p)4 × (q)2/ s × t = 6 ×(3/4)4 × (1/4)2 = 6 ×5 ×4 ×3/4×3/4×3/4×3/4×1/4×1/4/2×4 = 15×81/256 ×1/16 = 1215/4096 = 0.2966 = 0.297 Through binomial expansion: (p+q)6 = p6 + 6p5q + 15 p4 q2 + 20 p3 q3 + 15 p2q4 + 6 pq5 +q6 P = 15p4q2 = 15 × (3/4)4 × (1/4)2 = 15 × 81/256 ×1/16 = 1215/4096 = 0.2966 = 0.297 Example 6: The four babies were born in Aligarh general hospital (a) what was the chance that two will be boys and two girls (b) what was the chance that all four would be girls? Solution: The probabilities of boys and girls were ½ = 0.5 P=½ q=½ (a) n = 4. s = 2 and t = 2 (b) (s) for girls and (c) (t) for boys (d) P = n × ps × qt/s×t (e) = 4 × (1/2)2 × (1/2)2/2×2 (f) = 4.3. (2) ×1/4 ×1/4/2.1(2) (g) =6×¼×¼ (h) = 3/8 Probabilities was 3/8 (c) (p+q)4 = p4 + 4 p3q + 6 p2q2 + 4 pq3 + q4 (d) p = q4 (1/2)4 = 1/8

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Example 7: There are eight children in a family, where both parents are heterozygous for albinism what mathematical expression predicts the probability that six are normal and two are albinos? Solution: As both parents are heterozygous the probabilities of normal is ¾ and albinos ¼ i.e., p = ¾ q=¼ n= 8 s = 6 and t = 2 (a) P = n × ps × qt/s×t = 8 ×3/4 × (1/4)2/6 (2) 8.7 (6) ×(3/4)6 × (1/4)2 = 28 × (3/4)6 ×(1/4)2 = 28 × 3/4×3/4×3/4×3/4×3/4×3/4 ×1/4×1/4 = 7×729/16384 = 5103/16384 = 0.31146 Example 17: A multiple allelic system is known to consist of seven alleles. Assuming that this is a diploid species, how many different genotypes could exist in the population? Solution: Number of possible genotypes = Number of different allelic combination (heterozygotes) × number of genotypes with two same allele (homozygotes) = n + n /k (n – k) n=7 k = 2 (heterozygotes) = (7)/ 2 (7–2) +7 = 7.6 (5)/2.5 +7 = 21 +7 = 28 genotypes Poisson Distribution The poison distribution was derived by French mathematician Simeon Denis Poisson (1837) and is known as Poisson distribution. It represent the Poisson distribution of discrete, random variables of rare events whose probability occurrence is very small but the number of events/trials is very large and may approach infinity. It is basically applicable to such events where binomial expression formula can be used in determining theoretical probabilities. So the definition of Poisson distribution is a discrete probability distribution of rare events and the mean and the variance is equal. Characteristics • The events are independent and random. • It is limited form of binomial distribution.

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It may be expected in cases where the chance of any individual event being success is small. • It has a single parameter, the mean of the distribution. • The mean and the variance are equal. • It is a discrete probability distribution because it is a probability distribution of whole number (0, 1, 2, 3….n) of events. • It is positively skewed and declines with the rise of value of mean. • It is leptokurtic which decreases with the increases of mean. Condition under the Poisson distribution is used • It is applicable when the observation or number of events is very large but the probability of success is very small. • The random variables should be discrete. • A dichotomy exist, i.e., the happening of the events must be divided into two classes viz., Success or failure occurrence or nonoccurrence. • P should be small or it case to zero. It is independent it means happening of one events does not affect the happening of other event. Computation of Poisson distribution The random variable(X) is applicable for probability of distribution so it’s said to have Poisson distribution. P (X) = e-m mx/x P = Probability of success x = variables (such as 0, 1, 2, 3 and n) e = constant 2.7183 (base of natural logarithm m = finite positive constant and is known as parameter of Poisson distribution. Number of 0 success (X)

1

2

3

r

n

Total

Probabilities e-m P(X)

e-m m/1

e-m m2/2

e-m m3/3

e-m mr/r

e-m mn/n

1

P (0) + p(1) + p(2) + p(3) + p(r) + p(n) + ………α = e-m + e-m m/1 + e-m m2/2 + e-m m3/3 + e-m mr/r + e-m mn/n + ………α = e-m (1 + m/1 + m2/2 + m3/3 + mr/r + mn/n + ………α = e-m em = e 01 = 1 Mean = m = p Standard deviation = √m Skewness given by (ß1) = 1/m Kurtosis given by (ß2) = 3 + 1/m Variance = m Examples of Poisson Distribution

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The number of fish death in tanks in one week due to some pesticide treatment in water. • The number of bacterial colonies in a given culture of per unit area on microscopic slide has been seen under microscope. • The emission of radioactive particles (alpha = α) • The number of mistakes have committed by a good typist per page. • The numbers of buses are passing through a certain road (M.G Road Agra). • The number of diseases or death by cancer or heart attack in any cities like in (Agra) hospitals in one year. Example 1: In Biotechnology of 520 pages, 390 typological error occur. Assuming Poission law for the number of pages error per page, find probabilities that random sample of 5 pages will contain no error? Solution: Here n = 5, Book has 520 pages Typological errors 390 pages Therefore probabilities (P) = 390/ 520 = 0.75 Mean np = 5 × 0.75 = 3.75 Using Poisson probabilities law P (r) = e-m mr/ r = e-0.75 mr/r Probabilities error zero, Therefore P(0) = e-0.75 (3.75)0/0 = e-0.753.75 Example 1: The Biostatistics book with 585 pages contains 43 typological errors. If these errors are randomly distributed throughout the book, what is the probability that 10 pages, selected at random will be free from errors? Solution: Here n = 10 Book has 585 pages Typological errors 43 pages Therefore probabilities P = 43/585 = 0.0735 Mean (m) np = 10 × 0.0735 = 0.735 Poisson distribution (Pr) = e-m mr/r = -0.735 × (0.735)r/ r Probability zero error P (0) = e-0.735 × (0.735)0/0 = e-0.735 ×1 = e-0.735

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= 0.4795 Example 3: If the mean of the Poisson distribution is 4 then find the S.D, ß1, ß2, µ3, µ4 Solution: Here m = 4 Variance = m = 4 S.D = √4 =2 Skewness (ß1) = 1/m =1/4 =0.25 Kurtosis (ß2) = 3 +1/m =3+¼ 0.325 µ3 = m =4 µ4 = m +3m2 = 4 + 3 (4)2 = 4 +3.16 = 4 +48 =52 Example 4: The following data are obtained from vector cytogenetic research laboratory of Dr. M.P.S College Agra. Events (x1)

Frequency (f)

0

20

1

26

2

16

3

4

4

2

• What is mean? • What is e-m? • What is P(0)? Solution: Mean (X) = ∑ fx/ ∑ f = 0+26+32+12+18/20+26+16+4+2 = 78/68 1.147 =1.15 (X) = m = 1.15 e-1.15 = 0.32

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P(0) = e-m (1.15)0/0 = 0.32 × 1 /1 = 0.32 Example 5: In a family of 8 children where both parents are heterozygous for albinism, what mathematical expression predicts the probability that six are normal and two are albino? Solution: Both parents are heterozygous, the probabilities of normal is 3/4 and albinos 1/4. i.e., p = ¾ q=¼ n=8 s = 6 and t = 2 P = n ×ps qt/s(t) = 8/6 (2) × (3/4)6 × (1/4)2 = 8.7 (6)/ 2.1 (6) × (3/4)6 × (1/4)2 = 28 × (3/4)6 × (1/4)2 = 28 × 3/4×3/4×3/4×3/4×3/4×3/4 ×1/4×1/4 = 7×729/ 1638 = 5103/16384 = 0.31146

14.42. SKEWNESS The distribution is said to be symmetrical when mean, median and coincide. It has three parts left tail and middle part also. It has also right and left tail are equal length. It is used to denote the extent of a symmetry in the data. When the frequency distribution is not symmetrical it is said to be skewed. The meaning of skewness is “ lack of symmetry.” A symmetrical distribution has therefore zero skewness. Characteristics 2. It may be positive or negative. • Positive skewness: • The curve of the distribution has longer tail toward the right it means the higher values of the variable. • Mean > Median > Mode • If the curve of the distribution has a longer tail towards the left, i.e., the lower values of the variable • Mean < Median < Mode 2. Here Mean, Median and mode are failed to coincide. Both median and mean are displaced from the mode toward the skewed tail.

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Mean > Median > Mode (Positive Skewed) Mean < Median < Mode (Negatively Skewed) Here the first quartile is displaced toward the skewed tail. Therefore Q3 –Q2 > Q2 –Q1 (Positively Skewed) Q2 – Q1 > Q3 –Q2 (Negatively charged). Measures of Skewness • It indicate not only the extent of skewness in numerical expression but also the direction, i.e., the number in which the deviations are distributed. • It is normally measures of symmetry are called measures of skewness. • The absolute measures are known as measures of skewness. • It tells us the extent of symmetry whether it is positive or negative. Absolute skewness = Mean – Mode Mean > Mode (Positive skewness) Mode > Mean (Negative skewness) It is also known as coefficient of skewness and it is given by ß1 = µ23/µ 32 µ3 = 3rd moment and µ2 2nd moment. There are important measures of relative skewness. 1. Karl Pearson’s coefficient of skewness Sk = Mean – Mode/ Standard deviation Sk = 3 (Mean – Median)/Standard deviation 2. Bowley’s quartile coefficient of skewness Sk = Q3 – 2Q2 + Q1/ Q3 – Q1 (Q1 = First quartile; Q2 = Second quartile) 3. Kelly’s coefficient of skewness Sk = P90 + P10 – 2 Median/P90 – P10 (P10 = 10th Percentiles; P90 = 90th Percentiles

14.43. KURTOSIS AND MOMENT It is used to describe the degree of peakedness of a frequency distribution compared to that of normal distribution. Characteristics 1. It is measure the peakedness of a normal curve. 2. It is also called as measures of convexity of the curve. 3. It introduced the three broad patterns. 4. If the peakedness viz., (Leptokurtic, Mesokurtic and Platykurtic). • The curve is neither flat nor peaked is known as mesokurtic, i.e., normal curve. • The curve which has higher and sharper peaked is called as mesokurtic

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it means the normal curve. • The curve has higher and sharper peaked and narrow body then the normal curve is known as Leptokurtic. • The curve is flatter and its center, broader in the body and thinner at tails than normal curve is called as Platykurtic. Measures of Kurtosis The frequency of distribution is based upon the fourth moment about the mean of the distribution. ß2 = µ4/µ22 = µ4/ σ4 ß2 = 3 Mesokurtic ß2 < 3 Platykurtic ß2 > 3 leptokurtic

Importance of Skewness • •

It tells the direction and extent of asymmetry in a series. It provides us an idea about the nature and degree of concentration of items. Dispersion

Skewness

It spread the individuals values It shows the departure from symmetry, about the mean it means central i.e., direction of variation. value. It shows the degree of variability.

It shows the value is higher or the lower concentration.

It is types of averages of deviation- It is not the average but it is measured average of the second order. by the use of mean, median and mode. It judges the truthfulness of the It judges the truthfulness of the central central tendencies. tendencies.

Significance It tells us the extent to which a distribution is more peaked or more flat topped than normal curve. It also depend the shape of the top of a frequency curve. Moments It is used in mechanics, physics, etc. It is also applied in statistics, it describe the various characteristics of frequency distribution viz., central tendency, dispersions and skewness and kurtosis. It is also can be defined as arithmetic mean of various powers of deviations taken from the mean of distribution. Role Moments: First moments (µ1) of frequency It is always zero, i.e., It measures mean of the distribution = ∑ X – X/ N µ=0 distribution µ1 = X = 0

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Second Moments (µ2) of µ2 = σ2 frequency distribution about the mean is the variance of the distribution = ∑ (X –X)2/N

It measures the variance, i.e., the spread of the different terms in a distribution.

Third Moments (µ3) = ∑ (X- It deals with skewness It gives an idea about the X)3/N degree of skewness present Fourth Moments (µ4) = ∑ (x – It is highlights on the It measures kurtosis. x)4/N height of frequency distribution whether it is more peaked or flat topped than normal

Example 1: The coefficient of skewness = 3, Mean = 90, Median = 80; Find the value of S.D. Solution: Sk = 3 (Mean – Median)/S.D S.D = 3 (Mean – Median)/Sk S.D = 3 (90 -80)/ 3 = 3 ×10/ 3 = 10

14.44 SET THEORY AND PROBABILITY It is well defined collection of all possible distinct objects is called a set. The object of the set is called as elements or members. The sets are usually denoted by cpaitals letters (e.g., A, B, C and D) and their elemnts are denoted by small letters (e.g., a, b, c and d). The elements are enclosed in within curly backers (……). It is separated by commas. Example 1: The collections of all consonants of English alphabet and collection of all odd numbers less than 50. Finite and Infinite Set Finite set: It contains limited numbers of different elements is called as finite eg., A = (a, b, c d, e) is finite because it has 5 elements Infinite set: It contains unlimited numbers of different elements is called as infinite e.g., (2, 4, 6 and 8), it has no countable elements. Null set It does not contains any elements at all is called as null set. It is also called void set or empty set. There is only one such set

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It is denoted by Examples 1: A person who can jump to a height of 5 miles is the null set because none can jump to such height. Unit Set The set having only one element and it is also known as single tone set. Equal Set The two sets viz A and B are called equal if they have same elements. When A and B are equal, we write A = B if they are not equal we write A ≠ B. Equivalent Set The two sets viz A and B are equivalent if the number of elements, i.e., cardial numbers are equal. e.g., A = (2, 4 and 6) and B= (a, b and c) Here n (A) = n (B) = 3. Cardinal Set The distinct elements in a finite set is called its cardinal number. It is denoted by n (A). The cardial number null set is zero. In cardial number infinite set is not defined. E.g.: A = (2, 3) has 2 elements, so n (a) =2 B = (a, e, I, o, u) has 5 elements so n (A) =5 Subsets and Superset The two sets viz., A and B if each element of set A is also an element of a set B, then set A is called a subset of B and set B is the superset of A. This is read as “B” contains “A” or “A” contains “B” Example: The set A = (2, 5) is subset of the set B = (2, 5, 7). Here all the elements of A are also the elements of B On the other hand B is a superset of set “A.”

14.45. COMPARATIVE STATISTICS In this, we are compared variables. We can find out variables are basically same means they could originate from the same population or if they are significantly different means they have a different origin. So, there are two tests ANOVA (analysis of variance) and t-test (Figure 14.11).

14.46. CHI SQUARE TEST The calculation of he quantity which is used to compare an “observe” ration with an “expected” or “theoretical” ration and to to determine the how closely

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the former fits the letter so it involve all the statistical test for the calculation or observation of the hypothesis to be significant or not significant value. In scientific research, first we make a hypothesis and do experiments after completion of the experiment we analysis the data that is correct or not so chi square test for the analysis of our observation. So the measure of chi-square enables us t find out degree of discrepancy between “observed” frequencies and “ theoretical or “expected” frequencies and thus to determine where the observed and theoretical frequencies is due to the error of sampling or due to the chance. It is computed on the basis of frequencies in a simple and thus the value of chisquare so obtained is a statistic. Chi-square is not a parametric test as its value is not derived from the observations in a population. Hence chi-square test is a Non Parametric test. Chi square (X2) test: A statistical test for determine the number of observations which is derived from those “expected” or “theoretical” number under a particular hypothesis. X2 = i = 1 ∑ (1 (O – E) 1 -1/2)2/E where ½ = 0.5 Yates correction O = observed frequencies E = expected frequencies Important characteristics of chi-square test • Chi square will be zero if each pair is zero and it might be assume any value extending to infinity, when the differences between the observed frequency and expected frequency in each pair are unequal. • Thus chi-square lies between O and ∞. • It is a statistics not a parameter. • It is always positive as each pair is squared up. Useful Points Hypothesis test It is the procedure which specifies a set of “rue for decision” whether to “accept” or “reject” the hypothesis under consideration (i.e., null hypothesis).

14.47. STUDENT “T” DISTRIBUTION TEST The t-test is used with small sample (n < 30) and it was worked out by W.S Gossett whose pen name was “student.” However the test is called as student t- test distribution. It may be defined as quantity representing the difference between the sample

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mean and true mean or population mean expressed in terms of the standard error. T = Difference between sample mean Standard error of the difference between means T = │X1 –X2│/SE where X1 and X2 = Mean SE = Standard error Type • Paired t-Test • Unpaired t-Test Condition for applying “t” test • Random sample are collected from normal population, • The population variances are regarded as equal for the testing the equality of two population means. • Samples are less than 30. • Some adjustments in degrees of freedom for “t” are made in case of two samples. “t” distribution properties • The “t” distribution curve varies with the degrees of freedom. • It is symmetrical distribution with mean zero. • It is asymptotic to X-axis, i.e., it extends to infinity on either side. • The graph is similar to that of normal distribution • It has greater spread than normal distribution. • The larger the number of degrees of freedom the more closely “t” distribution resembles standard normal distribution. “t” distribution applications • It observed sample correlation coefficient or differences between the mean of two sample. • The difference between two sample mean when the population variance being equal and unknown. • The single mean when the population variance is unknown. • Calculate the t- value: T = │X1 –X2│/SE X1 and X2 = Mean Sx1 and Sx2 = SD n1 and n2 = size of sample SE of (X1 – X2) = SD √ 1/n1 + 1/n2 SD = √ ∑ (X1 – X2)2 + ∑ (X2 – X2)2/ N1+N2 -2 • Determine the pooled degree of freedom from the formula df = (n-1) + (n2 -1) = n1+n2–2

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• Compare the calculated value with the table value at particular degrees of freedom. Example: There are 13 children were given a usual diet plus vitamins “A” and “D” tables. While the second comparable group of 12 children was taking the usual diet. After 12 months, the gain in weight in pounds was noted as given in the table. Can you say that Vitamins A and D were responsible for the difference? A

5

3

4

3

2

6

3

2

3

6

7

5

3

B

1

3

2

4

2

1

3

4

3

2

2

3

─

Solution: Null Hypothesis: Vitamins (A and D) are responsible for the gain weight difference Alternative hypothesis: Vitamins are not the responsible for the gain weight differences. S.no

Gr A (x)

(X –X) = D

(X-X)2 = D21

Gr B (Y)

(Y-Y) = D2

(Y-Y)2 = D22

1

5

5– 4 = 1

1

1

1 – 2.5 =- 1.5

2.25

2

3

3– 4 = -1

1

3

3 – 2.5 = 0.5

0.25

3

4

4– 4 = 0

0

2

2 – 2.5 = -0.5

0.25

4

3

3– 4 = -1

1

4

4 – 2.5 = 1.5

2.25

5

2

2– 4 = -2

4

2

2 – 2.5 = 0.5

0.25

6

6

6– 4 = 2

4

1

1 – 2.5 = -1.5

2.25

7

3

3– 4 = -1

1

3

3 – 2.5 = 0.5

0.25

8

2

2– 4 =-2

4

4

4 – 2.5 = 1.5

2.25

9

3

3– 4 = -1

1

3

3 – 2.5 = 0.5

0.25

10

6

6– 4 = 2

4

2

2 – 2.5 = -0.5

2.25

11

7

7– 4 = 3

9

2

2 – 2.5 = -0.5

0.25

12

5

5– 4 = 1

1

3

3 – 2.5 = + 0.5

0.25

13

3

3– 4 = -1

1

∑ 52

∑ D 1 = 32 2

─

0.25

∑ 30

∑ D22 = 11

GrA

GrB

n = 13

n = 12

X = 14

Y = 2.5

D21 = 32

D22 = 11

SD (A –B) = √∑ (X –X)2 + ∑ (Y-Y)2/ n1 + n2 – 2 = √ d21 + d22/ 13 +12 -2 = √ 32 +11/23 = √43/23

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SE = SD √ 1/ n1 + 1/ n2 =√ 43/23 ×b√ 1/13 + 1/12 = √43/23 × √12 +13/156 = √43/23 × √25/156 = √1.87 × √0.160 = 1.37 ×0.4 = 0.548 = 0.55 t = X – Y/ SE 4 -2.5/0.55 1.5/0.55 = 2.72 5% level of significance it means 0.05. Critical value is calculated for df 23 is 2.07. Decision (t) = 2.72 │ t│ = 2.72 > t0.05, 23 = 2.07.

14.48. Z-TEST The deviation from the mean in a normal distribution r curve is called relative or standard normal deviate and is given the symbol “Z.’ It is measured in terms of SD and indicates how many an observations is bigger or smaller than the mean in units of SD. So “Z” will be ration Z = Observations – Mean/SD = X-X/SD It is applying to the sampling variability and the difference between a sample estimate and that of population is expressed in terms of SE instead of SD. The score of the value ration between the observed difference and SE is called “Z.” Condition for “Z” • It must be quantitative. • It should be assumed to follow normal distribution. • It must be randomly collected the data. • The sample size must be larger than 30. Test of significance of differences between a sample mean (µ) and population mean (X) Z = X – µ/ SE (X) Test of significance and difference between the two sample means Z = X1 –X2/ SE (X1 – X2)

14.49. F-TEST OR FISHER’S F TEST

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The F – test was originated by RA Fisher. It is known as F-test. It is also called as variance ratio test as comparison of sample variance involves in this test. F –Test: hypothesis variances derived from two samples F = σ21/ σ22 Assumption of F-test: • It should be equal for all group. • It should be independent of each value. • It should be normally distributed. Uses of F-Test • The two independent estimates of the population variances are homogeneous. • Two independent samples have been drawn from the normal populations with same variances (σ2). • It is equality of population variances • It gives variance ration at different levels of significance at df = (n1–1). • F- ration value is smaller than the tale value so the null hypothesis is accepted • It indicates the samples are drawn from the same population. • It is used to calculate F Statistics.

14.50. T TEST One Tailed t Test The statistical hypothesis where either alternative hypothesis is one sided is called one tailed test or one sided test. • It may be right or left tailed test. • If we want to know one particular drug is than the other. • It will be one tailed test. Two-Tailed t Test It is a test of statistical hypothesis based on rejected region represented by both sides of the standard normal curve. Example: The nourished children or healthy children is different look from that of unnourished or unhealthy children. Example: The sample of mean of 1600 IQ level children was 99. It is likely that this was a random sample from a population with mean I.Q 100 and standard deviation is 15. Solution: Null hypothesis: the sample has not be drawn from the population with mean I.Q 100. Alternative hypothesis: sample has been drawn from the population hypothesis.

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Here n = 1600 X = 99 µ = 100 SD = 15 SE = SD/√n = 15/ √1600 = 15/40 = 0.375 │Z│ │X -µ│/ SE (X) = │99 -100│/0.375 = │2.67│ Critical value is not given so we take α = 0.05. Decision: calculated value of Z is 2.67 since │Z │ is 2.67 > Zα 1.96 So the null hypothesis is rejected, sample has not been drawn from population with mean 100 and SD is 15.

14.51. ANOVA (ANALYSIS OF VARIANCES) It is powerful statistical procedure for determining if differences in means are significant and for dividing the variance into components. Variance (σ2): It is an absolute measure of dispersion of raw scores around the sample (group) mean and the dispersion of the scores resulting from their varying differences (error terms) from the means. The square of the standard deviation is called the variance and is denoted by the (σ2). Mean Square: The measure variability are used in the analysis of variance is called a “Mean square.” Sum of square deviation from mean divided by degrees of freedom. Mean square = Sum of square deviation from mean/Degrees of freedom Assumption in the analysis of variances: It effects of various components are additive. It occurs random and it is independent of each other in the groups. In this the population is normally distributed with common variance. The samples are independently drawn. Technique for the analysis of variance One way ANOVA: The single independent variable is involved Eample: the effect of pesticides (independent variables) on the oxygen consumption (dependent variable) in a sample f insect. Two way ANOVA: The two independent variables are involved. Example: There are number of group of pesticides involve for the oxygen consumption of sample of insect. Procedure: • It is more convenient.

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It is based on the short cut method on the sum of the squares of the individuals values are usually used. The procedure of the calculation in direct method are lengthy as well as time consuming and this is not popular in practice for the all experiments.

14.52. NON-PARAMETRIC STATISTICS Parameter It is the numerical index or summary value like mean, median and standard deviation or variance of a variable for the entire population. Non-parametric test The test or methods are mathematical procedures concerned with the treatment of standard problems when the assumption of normality is replaced by general assumption concerning the distribution function. It is also called the distribution free. Parametric test The most commonly used statistical methods are called parametric because they are involved in testing the values of parameter (mean, median or standard deviation). Characteristics • It can be computed by very simple method. • It does not require normal distribution of the variables. • It can be used for very small sample. • It works out without using any pre-computed statistic as an estimate of parameter. • It can be done with very little assumption. Merits and demerits of non-parametric test Merits • It can be applied in all types of data. • It does not need pre-computed statistics. • It has a greater range of applicability. • It does not require laborious and lengthy calculations. • It is generally simple to understand and very easy to computed and applies. Demerits • The procedure has lack of power. • It is often wasteful of information and less efficient.

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• It sometimes pays for freedom from assumption. • This procedure has lack of power. Types of non-parametric test Mann-whitney u-test or rank sum test • The test is also known as U-test. • The method is used to determine the significant difference between the two independent sample groups. Kruskal-Wallis test or H-test • It is the rank dependent one way ANOVA and interpreted using critical chi-square values. • It is a kind of rank sum test. The sign test for paired data • It is discarded if the paired observation difference is zero. • It is based on the direction (+ or – sign) of a pair of observations and not on their numerical magnitudes. One sample run test • This test deals with the randomness with which the sample items have been selected. • It is based on the order in which the sample observations are obtained. Kolmo Gorove-Smirnov test • It is worked out to determine whether there is a significant difference between the observed frequency distribution and a theoretical distribution. • It is also known as K-test, i.e., another method of measuring goodness of fit of a frequency distribution. • It is more powerful and easier to apply. Kenoal test for concordance • It is used to test the significance of more than two sets of ranking individuals. • It is applicable when two sets of ranking individuals are available. Median test for independent sample The test is used to find out the significance of differences between means of two or more independent groups using a common median of those groups. Wilcoxon signed rank test It accounts for the magnitude of differences between paired values and not only their sign. It is useful in comparing the two populations.

14.53. IMPORTANT FORMULAS

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Average Formula: Let a1, a2, a3, a4, a5…….an be set of numbers = (a1+a2+a3+a4+a5….+an)/n Adding Formula: a/b+c/d = ad+bc/bd Subtracting formula: a/b – c/d = ad – bc /ad Multiplying fractions: a/b * c/d = ac/bd Dividing fractions: a/b/c/d = a/b / c/d = a/b * d/c = ad/bc

15 Software Used for the Analysis of Observations and Data

15.1. SIGMA PLOT Sigma plot is used for prepare graph, analysis of variance and most of presentation of data for analysis. This software can read multiple formats, we can directly paste data from excel to Sigmaplot worksheet and make graph easily.

15.2. SPSS (STATISTICAL PACKAGE FOR THE SOCIAL SCIENCES) It is a widely used program for statistical analysis in science and various research fields (market researchers, education researchers, and health researchers).

15.3. ORIGIN PRO SOFTWARE It is used for data analysis and graphing software. Nowadays origin software is used to create attractive graphs with different colors and more easy to other software. It is an easy-to-use interface for beginners, with high-quality graphing features. Graph qualities are much higher than above-mentioned software. Origin simply starts with a built-in graph template and then we can easily customize each and every element of a graph according to our need. We can easily add additional axes, panels or layers to graph page in origin. We can also save the settings of a graph as a custom template for their repeat use. It is column oriented which is not common for the above-mentioned software. Therefore, it provides several statistical analysis tools like descriptive statistics (basic statistics) and one-way and two-way analysis of variance (ANOVA),

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etc. You can also see in screen short below for easy to present data as more attractive diagram. Open the software Origin Pro

Maximize the data sheet so you will enter your data.

Now you found long data sheet for enter many values

If you want to increase the number of cell than click on “+” side which is under circle.

See the number of cell when you are click on “+”

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Enter the data on data sheet

Select cell for set as an “error” for one treatment

Click on cell foe “set as a error” and right click then you will find one new window and select as “set as”

Select “set as” then select “Y-error”

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Select cell for another treatment and same as perform before for first treatment

Now select all the data cell for make graph

Now go to graph symbol which is already given below the software line.

Choose any graph symbol and after that you will find graph on another sheet which is called graph sheet.

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After graph formation click on left side for set numbers according to your choice and bold them.

See in circle for set numbers and given data values

Language set as “time roman” see in circle how we shall make.

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After the data number set, select the title on left side “X” which is under circle.

So, you enter words what you want in text and set the language same as tell you before.

Now, after that we want to set the values which are given in below the line graph see in circle. Select the number and will do same as above.

We want to set text in box, then you have to select and then enter the words and click on enter button.

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See in circle.

Now as above given the name of treatment and select that box and write what you want in text.

See in circle, if you want that box set according to you on graph than select and move and place them.

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Now you want to increase the width of error symbol than you have to click on that and set size and click on “OK” button which is give below line of window, see in circle.

See in circle of error width.

Now you want to set bar size and change color than click on bar, select group, select independent, select spacing, pattern and change according to you.

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See in circle

See in circle, if you want to change in color, then select the color and click on “OK”

If you also want to change in style of bar than click on “None” and select the style of bar and click on “Ok,” See in circle

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If you want to increase width of bar size than click on width and select the size number see according to red line.

Now you will find attractive graph and then you want to copy that graph, go for “Edit” and choose “Copy Page”

Now you want to graph with jpg picture than you have to go on “File,” then “Export graphs,” then “Open dialog”

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After that, new window open see in circle

Select “jpg” see according to red line

Set pixel and then click on “Ok” button now, you go to document file in computer, open origin file, select “user file” and now you will find picture. Take that picture and paste where you want example in Microsoft window or Microsoft power pint slide.

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After set as picture then you find new open window and choose “Hide” see in circle

Now, if you want to change style in bars, follow above given steps.

See another attractive graph and make according to red lines.

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Now you will find graph which is “jpg form.” See attractive graph

Now you want to do data analysis to check your data will significant or not significant. You have select “analysis,” which is give above side on window and then descriptive statistics, then correlation coefficient after that click on “OK” which is give in circle.

Now you will find report of your data and analysis to check the valve of significant and set according to you. Same as above you also do “ANOVA, T-test” and many other statistics apply on your data.

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After that, you want to save origin file then you will go “File” select “Save project as”

Get new window then write a file name and then save where you want for further analysis.

Figure 15.1. Screenshot represents the Origin Pro software

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Important Notations Symbols α

A Ս B A ∩ B Ac B (n, p) X2k X2k, ß (R) X2k, ß (L) C1 C2 C3 C4 C5 C6 Ս

Fl1.l2, Fl1.l2, ß HA H0 µ µX

Mk (n, p1,……,pk)

N Rx σ

∑ σx

X1, X2, …Xn

Notation It uses to denote the level of significance for hypothesis testing problem. Union of events A and B Intersection of events A and B Complement of event A Binomial experiment where n = number of trials and p = probability of success in a single trial. Chi-square distribution with k degrees of freedom (df) 100 (1- ß)th percentage point of the X2k distribution. 100 ß th percentage point of the X2k, distribution. Factor for control limits of X-chart based on range. Factor for limits of X-chart based on standard deviation. Factor for lower control limit of R-chart Factor for upper control limit of R-chart Factor for lower control limit of s-chart Factor for upper control limit of s-chart Empty event (set) or Null event (set). (Snedecor’s) F-distribution (curve) with l1 and l2 degrees of freedom (df). 100ßth percentage point of the Fl1.l2-distribution (curve) Alternative hypothesis Null hypothesis Mean of probability distribution (or a population) Expected value of a random variable X Multinomial experiment where n = number of trials and pi probability of getting the ith outcome in a single trial with p1 + p2 + …+ pk =1. Population size Range of random variable X Standard deviation of a probability distribution (or a population). Stands for summation of certain quantities. Standard deviation of random variable X. Observation in a raw dataset.

INDEX A Absorbance 261, 277, 279, 281 Acetobutylicum 5, 10 Actinomycetes 6, 20, 21 Agricultural chemical 4 Alpha diversity 41 Aluminum 253, 254 Aminobenzyloxymethyl 90 Anoxyphotobacteria 20 Antaradiography 159 Antibiotic penicillin 30 Antidigoxigenin fluorescein 86 Antirabies virus 177 Aquatic application 4 Aquatic plants 229 Archaea 15, 22 Archaebacteria 19, 21 Archaeobacteria Bacteria 21 Autoradiography 85, 90, 92, 96, 106

B Bacillus anthracis 15, 26 Bacterial cells 181 Bacterial taxonomy 20 Bacteriology 18, 21

Bacteriophages 136 Bacterium 261, 265, 273 Baculovirus 144 Biofertilizers 31, 40 Biology 1, 3, 6, 8, 9, 10, 11, 16, 29, 31, 32, 33, 34, 35, 36, 37 Biopesticides 31, 40, 144 Bioremediation 9, 16, 34, 40 Biotechnology 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 26, 27, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 Biotinylated 85 Bovine Somatotropin (BST) 2 Bradford method 275

C Cell suspension 262, 263 Cellular machinery 24 Center for Cellular and Molecular Biology (CCMB) 167 Central Electricity Authority 228 Chamberland 26 Chemiluminescent 84, 86 Chlorophyll 228

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Chromosome 56, 58, 60, 69, 70, 175, 183 Cladosporium 35 Clostridium 5, 10 Commercial seed production 49 Complementary DNA (cDNA) 95 Complex compound 215 Complex relationship 10 Consultative group on International Agricultural Research (CGIAR) 46 Corticotropin 176 Corynebacterium 26, 29 Cosmids 139 Coxsackie 16 Culture manipulation 190 Culture media 251, 254, 257 Custom template 401 Cytochemical 85, 86 Cytokinin 202 Cytoplasm 205, 208 Cytoplasmic genetic information 207 Cytosine monophosphate (CMP) 57

D Dansyl chloride 282, 283 Deoxyadenosine 52 Deoxyadenosine triphosphate (dATP) 52 deoxyguanosine triphosphate (dGTP) 52 Deoxynucleotides 52 Deoxynucleotide triphosphates (DNTPS) 152 Deoxyribonucleotide 51, 52, 85 Deoxyribotids 51 Department of Science and Technology (DST) 36

Digoxigenin 86, 163 Disinfection 251

E Ecogeographica 43 Ecological hazard 9 Ecotropical 15 Elcctrophoresis 98 Ellipsoidal structure 56 Elongation 198, 202 Embryogenic 204, 205, 211 Embryogenic cells 204, 205 Embryology 32 Endonucleases 76, 77, 91, 95 Endophytic 15 Energy sources 228, 229 Enzymable methods 193 Enzyme hypothesis 58 Enzymes 1, 2, 5, 7, 14, 16, 30, 31 Enzyme technologies 5 Equal volume 262 Ethanol precipitation 271, 272 Ethidium 268, 271 Ethidium bromide 77 Eukaryotic 15, 21, 22, 23 Eukaryotic chromosomes 56 External application 4 Extrachromosomal 132

F Fermentation medium 25 Fermentation process 5, 28 Financial resources 9 Firmicutes 20

G Galactosidase fusion proteins 138 Gasoline 5 Gene cloning 150, 153, 164, 167

Index

Genetically modified organism (GMO) 7 Genetic engineering approval committee (GEAC) 244, 245 Genetic manipulation 6 Genetic material 16, 19, 22, 29, 40, 46, 51, 54, 57, 63, 123, 127 Genomic 150, 151 Gentotypes 205 Germplasm 40, 43, 45, 49 Global partnership 47 Graphing software 401 Greenhouse gas 3 Guanosine monophosphate (GMP) 57

H Haemophilus 75 Halobacterium salinarum 55 Hamberlandt 196 Hazard 40 Helminths 94 Hemophilia 180 Heterocyclic 51 Heterogeneous mixture 153 Heterokarytic 207 High fructose corn syrup (HFCS) 6 Homopolynucleotides 53 Human genome 179 Human globin genes 180 Human growth 2, 30 Human hormone 2 Human welfare 10 Hybrid heterosis 205 Hybridization technique 96 Hybridized unhybridized 184 Hydrocarbon 231, 232 Hydrochloric acid 278

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Hydrogen bond 54, 57 hypothalamus 175, 176

I Immunoassays 87 Immunoglobulins 182 Indole acetic acid (IAA) 198 Institutional biosafety committees (IBSCs) 244 Interdisciplinary synthesis 31

L Laboratory 251, 253, 261, 273, 284 Lactoserum 231 Lignocellulosic 231, 233 Liquid medium 203, 204, 210, 211 Logarithmic value 261

M Mammalian cells 187 Mathematical function 297 Matrix interaction 188 Mediapreapartion 198 Mendosicutes 20, 21 Mericlones 203 Meristematic 202 Meristemming 203 Mesenteroides 5 Metabolism 63 Metallic 252 Metalloenzymes 236 Method human embryos 31 Microbes 14, 15, 16, 251, 262, 265 Microbial cells 63 Microbial fermentation 3, 6 Microbial growth 19 Microbiology 1, 11, 15, 16, 18, 25, 26, 27, 28, 31, 38

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Microorganism 1, 2, 4, 5, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 25, 27, 33, 34, 40 Microsatellites 69, 70, 72 Microscopic organism 18, 23 Mineral leaching bacteria 5 Molecular 3, 6, 9, 10, 11, 16, 17, 31, 32, 36, 37 Molecules 54, 56, 57, 60, 62, 63, 65 Monoclonal antibodies 87 Monosomics 92 Morophology 20 Multidisciplinary strategies 31 Multiple cloning site (MCS) 139 Myceteae 19 Mycology 22

N National Biotechnology Board (NBTB) 36 National Hydroelectric Power Corporation (NHPC) 228 Nomenclature 15, 19 Nonphotosynthetic 20 Nuclear energy 227 Nuclear membrane 19, 22 Nucleic acid technology 85 Nucleotides 53, 54, 57, 65, 69, 70 Nucleotides undergo 53 Nutrient medium preparation 197

O Obligation 48 Organic acid 5, 14 Organism 2, 4, 6, 7, 11, 15, 19, 26, 28, 33, 35 Oxyphotobacteria 20

P Partnership 38, 48 Pathogenicity 26, 28 Peptidoglycan 21 Phamacogenomics 35 Phamacologic 35 Phosphate 51, 52, 53, 61 Phosphodiester 51, 54, 56, 61 Phosphogluconate 221, 222 Phosphonic 278 Phosphorus 198 Photosynthetic eukaryotes 23 Phylogeny 19 Physiological function 58 Phytochrome gene 68 Phytosanitary 45 Phytostabilization 235 Plant breeders rights (PBRs) 48 Plant genetic resources (PGR) 47 Plant product 45, 48 Plant Protection 49 Plant protection Advisor 49 Plant Quarantine (PQ) 49 Plant variety rights (PVR) 48 Plasma membrane 238 Plasmids 132 Polyacrylamide gel electrophoresis (PGE) 90 Polycistronic 59 Polylinker 132, 133, 138, 139, 144 Polymerase chain reaction (PCR) 64, 87, 106 Polymeric compound 51 Polypeptide chain 59 Polypeptide hypothesis 59 Population dynamics 9 Productivity 10, 47 Prokaryotes 19, 20

Index

Protein fragment 89 Proteogenomics 35 Protoplast 205 Protozoa 15, 18, 19, 24 Protozoan 181 Protozoology 18 Pteridophyta 229 Pulsed field gel electrophoresis (PFGE) 88, 98 Pyrimidines 51, 53, 57

Q Quantitative information 295

R Rabis virus 177 Restriction enzyme 75, 77, 79, 80, 83, 88, 89 Restriction fragment length polymorphisms (RFLPs) 78, 92 Reverse transcriptase 152 Review committee on genetic manipulation (RCGM) 245 Rhizogenes 136 Ribonuclease 61 Ribonucleoprotein 60 Ribonucleotides 53, 57

S Saccharomyces cerevisiae 5 Salt concentration 271, 272 Scotobacteria 20 Scquence 97 Semiconservative mechanism 54 Shibasaburo 26, 29 Shikonin 196 Sigma plot 401 Sigmoidal growth 262

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Simple sequence repeats (SSR) 69 Single-cell protein (SCP) 6 Society organization 48 Sodium dodecyl sulfate (SDS) 263 Sodium hydroxide 14, 27 Sodium molybdate 278 Somatic hybridization technique 207 Somatostatin 2, 33 Spliceosome 60 Spontaneous 18, 19 Spontaneously 18, 19 Sporogenous 196 Streptomycetes 81 Subclone 97 Synthesized 2, 5 Synthetic medium 25, 28 Systematic Bacteriology 20

T Tatum proposed one-gene-one protein hypothesis 61 Taxomyces andreanae 15 Technology Information Forecasting and Assessment Council (TIFAC) 37 Tertracycline 156 Tetrahymena 61 Thermococcus litoralis 65 Thermophila 61 Thermoplasma 55 Tissue Plasminogen Activator (TPA) 5 Tobacco mosaic virus (TMV) 141 Totipotency 195 Toxicogenomics 35 Transformation 3, 150, 165, 166 Transgenic organisms 9 Transition Period 18

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Transposase enzyme 142, 146 Triphosphate 52, 53, 57, 65

U Ultramicroscopic 15, 23 United Nations Industrial Development Organization (UNIDO) 37

V Vector system 2 Vigorous growth 8

W Water hyacinth 230