Protocols in Livestock Genome Analysis 9789381226841, 9381226849

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PROTOCOLS IN LIVESTOCK GENOME ANALYSIS

PROTOCOLS IN LIVESTOCK GENOME ANALYSIS Authors Anuj Chauhan Subodh Kumar Arvind A. Sonwane Deepak Sharma Division of Animal Genetics Indian Veterinary Research Institute Izatnagar-243 122 (U.P.)

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About the Book The book Protocols in Livestock Genome Analysis Presents the Fundamental principle and detailed protocols of laboratory techniques with an adequate amount of thought been applied towards making it simplistic and effective. The book has been structured into seventeen chapters. It begins with Chapter 1, givingreaders some basic safety and precautions while working in molecular genetics laboratory that is exposed to hazardous agents. Chapter 2, 3 and 4 explains the most basic and fundamental procedures in molecular genetics laboratory i.e. isolation of genomic DNA, plasmid DNA, total RNA which are prerequisite for researchers conducting downstream genetics research like gene amplification or expression. Chapter 5 deals with quantification of DNA and RNA with specific examples. Chapter 6 deals with technique of polymerase chain reaction for amplification of a specific DNA sequences while Chapter 7 focuses on various variants of polymerase chain reaction that have been developed over the years for specific applications. Chapter 8 discusses the reverse transcriptionpolymerase chain reaction which is important prerequisite for gene expression studies and genetic characterization. Chapter 9 describes at length, agarose gel electrophoresis for the separation of nucleic acids and their analysis. Chapter 10describes single strand conformation polymorphism, a technique that is capable of identifying most sequence variations in a single strand of DNA. Chapter 11 details the restriction digestion of DNA molecules by restriction endonucleases. Chapter 12 describes real-time PCR that can detect their accumulation and quantify the number of substrate DNA molecules present in starting sample. Chapter 13 discusses the method of chromatin immunoprecipitation, a very important technique for elucidating the transcriptional regulation of gene expression. Chapter 14 deals with detailed analysis of microsatellite repeat sequences along with statistical tools for data analysis. Chapter 15 explains the very important procedures of DNA ligation,

competent cells preparation and transformation involved in cloning of any desired DNA sequence. Chapter 16 describes the set of steps for retrieving QTL & SNP information from online Animal QTL databases. Chapter 17 provides a simple understanding of DNA microarrays. Chapter 18 details the in situ hybridization method for localization of genetic material. Chapter 19 provides the recipe for preparation of buffers and solutions required for conducting various procedures outlined in previous chapters.

About the Authors Veterinary Research Institute, Izatnagar since past five years. The author is involved in post graduate teaching and research in the institute and has successfully completed projects on understanding key transcriptional factor binding sites in DNA governing immune response in cattle and gene therapy procedures in cattle results of which have been presented at prestigious scientific forums. He is currently involved in two ambitious projects aimed at finding predisposing DNA markers for tuberculosis and brucellosis in cattle. He is also associated with DBT sponsored program for upliftment of SC/ST communities through introduction of improved swine germplasm. Dr. Subodh Kumar is Senior Scientist at the Division of Animal Genetics in the Indian Veterinary Research Institute, Izatnagar. He has research and teaching experience of more than 15 years in the field of Animal Genetics and Breeding and has guided numerous Post Graduate and Doctoral degree students. He has 41 research papers and 104 GenBank submissions to his credit. He has successfully completed 8 research projects in various capacities. Presently, he is instrumental in discovering and exploring genes that are responsible for influencing sperm motility in cattle by the fund provided by the prestigious National Agricultural Innovation Project (NAIP). Dr. Arvind Sonwane is a Scientist (Senior Scale) at the Division of Animal Genetics in the Indian Veterinary Research Institute, Izatnagar for the past five years. With specialization in field of Animal Biotechnology, he is involved in post graduate teaching and research in the institute. He has successfully completed a key research project on Gene therapy in cattle with promising results and was also associated in projects on miRNAs profiling and finding transcription factor binding sites. He is currently involved in an ambitious project aimed at finding predisposing DNA markers for brucellosis in cattle. The author has been a key resource person facilitating the post

graduate research of a number of master’s and doctorate students passing out from the division. Dr. Deepak Sharma is Head, Division of Animal Genetics at the Indian Veterinary Research Institute, Izatnagar. He possesses vast experience in the field of Animal as well as Avian Genetics research and teaching. He has 130 refreed research publication and 8 books to his credit. He has also been instrumental in developing of three improved varieties of guinea fowl viz. Shwetambari, Chitambari, Kadambari, and two of egg type germplasm. He is recipient of prestigious ICAR Team Research Award for Biennium 1997-98 and Prof. Ramraksha Kiran Shukla Award. He is also member of several esteemed committees including DST’s National Awards committee for R&D Efforts in Industry and ISI committee for Poultry Housing and Management and Feed, ISI, New Delhi. He also acts as Editor to some prestigious research journals of the subject.

CONTENTS Foreword Preface Acknowledgement 1 Laboratory Safety and Precautions 2 Isolation of Genomic DNA 3 Isolation of Plasmid DNA 4 Isolation of Total RNA 5 Quantification of Nucleic Acids 6 Polymerase Chain Reaction 7 Variants of Polymerase Chain Reaction 8 Reverse Transcription-Polymerase Chain Reaction (RT-PCR) 9 Agarose Gel Electrophoresis 10 Single Strand Conformation Polymorphism (SSCP) 11 Restriction Digestion 12 Real Time PCR 13 Chromatin Immunoprecipitation Assay 14 Microsatellite Analysis 15 DNA Ligation, Competent Cells Preparation and Transformation 16 Retrieving QTL & SNP Information from Databases 17 DNA Microarray

18 In situ Hybridization 19 Preparation of Required Buffers and Solutions

Foreword Analysis of the genetic determinants of livestock production traits now relies heavily upon the immense research capabilities embodied in Molecular Biology. These entail elucidation of the DNA sequences that comprise the genes themselves and detailed examination of the function of genes (gene expression) and their products, in the tissues and cell types where they are active. Therefore, in the present era of accelerated scientific discovery, it has become imperative on the part of all stakeholders to keep themselves abreast of novel technologies developed in order to harness their full potential for livestock production and health as well. Division of Animal Genetics at IVRI has been serving as a leading National Centre in its field, and is progressing with a vision for enhancing animal production through genetic improvement of cattle, buffalo and other livestock species for better disease resistance/resilience as well as development and use of genetic markers for the improvement of fertility. The impressive track record of the Division in the areas of research and development, PG teaching, human resource development and consultancy services has been acknowledged in the form of prestigious National projects as well as awards given for the outstanding performance of the dedicated Faculty and meritorious students. It gives me immense pleasure to know that the committed scientists of Animal Genetics Division at this premier research institute have brought out a useful book entitled “Protocols in Livestock Genome Analysis” authored by

Drs Anuj Chauhan, Subodh Kumar, Arvind Sonwane and Deepak Sharma. I believe that this book would contribute significantly towards dissemination of scientific knowledge and serve as a valuable guide for researchers/teachers/postgraduate students working in the core domain and allied disciplines alike. On behalf of the entire IVRI family, I convey my appreciation and greetings to the dedicated scientists in the Faculty for taking this initiative of knowledgesharing for knowledge-gain in this emerging area of immense importance and urge the IVRI scientific fraternity to take more such initiatives in the interest of the profession, institute and the council, with an ultimate aim to benefit the stake holders in national and global community. (Gaya Prasad)

PREFACE The key motivation for writing this book was to offer researchers/teachers/postgraduate students, a practical guide on the important laboratory protocols frequently employed in molecular level research in the field of Animal Genetics. In the last two decades, genomics and biotechnology revolution has created a set of applied DNA-based technologies that have become available as a tool in hands of stakeholders to aid in making their selection decisions. Research is in progress to identify regions of DNA that influence expression of economic traits pertaining to production, reproduction and health etc. in livestock species. This book seeks to provide knowledge and expertise in hands of stakeholders about the techniques that are being currently used to carry forward such researches. Although literature is available that covers many molecular biology protocols, but we felt an evident requirement for a complete instruction manual that imparts knowledge on the principles and protocols of important and frequently utilized molecular laboratory techniques in exploring the livestock genome accurately in a user friendly style. The book provides step by step protocols and tips for uninterrupted flow of work in the laboratory. The most important part in this book is the Trouble shooting, which is usually not found in other books on similar lines. This aspect has been carefully written for all the chapters/ techniques and is largely based on our personal experiences. Though we have made every attempt to cite the most important publications in the concerned area, with emphasis on papers, books and electronic publications, still we realize that omissions and errors are very difficult to avoid and hence, apologize for any possible mistake. Authors

Acknowledgement We are pleased to extend our sincere appreciation and thanks to Dr. Gaya Prasad, Director, Indian Veterinary Research Institute for his valuable guidance and support. We acknowledge the assistance, encouragement and constructive counsel provide by the faculty of Division of Animal Genetics. We also wish to place sincere thanks to other scientific, technical, administrative, ministerial and supporting staff Division of Animal Genetics for their cooperation and assistance. All grace of Almighty God is thankfully acknowledged. Authors

CHAPTER-1 Laboratory Safety and Precautions In a molecular biology laboratory, there is a certain hazard associated with the use of a variety of chemicals and materials. One should learn and adhere to the general safety guidelines to ensure a safe laboratory environment. The risk involved in handling potentially hazardous laboratory reagents required for performing various molecular biology protocols and ways to counter them are outlined in this chapter along with the importance of wearing personal protective equipments.

Handling Laboratory reagents Acrylamide (unpolymerised) Is a potent neurotoxin and is absorbed through skin (effects are cumulative). Use gloves and a face mask when weighing. Ammonium persulfate Ammonium persulfate is extremely destructive to tissue of the mucous membranes and upper respiratory tract, eyes and skin. Inhalation may be fatal. Wear appropriate gloves, safety glasses, and protective clothing. Always use in a chemical fume hood. Wash hands thoroughly after handling. Chloroform Chloroform is irritating to the skin, eyes, mucous membranes, and respiratory tract. It is a carcinogen and may damage the liver and kidneys. It is also volatile. Avoid breathing the vapours. Wear appropriate gloves and safety glasses. Always use in a chemical fume hood.

Ethidium bromide Ethidium bromide is a powerful mutagen and is toxic. Avoid breathing the dust. Wear appropriate gloves when working with dye solutions. Formaldehyde (HCHO) It is highly toxic and volatile chemical. It is also a carcinogen. It is readily absorbed through the skin and is irritating or destructive to the skin, eyes, mucous membranes and upper respiratory tract. Avoid breathing the vapours. Wear appropriate gloves and safety glasses. Always use in a chemical fume hood. Keep away from heat, sparks and open flame. Phenol Phenol is extremely toxic, highly corrosive, and can cause severe burns. It may be harmful by inhalation, ingestion, or skin absorption. Wear appropriate gloves, goggles, and protective clothing. Always use in a chemical fume hood. Rinse any areas of skin that come in contact with phenol with a large volume of water and wash with soap and water. Polyacrylamide Is considered nontoxic, but it should be handled with care because it might contain small quantities of unpolymerised acrylamide. Silver nitrate (AgNO3) Strong oxidising agent and should be handled with care. It may be harmful by inhalation, ingestion, or skin absorption. Avoid contact with skin. Wear appropriate gloves and safety glasses. It can cause explosion upon contact with other materials. SDS (sodium dodecyl sulphate) SDS is toxic, an irritant, and poses a risk of severe damage to the eyes. It may be harmful by inhalation, ingestion, or skin absorption. Wear appropriate gloves and safety goggles. Do not breathe the dust. Sodium hydroxide (NaOH) Highly toxic and caustic and should be handled with great care. Wear

appropriate gloves and a face mask. All concentrated bases should be handled in a similar manner. TEMED (N,N,N’,N’-tetramethylethylenediamine) TEMED is extremely destructive to tissues of the mucous membranes and upper respiratory tract, eyes and skin. Inhalation may be fatal. Prolonged contact can cause severe irritation or burns. Wear appropriate gloves, safety glasses, and other protective clothing. Use only in a chemical fume hood. Wash thoroughly after handling. It is flammable; vapour may travel a considerable distance to source of ignition and flash back. Keep away from heat, sparks, and open flame.

Personal Protective Equipments Protective material should always be worn when performing experiments in molecular biology laboratory. Eye Protection : Wear safety glasses when performing for e.g. phenol : chloroform extraction, even when working in a fume hood. Phenol can cause severe burns to the eyes. Wear chemical splash goggles when there is a splash hazard. Wear UV protection during visualization of gel. The UV radiation cause injury with exposures as brief as three seconds in duration Gloves: Wear disposable nitrile gloves to protect against accidental hand contact. If accidental contact occurs, remove and discard contaminated gloves immediately. (The breakthrough time for a 4 mil nitrile glove is approximately 3 minutes for chloroform.) Protective Clothing : Wear standard laboratory apparel.

CHAPTER-2 Isolation of Genomic DNA The collection of pure high molecular weight genomic DNA from whole blood or cell cultures is a key starting step for researchers conducting downstream research such as PCR amplification or restriction digests. Blood is a complex mixture of cells, proteins, metabolites, and many other substances. About 56% of blood volume is comprised of cells, more than 99% of which are erythrocytes. Human and livestock species (except poultry) erythrocytes and thrombocytes (platelets, 0.5% of blood components) do not contain nuclei and are therefore unsuitable for preparation of genomic DNA. The only blood cells that contain nuclei are leukocytes (0.3% of cellular blood components). For the purpose of DNA isolation, Leukocytes are the cells we are interested in. There are many protocols for isolation of genomic DNA from blood samples including Salting out and DNA precipitation, Extraction with Organic Solvents like phenol, chaotropes, and DNA precipitation, Silica Resin-Based Strategies, anion exchange Based Strategies, and Cesium chloride density gradients. Salting-out is a conventional technique where proteins and other contaminants are precipitated from the cell lysate using high concentrations of salt such as potassium acetate or ammonium acetate. The precipitates are removed by centrifugation, and the DNA is recovered by alcohol precipitation. Using a cesium chloride (CsCl) density gradient, genomic DNA can be purified by centrifugation. Cells are lysed using a detergent, and the lysate is alcohol precipitated. Resuspended DNA is mixed with CsCl and ethidium bromide and centrifuged for several hours. The DNA band is collected from the centrifuge tube, extracted with isopropanol to remove the ethidium bromide, and then precipitated with ethanol to recover the DNA. Solid-phase anion- exchange chromatography is based on the interaction between the negatively charged phosphates of the

nucleic acid and positively charged surface molecules on the substrate. DNA binds to the substrate under low-salt conditions, impurities such as RNA, cellular proteins, and metabolites are washed away using medium-salt buffers, and high- quality DNA is eluted using a high-salt buffer. The eluted DNA is recovered by alcohol precipitation. Silica based method is based on the selective adsorption of nucleic acids to a silica-gel membrane in the presence of high concentrations of chaotropic salts. The protocol here describes the 'Phenol: chloroform extraction' method as described by Sambrook et al. (1989) with slight modification.

Principle of Phenol : chloroform extraction A phenol : chloroform extraction is a liquid-liquid extraction. A liquidliquid extraction is a method that separates mixtures of molecules based on the differential solubilities of the individual molecules in two different immiscible liquids. First, a volume of phenol is added to the aqueous mix containing the proteins and the DNA to be purified. Since phenol and water are immiscible, two phases form - a water (aqueous) phase and a phenol phase. DNA is polar because of its negatively charged phosphate backbone, and therefore is soluble in the upper aqueous phase instead of the lower organic phase (water is more polar than phenol). Conversely, proteins contain varying proportions of charged and uncharged domains, producing hydrophobic and hydrophilic regions. In the presence of phenol, the hydrophobic cores interact with phenol, causing precipitation of proteins and polymers (including carbohydrates) to collect at the interface between the two phases (often as a white flocculent) or for lipids to dissolve in the lower organic phase. Phenol is the denser of the two liquids so it sits on the bottom. Purified phenol has a density of 1.07 g/cm3 and therefore forms the lower phase when mixed with water (1.00 g/cm3). Chloroform ensures phase separation of the two liquids because chloroform is miscible with phenol and it has a higher density (1.47 g/cm3) than phenol; it forces a sharper separation of the organic and aqueous phases thereby assisting in the removal of the aqueous phase with minimal cross contamination from the organic phase. The phases are then mixed thoroughly. This forces the phenol into the water layer where it forms an emulsion of droplets throughout. The proteins in the water phase are denatured and partitioned into the phenol, while the DNA stays in

the water. The mixture is then centrifuged and the phases separate. The DNAcontaining water phase can now be pipetted off, and the phenol/protein solution is discarded. Commonly, the DNA is then de-salted and concentrated using ethanol precipitation.

Requirements Reagents and solutions Π RBC Lysis buffer (1x) DNA extraction buffer: Sodium dedecyl sulphate (SDS) Proteinase-K Tris saturated phenol Phenol : chloroform : isoamyl alcohol Chloroform : isoamyl alcohol 3M Sodium acetate Isopropanol Ethanol TE (pH 7.8) (optional) Equipments & labware Centrifuge Water bath Weighing Balance Micropipettes and tips Polypropylene and Eppendorf tubes Gloves

Procedure Collection of blood : In sterile polypropylene vials containing 0.5 M EDTA (0.5 ml/10 ml of blood) as an anti-coagulant, 5 -10 ml of blood is collected from each animal from the jugular vein under sterile conditions. After collection of blood, cap the vials tightly and mix gently to facilitate thorough mixing of blood with the anti-coagulant. Keep the blood vials in icebox containing ice/ gel cool packs till transported to the laboratory.

Samples are processed immediately for DNA isolation/ stored in deep freeze at -20°C till the isolation of DNA. The steps followed for genomic DNA isolation from the samples are as follows: 1. Transfer the fresh/thawed blood samples to 50 ml polypropylene centrifuge tube and centrifuge @3000 rpm for 20 min at room temperature. 2. Discard the reddish tinged supernatant, containing plasma and lysed RBC by careful pipetting. 3. Mix the pellet containing WBC and RBC with more than 2 volume of chilled RBC lysis buffer and keep in ice for 10 min after gently mixing it end to end, once or twice. 4. Again centrifuge @ 3000 rpm for 15 min at room temperature and discard the black tarry coloured supernatant, containing lysed RBC by pipetting. 5. Repeat step 3 and 4 times till the WBC pellet becomes free of the reddish tinge. 6. Once a clear 'Off white' coloured pellet of WBC is obtained, add DNA extraction buffer @ 3 ml per 10 ml blood and vortex to gently disperse the WBC pellet in the extraction buffer. 7. Incubate the WBC pellet mixed with DNA extraction buffer at 37°C for 30 min. 8. Subsequently add 10% SDS @ 200 μ! per 10 ml blood and mix gently by inverting the tube once or twice. Care should be taken while mixing, because after adding SDS, lysis of cell wall occurs and DNA now lies fully-exposed. As a result the contents of the tube now become viscous. 9. Add Proteinase-K (20 mg/ml solution) @ 20 μl /10 ml of blood, in two pulses. Add half of the requirement in first pulse and mix the contents of the tube gently, end-to-end and incubate at 50°C for 3-4 hrs. This is followed by the second pulse of remaining amount and overnight incubation at 50°C. 10. On the 2nd day, add equal volume of Tris-saturated phenol (pH >7.8) to the tube. 11. Keep the tubes on shaker and apply gently rotatory movements for 15 min to mix the contents thoroughly. 12. Subsequently, centrifuge the tubes @ 4000 rpm for 20 min at room

13.

14.

15.

16.

17.

18. 19. 20. 21. 22.

temperature. After centrifugation, the contents of the tubes gets separated in to two layers, the upper aqueous layer (containing DNA) and a lower heavier layer of phenol (containing proteins). A white, thin layer, of protein is also visible at the interphase of the two layers; transfer the upper aqueous phase to another 15 ml polypropylene tube with the help of 1 ml wide bore (3 mm diameter) micro tip. Ensure that the protein interface layer is not disturbed. Perform similar extraction (as in steps 10-13) once with phenol; Chloroform: Isoamyl alcohol (25: 24: 1) and once with chloroform: Isoamyl alcohol (24: 1). Finally, put the aqueous phase in a 50 ml polypropylene centrifuge tube and add 3 M sodium acetate @ 100 μ! per ml of aqueous phase to it and mix gently. Add more than 2 volumes of chilled isopropanol to the tube and mix gently by swirling the tube once or twice. Leave the tube at room temperature to allow the precipitation of DNA. Transfer the precipitated DNA into a sterile 1.5 ml micro¬centrifuge tube (using the wide bore micro tip of 1 ml capacity) along with 500 μl isopropanol and centrifuge 10,000 rpm for 10 min at room temperature. Discard the supernatant without disturbing DNA pellet. Wash the DNA pellet twice with 70% ethanol, similar to step 17 and 18. Finally, air dry DNA pellet for 1 h to remove traces of ethanol and subsequently dissolve in 200 μl TE buffer. Incubate the microcentrifuge tubes in water bath at 60°C for 2 h to inhibit DNAse activity and to dissolve pellet properly in the TE buffer. After 2 h of incubation, let the DNA to cool and stored at -20°C for further use.

Storage of DNA Isolated DNA may be stored at-20°C or-70°C in TE buffer/ DNase- free water. Tris-based buffers provide a safe pH of 7 to 8 and will not generate free radicals.

Integrity of DNA

The integrity and size distribution of total DNA can be checked by agarose gel electrophoresis and ethidium bromide staining. For details please refer to Protocol on Agarose gel electrophoresis.

Troubleshooting Problem

Possible cause

Suggested solution

Poor yield of nucleic acid

Poor phase separation

After the first aqueous phase has been transferred, add an equal volume of TE (pH 7.8) to the organic (phenol) phase and interface. Mix well. Separate the phases by centrifugation@ 4000 rpm for 20 min, extract aqueous phase and mix this second aqueous phase with the first, and proceed to next step

Phase High salt inversion concentrations - where aqueous phase is no longer on top Shearing

Diluting the aqueous phase and increasing the amount of phenol will correct this inversion

Excess Use large bore tips to transfer the DNA vortexing/pipetting from one tube to another. Avoid vortexing, repeated pipetting (especially through low- volume pipette tips)

References 1. Ausubel F.M., Brent R., K., R.E., Moore D.D., Seidman J.G., Smith J.A.and Struhl, K. (1987). Current Protocols in Molecular Biology, Volume 1, Wiley Interscience. 2. www.molecularstation.com/dna/genomic-dna-isolation/ 3. O'Neil, M. (2006). The Merck Index, Fourteenth edition. Merck & Co.

New Jersey. 4. Sambrook J. and Russell D. W. (2001). Molecular Cloning: A Laboratory Manual, Third edition, Cold Spring Harbour Laboratory Press, New York. 5. Sambrook J., Fritsch E.F. and Maniatis T. (1989). Molecular Cloning: A Laboratory Manual. Second edition. Cold Spring Harbor Laboratory Press, New York.

CHAPTER-3 Isolation of Plasmid DNA A plasmid is a small, circular, extra chromosomal double-stranded DNA molecule that is distinct from a cell's chromosomal DNA. Plasmids naturally exist in bacterial cells, and they also occur in some eukaryotes. Often, the genes carried in plasmids provide bacteria with genetic advantages, such as antibiotic resistance. Plasmids have a wide range of lengths, from roughly one thousand DNA base pairs to hundreds of thousands of base pairs. When a bacterium divides, all of the plasmids contained within the cell are copied such that each daughter cell receives a copy of each plasmid. Bacteria can also transfer plasmids to one another through a process called conjugation. Plasmids replicate independently of the bacterial chromosome and can exist in multiple copies in a single bacterial cell. Many plasmids have been isolated and analysed in recent years. Plasmids are extremely valuable tools in the fields of molecular biology and genetics, specifically in the area of genetic engineering. They play a critical role in such procedures as gene cloning, recombinant protein production and gene therapy research. In such procedures, a plasmid is cut at a specific site (or sites) using enzymes called restriction endonucleases. A foreign DNA element is then spliced into the plasmid. The resulting circular structure, a recombinant DNA molecule, is then introduced into bacterial cells (a process called transformation). The autonomous replication of the plasmid within the bacterial cells makes it possible to produce large numbers of copies of the recombinant DNA molecule for experimental manipulation or commercial purposes (such as the production of large amounts of insulin). Final step involves isolation of plasmid from the bacterial culture by breaking open the bacterial cells and separating the plasmid DNA from the chromosomal DNA (and all the other molecules inside the cell). When the plasmid has been isolated, the scientist

now has large amounts of the gene of interest that can be used for study and analysis of the gene. The most important considerations for a plasmid isolation protocol are: The plasmid DNA should be free of contamination from the host cell's chromosomal DNA. The plasmid DNA should be reasonably free of contamination from proteins and other constituents of the host cell. DNA should be pure enough to allow enzymatic reactions such as restriction enzyme digestion and sequencing reactions. The protocol should be rapid and give a high yield of plasmid DNA. The most commonly used method for plasmid DNA isolation is the alkaline lysis method. Alkaline lysis method of plasmid isolation was originally developed by Brinboim and Doly (1979). In this procedure, bacteria containing the desired plasmid are harvested from culture medium. Suspension of bacteria is made in isotonic solution which is subsequently subjected to lysis by an alkaline solution containing a detergent (SDS) and NaOH. While detergent serves to lyse cells and denature proteins, alkaline condition denatures genomic DNA, plasmid DNA as well as proteins. This mixture in subsequent step is neutralized by potassium acetate (pH 5.2). Neutralization results in renaturation of plasmid and genomic DNA. Since plasmid DNA is covalently closed, it reanneals properly and remains in solution in soluble form while genomic DNA reanneals randomly, resulting in the formation of precipitate. Precipitate is separated by high speed centrifugation. Plasmids from the supernatant can be recovered by precipitation using isopropanol or ethanol. The molecular and biochemical effects of each reagent used in the protocol are as follows: Glucose/Tris/EDTA (GTE) : The Tris buffers the cells at pH 7.9. EDTA binds divalent cations in the lipid bilayer, thus weakening the cell envelope. SDS/sodium hydroxide : This alkaline mixture lyses the bacterial cells. The detergent SDS dissolves the lipid components of the cell membrane, as well as cellular proteins. The sodium hydroxide denatures the chromosomal and plasmid DNA into single strands. The intact circles of plasmid DNA remain intertwined.

Potassium acetate/acetic acid : The acetic acid returns the pH to neutral, allowing DNA strands to renature. The large, disrupted chromosomal strands cannot rehybridize perfectly, but instead collapse into a partially hybridized tangle. At the same time, the potassium acetate precipitates the SDS (which is insoluble in potassium) from the cell suspension, along with proteins and lipids with which it has associated. The renaturing chromosomal DNA is trapped in the SDS/ lipid/protein precipitate. Only smaller plasmid DNA and RNA molecules escape the precipitate and remain in solution. Isopropanol : The alcohol rapidly precipitates nucleic acids, but only slowly precipitates proteins. Thus, a quick precipitation preferentially brings down nucleic acids. Ethanol : A wash with ethanol removes some remaining salts and SDS from the preparation. Ethanol also removes the remaining isopropanol, which has a higher vapor point than does ethanol. The ethanol-isopropanol evaporates more rapidly in the drying step. Tris/EDTA : this buffers the DNA solution. EDTA protects the DNA from degradation by DNase activity. Buffering DNA is important, as low pH ( 1) after insufficient incubation under suitable shaking conditions and temperature Cell lysis was

If cells have grown to very high densities or

inefficient (too many cells/ poor resuspension of bacterial cell pellet)

Poor DNA quality

a larger amount of cultured medium than recommended was used, the ratio of biomass to lysis reagent is shifted. This may result in poor lysis conditio ns, because the volumes of solutions I, II and III are not sufficient for setting the plasmid DNA free efficiently. Reduce culture volume or increase volumes of lysis solutions Thoroughly resuspend cell pellets before cell lysis. No cell clumps should be visible after resuspension.

Cell lysis time Make sure incubation after Solution II was too long addition does not last longer than 5 minutes Nicked or sheared plasmid DNA

Do not vortex sample after Solution II and Solution III addition. Also, make sure isolated plasmid DNA is properly buffered such as in TE, pH 8.0. Reduce culture volume if lysate is too viscous for gentle mixing.

Genomic/High molecular weight DNA contamination

Do not vortex the cell lysates after addition of Solution II and Solution III. Vortexing or aggressive mixing of lysates will shear genomic DNA resulting in contamination of plasmid DNA.

Nuclease Check buffers for nuclease contamination contamination and replace if necessary. Use new glass and plasticware, and wear gloves. Poor Salt Make sure DNA is thoroughly washed with downstream contamination 70% ethanol. Extra washes can be carried performance out to ensure clean DNA. of plasmid DNA Ethanol

Keep the given centrifugation time with

contamination ethanol, extend it if necessary. Make sure all the ethanol is dried from the plasmid tube Protein Make sure bacterial lysate is clear after contamination Solution III addition. Check culture volume against the recommended volumes and reduce if necessary.

Additional band below supercoiled plasmid DNA band

Suboptimal performance in sequencing reactions

TE buffer was used for DNA elution. Repurify plasmid DNA and elute in Nuclease-Free Water.

Denatured supercoiled plasmid DNA band

Incorrect incubation with the lysis buffer. Increased incubation time with lysis buffer II can cause denaturation of supercoiled plasmid DNA.

References 1. Ausubel F.M., Brent R., Kingston R.E., Moore D.D., Seidman J.G., Smith J.A. and Struhl K. (1998) Current Protocols in Molecular Biology. John Wiley & Sons 2. Bernard, R. Glick., Jack, J. Pasternak. (2010) Molecular Biotechnology, Fourth edition, American Society for Microbiology, Washington. 3. Craig, W. and Ralph R. (2000) Extraction and Purification of Plasmid DNA, The Nucleic Acid Protocols Handbook Pages, Humana Press, New York. 4. www.ncbe.reading.ac.uk/ncbe/protocols/DNA/PDF/ DNA07.pdf 5. Sabine, E. and Dirk, S. (2003) Isolation of Plasmids from E. coli by alkaline Lysis, Methods in Molecular Biology, Humana Press, New York 6. Sambrook J. and Russell D. W. (2001). Molecular Cloning: A Laboratory Manual, Third edition, Cold Spring Harbour Laboratory Press, New York. 7. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning:

A Laboratory Manual. Second edition. Cold Spring Harbor Laboratory Press, New York. 8. Sandy, P. and Richard, T., (2006) Principles of gene manipulation and Genomics, Seventh edition, Blackwell Publishing.

CHAPTER-4 Isolation of Total RNA RNA isolation from whole blood RNA purified from cells, tissues, whole blood or isolated blood cells is required for many of today's gene expression studies using real¬time polymerase chain reaction (RT-PCR) or micro-arrays. From a purely application point of view, total RNA might suffice for most applications, and it is frequently the starting material for diverse downstream applications. The preference for total RNA reflects the challenge of purifying enough poly(A) RNA for the application (mRNA comprises 1 kb fragments). It also sometimes helps to do a hot-start reaction (reduces non-specific annealing of primers) Component

Volume

10X PCR Buffer (15mM MgCl) 5.0 μl 2.5 mM dNTPs

5.0 μl

100gM Forward Primer

1.0 μl

100 gM Reverse Primer

1.0 μl

Template DNA

50-100 ng (say, 2 gl)

Taq polymerase (0.5U)

0.2 μl

Nuclease-free water

35.8 μl

Cycling Parameters (for Hot Start PCR) Step

Temp Time

Initial Denaturation

94°C

2 minutes

(add Taq at this stage, for hot-start) 35 Cycles

94°C

15-30 seconds

55°C

15-60 seconds

72°C

1 minute/kb

Final Extension

72°C

5 minutes

Hold

4°C

If standard conditions fail, run the first four cycles at 5-10° C lower than recommended, i.e. 42-46° C. (PCR primers with multiple mismatches will be extended, and hopefully some stick to your gene). If the primers are very degenerate (512 mixes or more), competitive inhibition can lead to problems. (Primers bind the correct template but are not extended by the polymerase because of unstable 3' ends.) This means that the first PCR cycles are very inefficient, and you sometimes have to run 50 cycles to see even faint band of your gene. Tips for running a successful degenerate PCR Keep the degeneracy of each primer low. Under 400 is great- under 1000 is ok but not good, and over 1000 isn't worth your time. In general, larger PCR reactions work better-tend to use 50uL reactions for degenerate PCRs Use 3-5 times the amount of primer you would normally use to increase the chances of the appropriate primer being in the reaction at any decent concentration. Upto 3 pl of each primer (at 10mM) for each 50 pl reaction can be used. Best results are obtained with nested degenerate PCR if possible. In this you have a minimum of 3, but best is at least 4 primers within the sequence. In the case of four, you will have two forward and two reverse primers. For the first PCR reaction you use the two "outer" forward and reverse primers. Then you take a portion of this first PCR and use it as template for the second reaction. This helps to reduce the number of amplicons and makes the reactions more specific to the gene you are looking for. The more primers you can design for a given gene, the better the

chances that one of the primer sets will work. Methionine (M) and Tryptophan (W) are the only amino acids that are coded for by a unique codon. Having these in your primer sequences is good. Try to avoid Serine (S) Arginine (R) and Leucine (L) as they each are coded for by six codons. This said; don't let the presence of some of these AAs keep you from using that region. But realize that a sequence of SSRLSR is not going to make a good degenerate primer. Amplifying a 200-600 bp region seems to be optimal.

Colony PCR Colony PCR can be used after a transformation to screen colonies for the desired plasmid. Colony PCR is an easy way to rapidly screen the bacterial colonies for positive clones that have been transformed with DNA and grown overnight to determine if they contain the insert of interest. Colony PCR is the most rapid initial screening method. Colony PCR involves lysing the bacteria and amplifying a portion of the plasmid. Primers are used which generate a PCR product of known size. Thus, any colonies which give rise to an amplification product of the expected size are likely to contain the correct DNA sequence. Colony PCR reaction Mix together the following on ice; always adding enzyme last. For multiple samples, make a large master mix and aliquot 25 gl in each PCR tube (also on ice). Component

Volume(per 25μ1 Reaction)

10X PCR buffer

2.5 μl

10 mM dNTPs

1.0 μl

10 gM Forward Primer

1.0 μl

10 gM Reverse Primer

1.0 μl

Taq DNA Polymerase (5u/10 gl) 0.25 μl

Nuclease-free water

to 25 μl

Use a micropipette tip to pick a single colony off a plate. Insert the tip into the PCR mixture and pipette up and down. The amount of cells should be small, just a touch will do, the small amount required to fill the end of the opening is sufficient. Sufficient mixing will result in complete cell lysis and high yields. Do not spin the tube down after adding the single colony — this might pellet the cells that were added. Perform PCR using your standard parameters. Cycling Conditions for a Routine PCR are as follows: Step

Temp

Time

Initial Denaturation 94°C

30 seconds

35 Cycles

15-30 seconds

94°C

55-64°C (varies with primers) 15-60 seconds 72°C

1 minute/kb

Final Extension

72°C

5 minutes

Hold

4°C

Run the amplified PCR products on 1% agarose gel with DNA marker of appropriate size and visualize by UV.

Quantitative PCR (Q-PCR) Real-time or Quantitative PCR use the linearity of DNA amplification to quantify the number of copies of nucleic acids during PCR This PCR uses fluorescence detection systems which are generally of two types: intercalating agents and labeled probes with fluorophores. For detailed protocol of this technique please refer to protocol on Real Time PCR.

Reverse Transcription PCR (RT-PCR)

RT-PCR is a technique which combines the methodologies of reverse transcription and PCR. Starting template is RNA from which cDNA is synthesized followed by amplification in PCR to provide a rapid, sensitive method for analyzing gene expression. RT-PCR is used to detect or quantify the expression of mRNA, often from a small concentration of target RNA. For detailed protocol of this technique please refer to protocol on Reverse Transcription PCR.

References 1. Bassam, B. J. and Caetano-Anolles, G. (1993) Automated "hot start" PCR using mineral oil and paraffin wax. BioTechniques 14: 30-34. 2. Chou Q. (1992) Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications. Nucleic Acids Res. 20: 1717¬1723 3. D'Aquila R.T., Bechtel L.J., Videler J.A., Eron J.J., Gorczyca P. and Kaplan, J.C. (1991) Maximizing sensitivity and specificity of PCR by pre¬amplification heating. Nucleic Acids Res. 19:3749. 4. Don R.H., Cox P.T., Wainwright B.J., Baker K. and Mattick J.S. (1991) "Touchdown" PCR to circumvent spurious priming during gene amplification. Nucleic Acids Res 19 (14): 4008 5. Erlich H.A., Gelfand D. and Sninsky, J.J. (1991) Recent advances in the polymerase chain reaction. Science 252:1643-1651. 6. Hecker K. and Roux K. (1996). "High and low annealing temperatures increase both specificity and yield in touchdown and stepdown PCR". Biotechniques 20 (3): 478-85. 7. Korbie D.J and Mattick J.S. (2008) Touchdown PCR for increased specificity and sensitivity in PCR amplification. Nat Protoc. 3 (9):14526. 8. Roux K.H. (2009). Optimization and troubleshooting in PCR. Cold Spring Harb Protoc doi: 10.1101/pdb.ip66 9. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual. Second edition. Cold Spring Harbor Laboratory Press, New York.

CHAPTER-8 Reverse Transcription- Polymerase Chain Reaction (RT-PCR) Reverse transcription is a process of synthesizing DNA from a template of RNA. This process is performed by certain retroviruses whose genetic code is made up of single-stranded RNA molecules. It also requires a special enzyme known as a reverse transcriptase enzyme. When these viruses infect a cell, they inject it with their RNA. Instead of being utilized in protein synthesis, this RNA goes through the process of reverse transcription, and is converted into a single-stranded DNA molecule. This single-stranded DNA is further converted into a double¬stranded DNA that then becomes integrated into the cell's genome. When these foreign genes are expressed, the cell's normal functions are altered and it becomes a manufacturing site for more viruses. Viral reverse transcriptase enzymes are utilized by molecular biologists in vitro to copy RNA molecules into DNA. This is carried out by annealing a short single-stranded primer to the RNAs of interest, which provides an initiation point for the reverse transcriptase enzyme. In the presence of deoxynucleotide triphosphates, reverse transcriptase will extend the primer to make a DNA strand complementary to the RNA template (cDNA). The resulting cDNA can be used in RT-PCR reaction. RT reaction is also called first strand cDNA synthesis. Three types of primers can be used for RT reaction: oligo (dT) primers, random (hexamer) primers and gene specific primers with each having its pros and cons. If mRNAs are the RNAs of interest, typically a DNA poly-T oligonucleotide primer is hybridized to the poly-A tails of the mRNAs. Oligo(dT) primers hybridize to the poly(A) tail present in most eukaryotic mRNAs and will initiate reverse transcription from the very beginning (3' end) of the mRNA. This is important advantage if the cDNA shall be cloned. But for expression analysis this may be a

disadvantage, because the transcription may not reach the PCR target sequence if the mRNA is not intact because of degradation, and a bias for amplicons located close to the mRNA 3' end may be introduced. Random sequence primers are short oligomers of all possible sequences. They are usually six (random hexamers) or nine (random nonamers) bases long. Random sequence primers will copy all RNA, including tRNA, rRNA, and mRNA. It is the priming strategy of choice if rRNA shall be used as reference and for total reverse transcription of prokaryotic mRNA. For total reverse transcription, when it is important to copy as many of the different mRNAs as possible, one may use a blend of random oligomers and oligo(dT) primers. Third option is to use sequence specific primers. This is the preferred strategy when a limited number of mRNAs shall be analyzed. The hybridization of specific primers to mRNA is highly sequence dependent because of the folding of mRNA to secondary and tertiary structures. There are several commercially available kits available for RT-PCR (Fermentas, Thermo scientific, Applied Biosystems, Qaigen, Invitrogen etc.); the protocol outlined below utilizes the Fermentas RevertAid First Strand cDNA Synthesis Kit for reverse transcription.

Materials required Template RNA Fermentas RevertAid Premium First Strand cDNA Synthesis Kit including: RevertAid™ Premium Enzyme Mix 5X RT Buffer 250 mM Tris-HCl (pH 8.3 at 25°C), 375 mM KCl, 15 mM MgCl2, 50 mM DTT 10mM dNTP Mix Oligo(dT)18 Primer100 μM, 0.5 μg/ μ! Random Hexamer Primer 100 μM, 0.2 μg/ μl Water, nuclease-free DNase I, RNase-free PCR tubes, micropipettes and tips Thermal cycler

Water, nuclease-free

Procedure Removal of genomic DNA from RNA preparation Total cellular RNA isolated by standard methods is suitable for use with the kit. Purified RNA must be free of salts, metal ions, ethanol and phenol to avoid inhibiting the cDNA synthesis reaction. Trace contaminants can be removed by ethanol precipitation of the RNA followed by two washes of the pellet with cold 75% ethanol. For RT- PCR applications, template RNA must be free of DNA contamination. Prior to cDNA synthesis, RNA can be treated with DNase I, RNase- free to remove trace amounts of DNA. Always perform a control (RT- minus) reaction which includes all components for RTPCR except for the reverse transcriptase enzyme. Proceed as outlined below: 1. Add to an RNase-free tube: RNA

1μg

10X Reaction Buffer with MgCl 1 μl DNase I, RNase-free

1μl (1 u)

Water, nuclease-free

to 10 μl

2. Incubate at 37°C for 30 min. 3. Add 1 μl 50 mM EDTA and incubate at 65°C for 10 min. RNA hydrolyzes during heating with divalent cations in the absence of a chelating agent. Alternatively, use phenol/chloroform extraction. 4. Use the prepared RNA as a template for reverse transcriptase.

First Strand cDNA Synthesis 1. Add the following reagents into a sterile, RNase free tube on ice in the indicated order: Component Volume (1 rxn)

Total RNA

2.0 ul (1 pg - 5 pg)

Oligo (dT)18 primer / Random 1.0 ul (100 pmol) hexamer primer 10 mM dNTP Mix

1.0 ul

Water, nuclease-free

to 15.0 ul

2. Add the following components to the reaction tube in the indicated order: Component

Volume (1 rxn)

5X RT Buffer

4.0 μ

RevertAid™ Premium Enzyme Mix 1.0 μl Total volume

20.0 ul

3. Mix gently and centrifuge. 4. Incubate: If an oligo (dT)18 primer is used-30 min at 50°C. If a random hexamer primer is used-10 min at 25°C followed by 30 min at 50°C. 5. Terminate the reaction by heating at 85°C for 5 minutes. The reaction product of the first strand cDNA synthesis can be used directly in PCR, or stored at -20°C for up to one week. Avoid freeze/thaw cycles of cDNA.

Control Reactions for RT-PCR The following negative control reactions should be used to verify the results of the first strand cDNA synthesis. Reverse transcriptase minus (RT-) negative control is important in RTPCR reactions to assess for genomic DNA contamination of the RNA sample. The control RT- reaction should contain all reagents for the

reverse transcription reaction except for the RevertAid™ Premium Enzyme Mix. No template control (NTC) is important to assess for reagent contamination. The NTC reaction should contain all reagents necessary for the reverse transcription reaction except for the RNA template.

PCR The product of the first strand cDNA synthesis reaction can be used directly in PCR. The volume of the first strand cDNA synthesis reaction mixture should not comprise more than 1/10 of the total PCR reaction volume. Normally, 2 pl of the first strand cDNA synthesis reaction mixture is used as template for subsequent PCR in a 25 pl total volume. The protocol for PCR is carried out as described previously.

Avoiding ribonuclease (RNase) contamination RNA purity and integrity is essential for synthesis of full-length cDNA. RNA can be degraded by RNase A, which is a highly stable contaminant found in any laboratory environment. To prevent contamination both the laboratory environment and all prepared solutions must be free of RNases. Recommended practices to avoid RNase contamination: Use certified nuclease-free labware or DEPC-treat all tubes and pipette tips to be used in cDNA synthesis. Wear gloves when handling RNA and all reagents, as skin is a common source of RNases. Change gloves frequently. Use RNase-free reagents, including high quality water. Keep the kit components tightly sealed when not in use. Keep all tubes tightly closed during the reverse transcription reaction.

Troubleshooting Reverse Transcription-PCR Problem

Possible cause

Suggested solution

No or low

RNA was degraded

Verify the integrity of the RNA by

yield of firststrand cDNA

denaturing agarose gel electrophoresis to ensure it is not degraded Isolate the RNA in the presence of a ribonuclease inhibitor, and ensure that all RT-PCR reagents and labware are free of RNases. Resuspend RNA in DEPC-treated water or elution buffer made with DEPC-treated water

RNA Increase the concentration concentration of the is too low template RNA Presence of RT inhibitors (SDS, EDTA, guanidinium chloride, formamide, Na2PO4, or spermidine)

Remove RT inhibitors during RNA purification with an additional 70% (v/v) ethanol wash following ethanol precipitation.

Suboptimal annealing of primers to RNA

Check annealing temperature If using random primers, incubate the reaction at 25°C for 10 minutes prior to increasing the temperature to 42°C for cDNA synthesis.

RNA was degraded

Analyze RNA on a denaturing gel before use to verify integrity. Use aseptic

technique for RNA isolation. Process tissue immediately after removal from animal. Store RNA in 100% formamide cDNA Add more cDNA concentration synthesis product to is too low the PCR (up to 5 μl) The primers or template are sensitive to remaining RNA template

Treat first-strand cDNA with RNase H before PCR

Poor PCR primer design

Avoid complementary sequences at the 3" end of primers. Avoid sequences that can form internal hairpin structures. Design primers with similar Tms

The RNA preparation may be contaminated with genomic DNA

Verify the presence of contaminating DNA by performing RT-PCR in the absence of reverse transcriptase

Please refer to section troubleshooting PCR in chapter for other probable problems and possible solutions with regard to PCR amplification

References 1. Bustin S.A. (2002). "Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems". J. Mol. Endocrinol. 29 (1): 23-39. 2. Gerard, G. F. and D'Alessio, J. M. (1993) Methods in Molecular Biology. Totowa, NJ: Humana Press, Inc. 3. Maniatis T., Fritsch, E.F. and Sambrook J. (1989) Molecular Cloning: A Laboratory Manual Cold Spring Harbor Press, Cold Spring Harbor, NY, USA. 4. RT-PCR Troubleshooting-Invitrogen accessed from www.invitrogen.com/site/us/en/home/References/protocols/nucleicacid-amplification-and-expression-profiling/pcr-protocol/pcr-and-rtpcr.html 5. Sambrook, J. and Russell, D. W. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. 6. Sambrook, J.; Fritsch, E.F. and Maniatis, T. (2000) Molecular Cloning: A Laboratory Manual. Second edition. Cold Spring Harbor: Cold Spring Harbor Laboratory Press. 7. Xiang, C. and Brownstein, M. J. (2003) Methods in Molecular Biology, Vol 224: Functional Genomics: Methods and Protocols.

CHAPTER-9 Agarose Gel Electrophoresis Agarose gel electrophoresis is a commonly used technique for the separation and analysis of nucleic acids. It is a procedure that separates molecules on the basis of their rate of movement through a gel under the influence of an electrical field. Nucleic acids have a consistent negative charge imparted by their phosphate backbone, and migrate toward the anode. In this method, DNA is forced to migrate through a highly cross-linked agarose matrix in response to an electric current. Agarose is a linear polymer made up of alternating residues of D-and L-galactose joined by a (1—>3) and a(1—4) glycosidic linkages. Agarose is insoluble in cold water but it dissolves in boiling water and upon cooling forms side chains aggregates forming a 3D mesh of channels 50-200 nm in diameter. Agarose melts at around 900C and gels at about 400C. Gelation involves a shift from a random coil in solution to a double helix in the initial stages of gelation, and then to bundles of double helices in the final stage. The average pore size varies with concentration and type of agarose, but is typically 100 to 300 nm. Agarose offers several advantages as a medium of DNA resolution by electrophoresis first being its ability to form a matrix which allows rapid diffusion of high molecular weight macromolecules without significant restriction by the gel. Secondly agarose gels have high gel strength, allowing the use of concentrations of 1% or less, while retaining sieving and anticonvective properties. Thirdly rapid staining and destaining can be performed with minimal background. Fourthly, agarose is nontoxic and, unlike polyacrylamide, contains no potentially damaging polymerization byproducts. There is no free radical polymerization involved in agarose gelation. Finally, Agarose gels are thermoreversible. Low gelling and melting temperature agarose permit easy recovery of samples, including sensitive

heat-labile materials. The DNA to be analyzed is forced through the pores of the gel by the electrical current. The larger the fragment of DNA, the more easily wills it become entangled in the matrix and, therefore, the more slowly will it migrate. Small fragments, therefore, run more quickly than large fragments at a rate proportional to their size. The relationship of size to migration rate is linear throughout most of the gel, except for the very largest fragments. Large fragments have a more difficult time penetrating the gel and their migration, therefore, does not have a linear relationship to size. The matrix can be adjusted, though, by increasing the concentration of agarose or by decreasing it. A standard 1% agarose gel can resolve DNA from 0.2-30 kb in length. The variables that influence the migration rate through a gel includes size of the DNA, the strength of the electrical field, the concentration of agarose in the gel and ionic strength of the running buffer. During electrophoresis one of the parameters (watts, volts, current) is held constant and the other two are allowed to vary as the resistance of the electrophoretic system changes. Agarose gels for DNA separation are run at constant voltage or constant current whereas sequencing gels are usually run at constant wattage to maintain a uniform temperature. When the current is held constant, the samples will migrate at a constant rate. Voltage and wattage will increase as the resistance increases, resulting in an increase in heat generation during the run. If a break occurs in the system such as a damaged lead or electrode or a buffer leak, the resistance of the gel will be greatly increased. This will cause a large increase in wattage and voltage resulting in the generation of excessive heat. It is even possible for the system to get hot enough to boil, or start the apparatus to scorch or burn. When voltage is set constant, current and wattage will decrease as the resistance increases, resulting in a decrease of heat and DNA migration. Since the heat generated will decrease, the margin of safety will increase over the length of the run. If a problem develops and the resistance increases dramatically, the current and wattage will fall since the voltage cannot increase. Even if the apparatus fails, the worst that is likely to happen is that the resistance will increase so much that the power supply will not be able to compensate, and it will shut off. The two buffers commonly used for DNA electrophoresis are Tris-acetate with EDTA (TAE; 40 mM Tris-acetate, 1 mM EDTA) and Tris-borate with EDTA (TBE; 89 mM Tris-borate, 2 mM EDTA). Because the pH of these buffers is neutral, the phosphate backbone of DNA has a net negative charge and

migrates towards the anode.TAE has low buffering capacity and may require recirculation for extended electrophoretic times (>6 hours) whereas TBE has high buffering capacity and no recirculation is required for extended run times. TAE is preferred when electrophoresis of large (>12 kb) DNA fragments is required and DNA is to be recovered from the gel while TBE is preferred for electrophoresis of small ( 5 °C

keep melting temperatures

irreproducible producing unequal

within 2 °C of each other; keep

data; later than

extension; annealing

the GC content to between 30%-

expected CT

temperature is too low; 50%; test assay performance

value

unanticipated variants

against carefully quantified

within the target sequence

controls

Actual CT is much earlier than anticipated

Genomic DNA contamination; multiple products; high primer- dimer production; poor primer specificity;performance against carefully samples of interest

DNAse-treat before reverse transcription; re-design primers to increase specificity; decrease primer concentration; increase annealing temperature; increase transcript naturally has high expression in ramp rate; test assay quantified controls

Multiple peaks are present in the plot

Primer dimers are present

Improve the stringency by raising the annealing temperature or lowering magnesium concentration. If the annealing temperature is increased above 60°C, a two- step protocol can be used in place of a three-step protocol. Try a hot-start reaction.

Primers are amplifying multiple genuine products (e.g., variants in cDNA template)

With variants from a cDNA, you may have to redesign the primers. If you run the PCR product on a gel, a significant difference in size (equal to an extra intron) will show up.

Plateau is Limiting much lower reagents;degraded than expected reagents such as the dNTPs or master mix

Check calculations for master mix; repeat experiment using fresh stock solutions

Technical replicates are not overlapping and have a

Calibrate pipettes; use positivedisplacement pipettes and filtered tips; mix all solutions thoroughly during preparation and during use; hold pipette

Pipetting error;insufficient mixing of solutions; low expression of target transcript

difference in

resulting in

CT values > 0.5

stochastic amplification

vertically when aspirating working with small volumes

Looping of data

Baseline adjustment

Reset Baseline to 3 cycles before

points during

includes too many

the first indication of

early cycles;

cycles; too much starting

amplification

high noise at material the beginning of recorded data Poor PCR

Samples may contain

Perform RNA purification

efficiency

PCR inhibitors

process on a sample using a new

(85%) - slope is

purification method. RNA with

below 3.6

a significantly lower A260/A280 ratio should be further purified by phenol-chloroform extraction, LiCl precipitation, or washing to remove residual salt Inaccurate sample and reagent pipetting

Accurate pipetting withlarly calibrated pipettes is required

References 1. Burkardt H.J (2000) Standardization and quality control of PCR analyses. Clin Chem Lab Med 38(2): 87-91. 2. Bustin S. (2002) "Quantification of mRNA using Real-Time reverse transcription PCR (RT-PCR); trends and problems", Journal of Molecular Endocrinology, 29: 23-39.

3. Freeman W.M., Walker S.J. and Vrana K.E. (1999) "Quantitative RtPCR:pitfalls and potential", Biotechniques, Vol.112 (22): 124-125. 4. Heid CA, Stevens J, Livak KJ, Williams PM (1996) Real time quantitative PCR. Genome Res 6: 986-993. 5. Hoogewijs D., Houthoofd K., Matthijssens F., Vandesompele J. and Vanfleteren J..R. (2008) "Selection and validation of a set of reliable reference genes for quantitative sod gene expression analysis in C. elegans.", BMC Mol Biol. 9:9. 6. Overbergh L., Giulietti A., Valcks D., Decallonne B., Bouillon R. and Mathieu C. J. (2003) "The use of real-time transcriptase PCR for the quantification of Cytokine Gene Expression", Biomol.Tech, Vol.14: 3343. 7. Pfaffl M.W. (2004) Quantification strategies in real-time PCR. In: Bustin SA (ed), A-Z of Quantitative PCR, pp. 87-120. La Jolla, CA: IUL Biotechnology Series, International University Line. 8. Schmittgen TD., Livak KJ. (2008) "Analyzing real-time PCR data by the comparative C(T) method." Nat Protoc. 3(6): 1101-8. 9. Souaze F., Ntodou-Thome A., Tran C.Y., Rostene W. and Forgez P. (1996) Quantitative RTPCR: limits and accuracy. Biotechniques 21(2): 280-285. 10. Wong M.L., Medrano J.F. (2005) Real-time PCR for mRNA quantitation. Biotechniques 39(1): 75-85.

Chapter 13 Chromatin Immunoprecipitation Assay In the post-genome era, attention has focused on the expression of genome sequences and how they are regulated. Gene expression is regulated at many levels. Regulation of gene expression i.e. how genes are turned on and off, or up and down, provides the molecular basis that drives normal cellular function, differentiation and metabolism, mechanisms of disease, and response of cells to stimuli such as drug treatments. Gene regulation occurs primarily at the level of gene transcription, whereby the genetic information encoded by the DNA is copied into an RNA transcript that is subsequently translated into protein. Gene transcription is controlled by the interactions between transcription factor proteins and their binding sites. Transcription factors are proteins that bind to the promoter elements upstream of genes and either facilitate or inhibit transcription. These DNA- protein interactions are crucial for vital cellular functions including gene transcription, DNA replication and recombination, repair, segregation, chromosomal stability, cell cycle progression, and epigenetic silencing. Detection of the specific DNA binding sites of transcription factors in the genome is therefore fundamental to understanding the complex and varied regulatory pathways that direct gene expression in the cell and govern all biological processes, including disease development. Chromatin immunoprecipitation (ChIP) assays are a powerful technique that allows detection of protein-DNA interactions in vivo. ChIP is a very versatile technique that enables investigators to identify regions of the genome associated with specific proteins within the context of the cell. Chromatin immunoprecipitation reveals an extraordinarily rich and dynamic chromatin environment. Moreover, it is an in situ technique that offers a more physiological representation of nuclear events involved in the processing of DNA. It has

been used to study both histones and non-histone proteins, such as transcription factors, within the context of the cell. Initially, the ChIP assay was used to study the association of hyperacetylated histones with specific DNA sequences to further understand the role of histone acetylation in transcription. However, more recent studies have used the ChIP protocol to characterize DNA sequences associated with specific transcription factors. In this technique, live cells are treated with formaldehyde to generate protein-protein and protein-DNA cross-links between molecules in close proximity on the chromatin template in vivo. A whole-cell extract is prepared, and the cross-linked chromatin is sheared by sonication to reduce average DNA fragment size to 100¬500 bp. The resulting material is immunoprecipitated with an antibody against a desired protein, modifed (e.g., acetylated, phosphorylated, methylated) peptide, or epitope (in situations where the protein of interest is epitope-tagged). DNA sequences that directly or indirectly cross-link with a given protein (or modi?ed variant) are selectively enriched in the immunoprecipitated sample. Reversal of the formaldehyde cross-linking by heating permits the recovery and quantitative analysis of the immunoprecipitated DNA. (Fig 1) describes basic principle of ChIP assay. The protocol uses ChIP assay kit from USB Corporation and follows manufacturers protocol with certain modifications.

Fig.1 Principle of Chromatin Immunoprecipitation assay.

Requirements Reagents & Solutions Π 37% Formaldehyde Π PBS (1X) Antibody to the protein of interest Proteinase K RNase A Protease Inhibitor (100X) Protein A agarose beads (pre-blocked) Lysis Buffer High Salt Buffer Lithium Salt Buffer TE Buffer, pH 7.5 Elution Buffer 5M NaCl 1.25M Glycine (10X) Phenol:chloroform: Isoamyl Alcohol Glycogen Ethanol (10% and 75%) Nuclease-Free Water Equipments and materials Sonicator Centrifuge and microcentrifuge Rotating platform Rotisserie Shaker Dishes or flasks (tissue culture) Gel electrophoresis apparatus Incubator Micropipettes and tips PCR apparatus Spectrophotometer Π Spin column Π Tubes (conical, 15-mL) Tubes (microcentrifuge, 1.5-mL)

Procedure

Cross-link protein-DNA complexes in vivo: 1. Cells are fixed with reversible cross-linking reagent formaldehyde added to a final concentration of 1% (675 11 37% formaldehyde to 25 ml PBS) forming protein-DNA complexes while mixing gently and thoroughly on a rotating shaker platform. Continue rotating at room temperature for 10 to 20 min. Add 2.5 ml of 1.25M Glycine to stop the cross linking. From this point on, keep everything on ice or at 4°C, including the centrifugation step. 2. Harvesting of cells: For adherent cells 1. Remove the growth medium.. 2. Wash cells twice with 10 ml of cold 1X PBS, and then scrape the cells into 1X PBS in a 15-ml conical tube. 3. Pellet the cells at 1500 rpm for 10 min at 4°C. For nonadherent cells 4. Transfer the cells to a 15-ml conical tube. 5. Pellet the cells at 1500 rpm for 10 min at 4°C. 6. Wash the cells twice with 10 ml of cold 1X PBS. 3. Centrifuge at 2000 rpm for 5 min to pellet cells. Resuspend cell pellet in 1 ml chilled PBS (freshly supplemented with 1X protease inhibitor) and transfer to a 1.5 ml microcentrifuge tube. 4. Centrifuge at 2000 rpm for 5 min to pellet cells, remove the supernatant. Record the packed cell volume which is used to correlate with cell number for the assay. Proceed to sonication or quick freeze the cell pellet in liquid nitrogen and store at-80°C for future use. Avoid multiple freeze-thaw cycles. When used, cells should be thawed on ice. Chromatin Fragmentation (Sonication) Resuspend cell pellet in 1 ml Lysis Buffer (freshly supplemented with 1X protease inhibitor) and transfer to a 15 ml conical tube. Sonicate chromatin to an average size of 200 to 1000 bp. If foaming occurs, centrifuge at 2000rpm for 5 min between sonication cycles. The size of the sheared DNA must be checked after cross-link reversal. In experiments in our laboratory, we have observed that 5S (5 cycles of sonication: each cycle pulse of amplitude 5 for 1 minute followed by a resting phase of 1 minute in ice) in Ultrasonicator

(Soniprep - 150) gave optimum shearing with DNA in a relatively tight distribution between about 100 bp and 500 bp. Immunoprecipitation of Protein : DNA Complexes 1. Chromatin Preclearing To a 1.5 ml microfuge tube, add the sonicated chromatin (200 μ!) and Lysis Buffer to a final volume of 600 μ?. A no antibody negative control tube (mock) is also included. Centrifuge at 20,000 x g for 10 min to remove any debris. Transfer supernatant to a fresh tube and add 50 μl of pre-blocked Protein A agarose beads to the supernatant followed by incubation on a rotisserie shaker for one hour. After centrifugation at 3000 x g for 5 min, transfer supernatant to a fresh tube and add specific antibody to protein of interset (1-4 μg) to the supernatant. No antibody is added for the negative control tube (mock). Samples are incubated on a rotisserie shaker for 4 hrs to overnight. The mock is a background check that is a control for non-specific interactions between chromatin and the agarose beads. 2. Capturing of Immune-Complexes (Antibody:Protein:DNA) Add 50 μl pre-blocked Protein A agarose beads to the sample and incubate on a rotisserie shaker for one hour and then centrifuge at 2000 rpm for 5 min. 3. Washing and elution of immune complexes Wash the Protein A agarose beads: immune-complex consecutively with the buffers in following order: Lysis Buffer (1 x 1 ml wash), High Salt Buffer (1 x 1 ml wash), Lithium Salt Buffer (1 x 1 ml wash),TE Buffer, pH 7.5 (2 x 1 ml washes). For each wash, incubate samples on a rotisserie shaker for 10 min then centrifuge at 2000 rpm for 5 min. From this point on, all manipulations are performed at room temperature. Add 120 μ! of Elution Buffer to the washed beads and incubate on a rotisserie shaker for 20 min at room temperature. After centrifugation at 3000 x g for 5 min, supernatant is transferred to a fresh 0.5 ml microcentrifuge tube. Repeat elution with another 120 μ1 of Elution Buffer and supernatants are combined. (The recovered volume is ~225 μ!) 4. Reversal of Formaldehyde Cross-Links Under salt (NaCl) concentration of 0.2 M reversal of cross-linking is

done at 65°C for 10 hrs to overnight in a thermal cycler 5. DNA Purification It was done by phenol extraction method. Purified DNA is then suspended in 50 μL of water or TE buffer and analyzed by PCR or real-time PCR.

Troubleshooting ChIP Problem

Possible cause

Suggested solution

Foaming - results in unequal shearing of DNA samples during sonication

Placing the tip end near the surface induces foaming

Position the microtip probe close (approximately at Vi inch from bottom) to the bottom of the tube to prevent foaming. If foaming occurs, centrifuge at 4000 rpm for 5 min.

Under-sheared chromatin and fragments larger than 800 bp high background and lower resolution

Excess cross linking of cells Cross-linking for longer than 10 min may inhibit digestion of chromatin

Time course at a fixed formaldehyde concentration can be done. Cross-linking time should be shortened to 10 min or less

Too many cells or suboptimal sonication/ shearing

Perform more shearing replications, turn up the sonication power, cross-link less, or use less cells

Not enough cells or too much sonication sonication power, cross-link

Perform fewer shearing replications, turn down the 150 bp or less - weak signal on PCR especially for amplicons greater than 150 bp in length. more, or more less cells.

Over-sheared Chromatin and fragments are too small shearing

Chromatin

Samples must be placed on ice in

degradation

between sonications. If the sonication is too long or powerful unwanted denaturing will take place

No product or very

Inadequate DNA

Add more DNA to the PCR

little product in the

added to the PCR

reaction or increase the

input PCR reactions

reaction or conditions

number of amplification

are not optimal

cycles

Not enough

For optimal ChIP results,

chromatin added to

add 5 to 10 μg chromatin

the IP or per immunoprecipitation chromatin is overdigested No product in the Not enough

Add 5 to 10 μg of chromatin

positive control

chromatin or

and 10 μl of antibody to

reaction

antibody added to each IP reaction & incubate the IP reaction or IP

with antibody over-night

incubation time is and an additional 2 h after too short

adding Protein G beads

Incomplete elution of

Elution of chromatin from

chromatin from

Protein G beads is optimal

Protein G beads

at 65°C with frequent mixing to keep beads suspended in solution

High background Non-specific in binding

Include a pre-clearing step,

non-specific antibody

to Protein A or G

whereby the lysed sample

controls

beads

is mixed with beads alone for 1 hr that are removed prior to adding the antibody.

The ChIP buffers may

Prepare fresh lysis and

be contaminated

wash solutions

Protein A or G beads give high background

Some Protein Aor G beads can give high background levels. Find a suitable source that provides the cleanest results with low background in the non¬specific control.

No product in the Not enough DNA Experimental added to the PCR Antibody-IP PCR reaction. reaction.

Add more DNA to the PCR reaction or increase the number of amplification cycles

Not enough Typically a range of 1 to 5 pg of antibody added to antibody is added to the IP the IP reaction reaction; however, the exact amount depends greatly on the individual antibody. Increase the amount of antibody added to the IP Antibody does not Find an alternate antibody source work for ChIP

References 1. A Beginner's Guide to ChIP - ABCAM. Accessed from http:// docs.abcam.com/pdf/chromatin/A-beginners-guide-to-ChIP.pdf 2. ChIP Assay kit- USB corporation. Accessed from www.usbweb.com. 3. Das, P. M, Ramachandran, K., vanWert, J. and Singal, R. (2004) Chromatin immunoprecipitation assay. BioTechniques 37:961-969 4. Das, P.M., Ramachandran, K., van Wert, J. and Singal, R. (2004). Chromatin immunoprecipitation assay. Biotechniques 6:961-9. 5. Michael, F. C., Craig, L. P., and Stephen T. S. (2009). Transcriptional Regulation in Eukaryotes: Concepts, Strategies, and Techniques, Second Edition. CSHL Press, Cold Spring Harbor, NY, USA, 6. O'Neill, L. P. and Turner, B. M. (2003) Immunoprecipitation of native chromatin: NChIP. Methods. 31(1):76-82. 7. Orlando, V. (2000). Mapping chromosomal proteins in vivo by formaldehyde cross linked-chromatin immunoprecipitation. Trends Biochem. Sci. 25:99 - 104. 8. Sambrook, J. and Russell, D. W. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York 9. Weinmann, A. S and Farnham, P. J. (2002) Identification of unknown target genes of human transcription factors using chromatin immunoprecipitation. Methods 26: 37-47. 10. Wells, J and Farnham, P. J. (2002) Characterizing transcription factor binding sites using formaldehyde crosslinking and immunoprecipitation. Methods. 1:48-56.

Chapter 14 Microsatellite Analysis Microsatellites are tandemly repeated sequences, where the repeating unit is 1 to 4 nucleotides long. One common example of a microsatellite is a (CA)n repeat, where n varies between alleles. The number of times the unit is repeated in a given microsatellite can be highly variable Microsatellites often present high levels of inter- and intra-specific polymorphism, particularly when the number of repetitions is 10 or greater, a characteristic that makes them useful as genetic markers. Microsatellite is used as a marker to identify a specific chromosome or locus. When being used as a marker, the specific number of repeats in a given microsatellite is not important, but rather the difference in the number of repeats between alleles that assumes significance. The variation in number of repeats affects the overall length of the microsatellite. A majority of microsatellites are present in introns or other non-coding regions of the genome. Certain microsatellite alleles are associated with certain mutations in coding regions of the DNA that can cause a variety of medical disorders. They have also become the primary marker for DNA testing in forensic contexts — both for human and wildlife cases. The reason for this prevalence as a forensic marker is their high specificity. Match identities for microsatellite profiles can be very high. In a biological/evolutionary context they are useful as markers for parentage analysis. They can also be used to address questions concerning degree of relatedness of individuals or groups. For captive or endangered species, microsatellites can serve as tools to evaluate inbreeding levels (FIS). From there we can move up to the genetic structure of subpopulations and populations (using tools such as F-statistics and genetic distances). They can be used to assess demographic history (e.g., to look for evidence of population bottlenecks), to assess effective population size (Ne) and to assess

the magnitude and directionality of gene flow between populations. Microsatellites provide data suitable for phylogeographic studies that seek to explain the concordant bio-geographic and genetic histories of the floras and faunas of large-scale regions. They are also useful for fine-scale phylogeniesup to the level of closely related species. Polyacrylamide gel electrophoresis, MetaPhor agarose gel electrophoresis and automated capillary electrophoresis are used for microsatellite analysis and all these methods produce comparable and reproducible results. The region containing the microsatellite is amplified by PCR using primers that flank the microsatellite. The size of the DNA amplified is determined by the number of repeats present in the microsatellite on that allele. The amplified DNA is then run out on a PAGE/agarose/sequencing gel that will separate DNA fragments based on size. Individuals typically have two alleles for all microsatellites If the number of repeats of one allele is different from the other, then two separate bands would show up on the gel.

Requirements Reagents and solutions Metaphor agarose TAE buffer Distilled water Equipments and Labware Electronic balance, Weigh boats Microwave oven 100 ml graduated cylinder 500 ml & 50 ml Erlenmeyer flask Electrophoresis apparatus & Power supply Vortex mixer UV transilluminator Micropipettes & tips

Procedure

Sample collection : Collect 10 ml venous blood in polypropylene tubes containing ACD as anticoagulant and brought to laboratory under low temperature. Finally, all the samples were kept at-20°C till DNA was isolated. Genomic DNA extraction : High molecular weight genomic DNA was prepared using phenol-chloroform extraction method (Clamp et al., 1993) with minor modifications. The quantity and quality of DNA was evaluated on spectrophotometer and through 0.8% agarose gel electrophoresis. DNA amplification: as per standard PCR protocols. Selection of microsatellite markers. The microsatellite marker analysis of livestock species is carried out using FAO recommended primer pairs. Preparing a 4 % MetaPhor® Agarose (volume 100 ml) 1. Take a 500 ml beaker (beaker should be 2-4 times the volume of the solution). 2. Take 100 ml 1X electrophoresis buffer and keep in refrigerator for chilling at 40C and put a stir bar to the beaker. 3. Weigh 4 g of MetaPhor Agarose and slowly sprinkle in the chilled buffer solution while the solution is rapidly stirred. Remove the stir bar if not Teflon coated. 4. Let the agarose soak in the buffer for 15 minutes before heating. This reduces the tendency of the agarose solution to foam during heating. 5. Cover the beaker with plastic wrap. 6. Pierce a small hole in the plastic wrap for ventilation. 7. Heat the beaker in the microwave oven on medium power for 2 minutes. 8. Remove the beaker from the microwave oven. 9. Gently swirl the beaker to resuspend any settled powder and gel pieces. 10. Reheat the beaker on high power (usually 2 minutes.) until the solution comes to a boil. Hold at boiling point for 1 minute or until all of the particles are dissolved. 11. Remove the beaker from the microwave oven. 12. Gently swirl the beaker to thoroughly mix the agarose solution. 13. Slightly cool the solution, then add 10 μ! of ethidium bromide and shake the solution to mix. 14. Cool the solution to 50°C-60°C prior to casting.

Once the gel is cast, allow the molten agarose to cool and gel at room temperature. The gel must then be placed at 4°C for 20 minutes to obtain optimal resolution and gel handling characteristics. MetaPhor agarose gel electrophoresis 1. Add 3μl 6x loading dye to the 20 μl PCR product and from this load 10μl on the Metaphor agarose gel submerged in chilled 1x TBE buffer. 2. Perform electrophoresis for about 3-4 hours at 6 V/cm using horizontal electrophoresis systems. Visualization of gel After electrophoresis, visualize the finely resolved product bands with U.V. light at 302 nm on a transilluminator. Scoring of gels/ Genotyping After gel electrophoresis and imaging use good quality gel photographs to score the all visible and unambiguously scorable fragments amplified by microsatellite primers. Each individual is genotyped manually by scoring the band (alleles). It can be done manually or it can be read from gel directly by a computer installed with software (for e.g. alpha digi doc software/ Science Lab. 2001 - Image Gauge Ver.4.0 software etc.). Alleles can be coded simply as their integer size in base pair in the case of microsatellites which heterozygous yield two band and those that are homozygous yields one band. Polyacrylamide gel electrophoresis can also be utilized instead of metaphor agarose gels to resolve microsatellite alleles and visualize the alleles by silver staining followed by scoring the gels. Microsatellite Data analysis Allele Number It is the total number of alleles for a given locus in a population. It can be determined manually from silver stained PAGE gels or ethidium bromide stained metaphor gels. Allele frequency The frequency of an allele, say a is the number of a alleles in the population divide by the total number of alleles and gives an indication of most or least prevalent alleles in the population. Effective allele number (Kimura and Crow, 1964)

The effective number of alleles is the reciprocal of the sum of squares of allele frequencies. Heterozygosity The heterozygosity, sometimes called the observed heterozygosity, is the proportion of heterozygous individuals in the data set. High heterozygosity means large genetic variability. Low heterozygosity means little genetic variability.

Polymorphic information content (PIC) The polymorphism information content (PIC) measures the probability of differentiating the allele transmitted by a given parent to its child given the marker genotype of father, mother, and child. It is the probability that one could identify which homologue of a given parent was transmitted to a given offspring, the other parent being genotyped as well. Probability that the parent is heterozygous and probability that the offspring is informative.

PIC refers to the value of a marker for detecting polymorphism within a population. PIC depends on the number of detectable alleles and the distribution of their frequency. F-statistic (Weir, 1990) It is a common statistical method of analysis of population genetic structure. F-statistics rely on allele identity information to describe genetic differentiation based on microsatellite markers. This information is often used to infer phylogenetic relationships or to obtain indirect estimates of gene flow. Three hierarchical F-statistics are defined below: 1. Inbreeding coefficient FIS= (Hs - Hi) / Hs It is the mean reduction in H of an individual due to non-random mating within a subpopulation i.e., a measure of the extent of genetic inbreeding within subpopulations. It can range from-1.0 (all

individuals heterozygous) to +1.0 (no observed heterozygotes) 2. Fixation Index FST = (Ht - Hs) / Ht It is the mean reduction in H of a subpopulation (relative to the total population) due to genetic drift among subpopulations i.e., a measure of the extent of genetic differentiation among subpopulations. It can range from 0.0 (no differentiation) to 1.0 (complete differentiation - subpopulations fixed for different alleles) 3. Overall Fixation Index Fit = (Ht - H)/Ht It is the mean reduction in H of an individual relative to the total population. Where, Hi = mean observed heterozygosity per individual within subpopulations Hs= mean expected heterozygosity within random mating subpopulations Ht = expected heterozygosity in random mating total population. The relationship between the three Fstatistics is: (1 - Fit) = (1 - Fb) (1 - Fst) Statistical softwares for microsatellite data analysis There are many freely available software packages that can be downloaded from internet. Some of the software packages commonly used in population genetics include POPGENE (http:// www.ualberta.ca/~fyeh/), Arlequin (http://lgb.unige.ch/ arlequin/), GENEPOP (http://wbiomed.curtin.edu.au/genepop/), Phylip (http://evolution.genetics.washington.edu/phylip/ getme.html), Microsatellite (http://oscar.gen.tcd.ie/~sdepark/mstoolkit/), TreeView (http://taxonomy.zoology.gla.ac.uk/rod/ treeview.html) Microsatellite data analysis with Popgene32 software Preparing input file Window-based text editor is used to prepare the required data file. Input file for consists of the header section and the data. The header section specifies (1) a job title delimited by /*...*/; (2) number of populations; (3) number of loci and (4) locus names. The body of data starts with, for each population, population ID # (optional), population name (optional). If you do not give population ID # or population name, you must leave at least one blank line between populations and POPGENE will generate population ID # automatically for you. But if you do, your population ID # and population

name must be unique for each population. In the demo procedure demonstrated here, microsatellite data on 14 microsatellite alleles of cattle is analysed. Analysis involved data on a population with 100 individuals each for 14 microsatellite alleles. In windows based text editor prepare input file as: /* Diploid Data of a population with hundred records (genotypes) & 14 loci */ Number of populations = 1 Number of loci = 14 Locus name: INRA063 INRA005 ILSTS005 HEL5 HEL1 INRA035 INRA032 INRA023 TGLA53 TGLA126

Then save the file in popgene software files as say, MICROCATTLE (as.dat/.txt) For microsatellite analysis in popgene software, in main menu click File and then Load Data and select Co-Dominant marker. Open the MICROCATTLE file.

Open Diploid Data Analysis dialog box to check: variables as columns, Single Populations.

In the queries following this step: Do you want to retain all loci for further analysis, Do you want to retain all population for further analysis, enter the significance level for LD test, enter the number of simulations for neutrality test. As per the desired statistic, click/fill in the desired stat. here we retained all loci for further analysis and our population was single.

References 1. Anonymous. 2004. MetaPhor agarose. Cambrex Bio Science Rockland, Inc., USA. www.cambrex. com/bioproducts 2. Nei M (1973) Analysis of gene diversity in subdivided populations. Proceedings of the National Academy of Sciences of the United States of America, 70:3321-3. 3. Nei, M., 1972 Genetic distance between populations. Am. Nat. 106: 283- 292 4. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor 5. Weir, B. S., and Cockerham, C. C. (1984) Estimating F-statistics for the analysis of population structure. Evolution 38:1358-1370. 6. Wright S (1965). The interpretation of population structure by Fstatistics with special regard to systems of mating Evolution,19:395-420. 7. Yeh, F.C.; R.-C. Yang, T. Boyle, Z.-H. Ye and J.X. Mao. 1997. POPGENE: the user-friendly shareware for population genetic analysis. Molecular Biology and Biotechnology Centre, University of Alberta, Canada.

Chapter 15 DNA Ligation, Competent Cells Preparation & Transformation DNA Ligation Ligation of DNA is an essential step in many modern molecular biology experiments. In living organisms, a set of essential enzymes, DNA ligases play critical role in DNA replication and repair. DNA ligases close nicks in the phosphodiester backbone of DNA. DNA ligases are essential for the joining of Okazaki fragments during replication, and for completing shortpatch DNA synthesis occurring in DNA repair process. There are two classes of DNA ligases. The first uses NAD+ as a cofactor and only found in bacteria. The second uses ATP as a cofactor and found in eukaryotes, viruses and bacteriophages. The smallest known ATP-dependent DNA ligase is the one from the bacteriophage T7 (at 41KDA). Eukaryotic DNA ligases may be much larger (human DNA ligase I is > 100KDA) but they all appear to share some common sequences and probably structural motifs. T4 DNA ligase is the most commonly used DNA ligase for molecular biology techniques and can ligate 'sticky' or blunt ends. Bacteriophage T4 DNA ligase is a single polypeptide with a M.W of 68,000 Dalton requiring ATP as energy source. The maximal activity pH range is 7.5-8.0. The enzyme exhibits 40% of its activity at pH 6.9 and 65% at pH 8.3. The presence of Magnesium ion is required and the optimal concentration is 10mM. T4 DNA ligase has the unique ability to join sticky and blunt ended fragments. Cohesive end ligation is carried out at 12°C to 16°C to maintain a good balance between annealing of ends and activity of the enzyme. If reaction is set at higher temperatures annealing of the ends become difficult, while lower temperatures diminishes the ligase activity. All T4 DNA ligase is inactivated by heating at 65°C for 10

minutes. Beside of these ligating complementary sticky ends, T4 ligase can ligate any two blunt DNA ends. Lack of cohesive termini makes blunt end ligation more complex and significantly slower. 10-100 times more enzyme is required to achieve similar ligation efficiency as that of cohesive end ligation. The enzyme has involved in the catalysis of the joining of RNA to either a RNA or DNA strand in a duplex molecule but this will not involved in the joining of single stranded nucleic acids. Ligation using T4 DNA ligase is usually performed between 4°C and 25°C. Cohesive ends have an annealing temperature below room temperature because they are only a few bases long. Low temperature ligations help stabilize DNA interactions, but at the expense of enzyme activity. The reduced activity of T4 ligase at lower temperatures can be partially compensated for by increasing the duration of the ligation reaction. In addition, because annealing temperature of the ends affects ligation efficiency, cohesive-end ligations with short or single base overhangs may benefit from higher concentrations of T4 ligase. In order to generate circular plasmids with the desired insert, and to limit the formation of undesired secondary products like concatemers, the total concentration of DNA in the ligation reaction should be less than 10 μg/ml. Ligations are regularly carried out in the presence of excess insert. For cohesive-end cloning, a relatively low insert: vector ratio of 3:1-5:1 should be effective but may need to be experimentally optimized.

Requirements Reagents and solutions T4 DNA ligase T4 DNA ligase buffer Vector DNA Insert DNA Nuclease free water Equipments and supplies Vials and Vial stand Micro pipette

Pipette tips Π Table top mini centrifuge Π Beaker Π Ice box

Procedure Conversion of molar ratios to mass ratios The following calculation will explain the conversion of molar ratios to mass ratios for a 3.0kb plasmid and a 0.5kb insert DNA fragment.

Example How much 0.5kb insert DNA should be added to a ligation in which lOOng of 3kb vector will be used? The desired vector: insert ratio will be 1:3.

The following ligation reaction of a 3kb vector and a 0.5kb insert DNA uses a 1:3 vector: insert ratio. Typical ligation reactions use 100- 200ng of vector DNA. Protocol described here uses the Promega T4 DNA Ligase, Blue/White Cloning Qualified kit. 1. Set up the following reaction in a microcentrifuge tube on ice. (T4 DNA Ligase should be added last). Component

10 μ? reaction

10X T4 DNA Ligase Buffer 111 Vector DNA (3 kb)

100 ng

Insert DNA (0.5 kb)

50 ng

T4 DNA Ligase

0.1-1U

Nuclease-free water

to 1011

2. Gently mix the reaction by pipetting up and down and microfuge briefly.

3. Incubate at 4°C overnight or 150C for 4-18 h or room temperature for 3 h. 4. Chill on ice and ligation mix is ready for transformation. There is considerable scope for alterations in the temperature and time needed for successful ligations. The optimal temperature for a ligation is a balance between the optimal temperature for T4 DNA Ligase enzyme activity (25°C) and the temperature necessary to ensure annealing of the fragment ends, which can vary with the length and base composition of the overhangs. Shorter duplexes (linkers less than 16 bases long) require lower temperatures as a result of their lower melting temperatures (Tm). In general, ligation reactions performed at lower temperatures require longer incubation times. The scientific literature reflects this variability in ligation conditions. Bluntend ligations generally are efficient at temperatures between 15-20°C for 418 hours, while sticky ends are ligated effectively at room temperature (22°C) for 3 hours or 4-8°C overnight.

Preparation of competent cells : calcium chloride method Competent cells are bacterial cells that have been specially treated to transform efficiently. Most types of cells cannot take up DNA efficiently unless they have been exposed to special chemical or electrical treatments to make them competent E. coli cells are more likely to incorporate foreign DNA if their cell walls are altered so that DNA can pass through more easily. Such cells are said to be "competent." Competence can be natural competence, a genetically specified ability of bacteria that is thought to occur under natural conditions as well as in the laboratory, and induced or artificial competence, arising when cells in laboratory cultures are treated to make them transiently permeable to DNA. Methods for preparing the competent cells derive from the work of Mandel and Higa who developed a simple treatment based on soaking the cells in cold Calcium chloride. There are two main methods for the preparation of competent cells. They are Calcium chloride method and Electroporation. Calcium chloride method is easy and fast, and provides reasonable transformation efficiencies (105-106 colonies per ug of DNA) which are sufficient for routine sub cloning experiments. Frozen vials can be kept at -70oC for months without significant loss of

viability or efficiency. In Calcium chloride method, Cells are made competent by a process that uses calcium chloride and heat shock. The exposure of a cell to ice-cold CaCl2 (0-5°C) and a subsequent heat shock (3745°C for 85-90 seconds) creates pores in the bacterial cell thereby allowing the uptake of plasmid DNA easily into the cell. This is because the Ca ions being positively charged attack both the negatively charged DNA and also the lipopolysaccharide membrane. Thus, the DNA can then pass through the cell on subsequent heat shock treatment. Cells that are undergoing very rapid growth are made competent more easily than cells in other stages of growth. So it is necessary to bring cells into log phase before the procedure is begun. The cells in rapid growth (log phase) are living, healthy, and actively metabolizing. The growth rate of a bacterial culture is not constant. In the early hours (lag phase), growth is very slow because the starting number of dividing cells is small. This is followed by a time of rapid cell division known as the log phase. The actual length of each phase depends on the temperature at which the cells are incubated. Rapidly growing cells are made competent more easily than cells in other growth stages. The competency of a stock of competent cells is determined by calculating how many E. coli colonies are produced per microgram of DNA added. An excellent preparation of competent cells will give ~108 colonies per ug. A poor preparation will be about 104/ug or less. Range of 105 to 106 is considered reasonable.

Requirements DH5a cells 0.1 M CaCl2 - pre-chilled LB broth Plasmid DNA 50 ml polypropylene tubes Eppendorf 1.5 ml tubes Shaking incubator Micropipettes and tips Centrifuge. Procedure

Preparation of competent cells 1. Revival of frozen culture: Frozen culture of bacterial cells (DH5a) is revived by culturing onto LB broth overnight at 37°C. 2. Transfer 200μ! of DH5a cells from LB broth to 20 ml of LB in a 0. 5liter flask. Incubate the culture for 3 hr at 37 °C with vigorous shaking. After the 1st hour check the culture's 0D600 every 15 - 20 min until it reaches 0.35. It is essential that the culture be in early log phase for the production of competent cells, this stage is typically reached at an OD600 of 0.35 to 0.4. 3. Transfer the bacterial cells to a sterile disposable, 50 ml polypropylene tube. Chill the cells by storing on ice for 10 min. It is very important to keep the cells at 4°C for the remainder of the procedure. Work with the tube sitting on ice as much as possible. The cells, and any bottles or solutions that they come in contact with, must be pre-chilled to 4°C. 4. Centrifuge the tubes at 6000 rpm for 10 min at 4 °C. Decant the supernatant. Stand the tube upside down for 30 s on a paper towel to drain away all media. 5. Resuspend the cell pellet in 5 ml of ice cold 0.1 M CaCl2 solution by gentle swirling and pipetting up and down with a pipette. 6. Transfer 200 μ! contents in 10 Eppendorf (1.5 ml) tubes and leave on ice for one hour. 7. Deep freeze the Eppendorf tubes till further use. Now the competent cells are ready for transformation. Cells stored at - 80°C can be used for transformation for up to ~6 months. Storage of competent cells for later use 1. Add 7.0 μl of DMSO per pre-chilled tube (with 200 μl contents) of resuspended cells. Mix by gently pipetting up and down. Store on ice for 15 min. 2. Add an additional 7.0 μl of DMSO to each pre-chilled tube (with 200 μl contents) of resuspended cells. Mix by gently pipetting up and down. 3. Immediately snap-freeze the cells by immersing in liquid nitrogen. Store tubes at -70 °C until needed.

Using frozen competent cells 1. Remove the number of tubes required from the -70 °C, thaw by warming with your hand. When cells are just thawed, place on ice. 2. Store cells on ice for 10 min.

Transformation of competent cells Transformation is the process of introducing foreign DNA (e.g. plasmids) into bacterial cells. After making cells competent or selecting the ideal competent cell strain, transformation is the next step. There are two ways to accomplish transformation successfully: by chemical transformation or by electroporation. Regardless of whether the transformation is being done for cloning or expression, the transformation procedures are the same. 1. Add DNA to cells. Mix the contents by pipetting gently up and down once. Store the tubes on ice for 1 hour. For instance for purpose of transformation of ligation mix into competent cells, add 5i l Ligation mix to freshly thawed competent cells (200i l) using a pre-chilled tip and mix gently. Include a negative control (cells to which no DNA is added). Treat the cells exactly the same way as the cells to which you added DNA. This control is plated on selective (expect no growth) and non-selective (expect growth) media. Include a positive control (one aliquot of cells transformed with 5 - 25 ng of purified supercoiled DNA carrying a selectable marker). This control is plated on selective media and used to calculate TE. 2. Following 1 hour of incubation on ice, Place the tubes at 42 °C for exactly 90 s and chill immediately on ice for 5 min. 3. Add 800 i l of SOC medium to tubes. Incubate the cells at 37 °C for 2 h with gentle agitation (150 rpm). 4. Centrifuge the cells at 40C for 5 minutes. Discard the supernatant. 5. Reconstitute the pellet in 200 i l of SOC medium. 6. These cells are then plated on LB agar plates containing antibiotic Ampicillin (100 mg/ml). For blue white screening of recombinants, the plates are also smeared with IPTG (200mg/ml, @ 40 i l/plate) and X-gal (80 i g/ml, @ 40 i

l/plate) before the plating of cells. After smearing with IPTG and X-gal, 50 i l of reconstituted cells (as in step 5) are poured on ampicillin positive agar plates. 7. Incubate the plates overnight at 370C. Then transfer the plate to 40C for enhancement of the blue colour. Recombinant clones are identified by the selection of blue/white colonies since the vector used in ligation is marked LacZ genetically. The lacZ gene is disrupted in recombinant; hence it could not utilize X-gal and gives white colour distinct from blue colour given by non¬recombinant colonies.

Identification of positive clones Even though blue/white screening can be used to determine if inserts are present, this technique can be used to determine insert size and/or orientation in the vector. Alternately, the presence of an insert and its size can be determined by growing each colony in liquid, the plasmid purified by a boiling or alkaline preparation protocol, digestion of the plasmid with restriction enzyme(s) that excises the insert, followed by separation by agarose gel electrophoresis.

Calculating transformation efficiency Transformation efficiency is the number of transformed cells per 1 μg of supercoiled plasmid DNA in a transformation reaction -colony forming units per microgram of DNA (cfu/ig). Procedure is outlined for calculating transformation efficiency given an initial concentration of .005μg/ μl plasmid and the following: 200μl CaCl2, 250 μl LB broth, 10 μl of plasmid added to culture, 100 μl of solution from the reaction tube spread on agar plate and 50 colonies on the plasmid ampicillin plate. Mass of the plasmid added to the plasmid transformation reaction tube. = Plasmid concentration x volume of plasmid added = .005 x10 = 0.05 μg plasmid in reaction Concentration of plasmid in the reaction tube = μg plasmid/total volume (broth + CaCl2)

= 0.05/ (200 + 10 + 250) = 0.0001 μg plasmid/ μ! Mass of plasmid in the solution added to the agar plate = 0.0001 μg plasmid/ μl x 100μl = 0.0^g plasmid Number of colonies on the LB/Amp plate per μg of plasmid = transformed colonies/ μg of plasmid = 50/0.01 = 0.01colonies per μg plasmid

Troubleshooting Problem

Possible cause

Suggested solution

Few or no colonies seen

Inefficient ligation

Review the cloning strategy, enzymes, and concentrations of both vector and insert. Set up a control for each step of the cloning procedure

Low DNA insert fragment concentration

Increase the insert fragment concentration. One can vary the molar ratio from 1:1 to 1:15 vector: insert

Wrong antibiotic concentration

Some plasmids with very low copy number require lower antibiotic concentrations. Check the antibiotic concentration that is optimal for your vector

Excess salts

DNA fragments should be dissolved in a low salt solution.

Ends of both vector and insert are dephosphorylated

Vector or Insert should have a 5" phosphate. If no phosphate group is present,

vector or insert can be phosphorylated using T4 Polynucleotide Kinase Presence of restriction enzymes or phosphatase remaining in ligation mix

Inactivate or eliminate any trace of restriction enzymes or phosphatase before ligating the DNA.

Poor transformation efficiency Contamination of DNA

Remove phenol, protein, detergents, and ethanol by ethanol precipitation or other method

Excess DNA

Use no more than 1 to 10 g of DNA in 5μL volume per 100 μL chemically competent cells

Improper storage of competent cells

Avoid repeated freeze/thaw cycles

Proper selection is not occurring

Check antibiotics used is the correct one & used at the proper concentration Ensure that the anti-biotic has not expired or been thawed for too long

Excess number of colonies

Calculation errors Selfligation of vector

Ensure that the correct dilution factors and DNA concentrations are used to calculate efficiency Use dephosphorylated vector

Satellite colonies

Degraded/expired Check expiration date of the

antibiotic antibiotic used. Avoid multiple freeze/thaw cycles Too many colonies on the plate

Plate the transformants at a higher dilution

Plates were Incubate the plates overnight at 37°C or incubated too long for 2-3 days at room temperature in the case of temperature sensitive plasmids Transformants Incorrect amplicon used

Gel purify PCR product contain incorr¬ect construct

Non-specific Gel purify PCR product PCR products are present Gel purify PCR product

Amplify PCR fragment using a high fidelity polymerase

Transformants Antibiotic levels are too low Increase the amount of do not contain antibiotic in agar plates plasmid Satellite colonies were used

Choose larger, well established colonies for analysis

References 1. Chung, C. T. et al. (1989) One-step preparation of competent E. coli: Transformation and storage of bacterial cells in the same solution. Proc. Natl. Acad. Sci. USA 86:2172-2175.

2. Sambrook, J., E. Fritsch and T. Maniatis (1989). Molecular Cloning: A Laboratory Manual. Second Edition. Cold Spring Harbor Press, Cold Spring Harbor, NY. 3. Sambrook, J. and D. Russell (2001). Molecular Cloning: A Laboratory Manual. Third Edition. Cold Spring Harbor Press, Cold Spring Harbor, NY. 4. Smith, H.O., D.B. Danner and R.A. Reich (1981). Genetic Transformation.Annual Review of Biochemistry 50: 41-68 5. Inoue et al. (1990). High Efficiency Transformation of E. coli Grown at 18 °C. Gene 96: 23-28 6. Hanahan, D. (1983). Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166: 557-580. 7. Hanahan, D., J. Jessee and F. Bloom (1991). Plasmid transformation of Escherichia coli and other bacteria. Method in Enzymology 204: 63-113. 8. Mahipal S, Arpita Y, Xiaoling M, Eugene A (2010). Plasmid DNA transformation in Escherichia Coli: effect of heat shock temperature, duration, and cold incubation of CaCl2 treated cells. Internal J. Biotechnol. Biochem. 6: 561-568. 9. Mandel M, Higa A (1970). Calcium-dependent bacteriophage DNA infection. J. Mol. Biol. 53: 159-162. 10. Nakata Y, Tang X, Yokoyama K (1997). Preparation of competent cells for high efficiency plasmid transformation of escherichia coli. Methods Mol. Biol. 69: 129-137 11. Sambrook, J. and D. Russell (2001). Molecular Cloning: A Laboratory Manual. Third Edition. Cold Spring Harbor Press, Cold Spring Harbor, NY. 12. Sambrook, J., E. Fritsch and T. Maniatis (1989). Molecular Cloning: A Laboratory Manual. Second Edition. Cold Spring Harbor Press, Cold Spring Harbor, NY. 13. Protocols and Applications Guide, Third Edition (1996) Promega Corporation.

Chapter16 Retrieving QTL & SNP Information from Databases A quantitative trait locus (QTL) is a polymorphic locus containing alleles that differentially affect the expression of a specific phenotypic trait. Quantitative traits are typically affected by more than one gene, and also by the environment. Many important livestock traits such as weight gain, milk fat content and milk yield are quantitative traits. The purpose of QTL research is to identify genes and gene variants that underlie these traits. QTL are identified via association of the studied traits with genetic markers. Research in QTL mapping has resulted in identification of several chromosomal regions containing genes predicted to be involved in manifestation of economic traits in livestock including production, reproduction and comμex diseases, such as mastitis, Johne's disease and many others. The eventual objective of QTL mapping is to identify the genes that underlie polygenic traits and to gain a better understanding of their physiological and biochemical functions. However, success has been limited in utilization of the wealth of QTL information for marker assisted selection. This is partly due to the inability to link QTL information to genomic information. One challenge is to find ways to integrate and visualize the QTL data that is produced by different types of QTL analyses, from different laboratories, and with different software tools. The recent availability of cattle, chicken and pig BAC finger printed contig maps and their emerging genome sequence information provide useful tools for the elucidation of QTL information. Integration of these map resources provides useful tools for positional identification of candidate genes. Hu et al., 2013 designed the Animal QTL database (QTLdb; http://www.animalgenome.org/QTLdb) including all publicly available QTL and single-nucleotide

polymorphism/gene association data on livestock animal species. Presently available structural genomics information for positional QTL information mining is integrated in this comprehensive QTL database tool. To date, there are 8,402 QTLs from 356 publications representing 626 different traits in pigs, 7,091 QTLs from 404 publications representing 441 different traits in cattle, 3,808 QTLs from 191 publications representing 296 different traits in chicken, 789 QTLs from 90 publications representing 219 different traits in sheep and 127 QTLs from 10 publications representing 14 different traits in rainbow trout in the database. Animal QTL database can be used to find QTL's for pig, cattle, sheep, chicken and rainbow trout by chromosome location/ trait classes. Researchers can utilize the database for finding single trait QTL on multiμe chromosomes or multiμe QTL on single chromosome. Further the database can be used to find experiment/ publication details that produced a QTL or all QTL from a particular publication. One may also find all locations that QTL for a trait may have been mapped to or find related traits from a known QTL trait. Most importantly DNA sequences/ SNP's associated with certain markers can also be viewed in Animal QTL database. Here in this exercise, we use the AnimalQTLdb to retrieve QTL and DNA sequence/ SNP in cattle for Somatic Cell Count (SCC) which is an indicator trait for mastitis in cattle. Steps in accessing and retrieving the information in the QTLdb Step 1 : Access the Animal QTLdb at http://www.animalgenome. org/QTLdb.

Step 2 : Select the Cattle QTL in main browser window

Step 3 : Select the option Browse - By cattle trait hierarchies

Step 4 : In option 2: select the health traits and in this select mastitis and then Somatic Cell count

As is clear, one can browse QTL for various economic traits in cattle by selecting the appropriate option in the browser window. Step 5: Somatic cell count QTL are disμayed in the browser Red QTL lines represent for significant and light blue lines for suggestive statistical evidence. SCC QTL on BTA 26 is selected is shown (encircled)

Step 6: Clicking the selected QTL opens the page with comμete QTL description including map information with peak location, key reference and study design for QTL identification.

Step 7: Go to SNP database (dbSNP) in NCBI (http://www.ncbi.nlm.nih.gov/snp) and search for peak loci, here BMS882 It gives the respective SNP listed in database, here rs (reference SNP) 134801808 present in Bos taurus is shown.

Step 8: In the detailed report page of SNP rs 134801808, retrieve the sequence by selecting 'Reveal marker in - Sequence view'.

The retrieved sequence can be utilized for several apμications including SNP mining in populations, bioinformatics analysis, population studies etc. References

1. Zhi-Liang Hu, Carissa A. Park, Xiao-Lin Wu and James M. Reecy (2013). Animal QTLdb: an improved database tool for livestock animal QTL/ association data dissemination in the post-genome era. Nucleic Acids Research, 41 (D1): D871-D879 2. Zhi-Liang Hu, Carissa A. Park, Eric R. Fritz and James M. Reecy (2010). QTLdb: A Comprehensive Database Tool Building Bridges between Genotypes and Phenotypes. Invited Lecture with full paper published electronically on The 9th World Congress on Genetics Apμied to Livestock Production. Leipzig, Germany August 1-6, 2010. 3. Sherry ST, Ward M, and Sirotkin, K. (1999). "dbSNP - database for single nucleotide polymorphisms and other classes of minor genetic variation". Genome Research 9 (8): 677-679.

Chapter 17 DNA Microarray Introduction Over the past century, scientists have studied cause-and-effect relationships between known genes and biological phenotypes. Recent technological advances have changed the landscape of biomedical research. The comμete genomes of several organisms are now available, and the expression of tens of thousands of genes may be quantified. There are several reasons why qualitative and quantitative determination of transcript patterns is of significantly important in molecular biology. By comparing the concentrations of individual transcripts present in samμes originating from different genotypes, developmental stages or growth conditions, genes can be identified that are differentially expressed and hence may have specific metabolic or morphogenetic functions (De Saizieu et al., 1998). Microarrays are simμe assays that measure the relative expression levels of different genes simultaneously (Lashkari et al., 1997). The most accessible approach for producing an oligonucleotide microarray is to synthesize individual oligonucleotides and subsequently immobilize them to a solid surface (Schena et al., 1998). For this immobilization to take μace, the oligonucleotides must be modified with a functional group in order to have attachment to a reactive group on a solid surface. Oligonucleotides can be attached to flat two-dimensional surfaces, such as glass slides, as well as to three¬dimensional surfaces such as microbeads and micro-spheres. Some of the modifications in oligonucleotides that help its attachment with surface or beads include amine-modified oligos covalently linked to an activated carboxylate group or succinimidyl ester, Thiol-modified oligos covalently linked via an alkylating reagent such as an

iodoacetamide or maleimide, AcryditeTM-modified oligos covalently linked through a thioether, Digoxigenin NHS Ester, Cholesterol-TEG, Biotinmodified oligos captured by immobilized Streptavidin etc. (Schena et al., 1996). Different modifications allow immobilization onto different surfaces: Modification

Surface treatment

Amino modified oligos

Epoxy silane or Isothiocyanate coated glass slide

Succinylated oligos

Aminophenyl or Aminopropyl-derivatized glass slide

Disulfide modified oligos

Mercaptosilanized glass support

Two-dimensional surfaces (Microarray slides) are usually silicon chips or glass microscope slides. Glass is a readily available and inexpensive support medium that has a relatively homogeneous chemical surface. Some of the three dimensional micro-spheres include, polystyrene micro-spheres, magnetic micro-spheres and silica micro¬spheres. Oligonucleotide length and melting temperature Identical length Oligos and equivalent hybridization buffers can be used to diminish the effect of GC content on melting temperature. Otherwise oligo can be designed with nearly identical predicted melting temperatures in traditional buffers such as 6xSSC. hybridisation buffer containing tertiary amine salts, like tetramethyl ammonium chloride (TMACI) or tetraethyl ammonium chloride (TEACI) to achieve GC content independent of hybridization (Khrapko et al., 2000). Oligonucleotide design Several softwares do exist for designing of oligonucleotides. Instead of using single software a combination of multiμe software (ARB phylogenetic software package, CalvOligo and Excel) can give the desired result. Labeling Amino Modified Oligonucleotides

This general procedure can be used to conjugate amino-modified oligonucleotides with active succinimidyl ester or isothiocyanate derivatives of various ligands, such as fluorescent dyes. At pH 9.0 the conjugation reaction occurs virtually exclusively at the free primary amine and does not involve the exocyclic amino groups of the nucleosides. 1. For a 250 nmole scale synthesis, resuspend the amino-modified oligonucleotide (i.e. approximately 100 nmoles of primary reactive amine) in 0.7 μ? of sterile distilled water. 2. Add 100 μ? of 10X conjugation buffer (1.0 μ Na HCOq)/ Na,COq, pH 9.0). 3. Freshly prepare a 10 mg/mL solution of active ester in DMF. Add 200 μ? of the solution to the reaction mixture. 4. Allow the mixture to stand at least 2 hours. 5. Remove the unreacted ligand by gel filtration. 6. Depending on the reactivity of the NHS-ester used, couμing efficiency can range from 20-80%. Labeled-oligo can be purified from unreacted oligo by preparative RP-HμC purification.

Preparation of DNA Microarray Printing Solution The DNA microarray printing solution consists of PCR products at a final concentration of greater than 100 ng/ml (preferably greater than 200 ng/ml). The concentration of the PCR products is determined using the suitable available method. PCR Amμification of DNA Materials and Reagents 96-Well polypropylene, V-bottom Microμate 5 mM dNTPs 25 mM MgCl2 Taq/ Pfu DNA Polymerase Appropriate DNA Primers 10X PCR buffer (300 mM Tricine pH 8.4, 500 mM KCl) Distilled water

PCR Products Clean up A. Ethanol Precipitation Method Materials and Reagents 3M sodium acetate pH 5.2 95% ethanol 80% ethanol 3X SSC, dilute from 20X SSC 3M Scotch aluminum tape μastic wrap Multichannel pipette Dry down 100 ml the PCR products to approximately 50 ml in a SpeedVac at low temperature setting (approximately 1% hour). Add 5 ml 3M sodium acetate pH 5.2 and 150 ml of 95% ethanol per well. Precipitate DNA at -20°C overnight. Centrifuge at 2,750 x g at 4°C for 60 min. Remove supernatant by rapidly inverting μates, then blotting on paper towel. Be careful otherwise pellets may be lost. Wash pellet with 100 ml 80% ethanol. Centrifuge at 4°C, 2,750 x g for 45 min. Remove supernatant by carefully inverting μates. Tap μates gently on paper towel to remove ethanol. Let μate sit open in a laminar flow hood to allow remaining ethanol to evaporate. This takes anywhere from 30min to 2 hrs. Use speedvac if needed, but do not overdryl Add 10-30 ml of filtered 3X SSC or other appropriate printing buffer. Seal μate with 3M Scotch aluminum tape. Wrap μate in wet paper towels, then μastic wrap. Store at 4°C for 48 hrs to allow DNA to go into solution. Store μates with DNA solutions at -20°C in a non-defrosting freezer. B. Millipore Multiscreen Method Materials and Reagents Multiscreen Vacuum Manifold

Multiscreen PCR μates 3X SSC, dilute from 20X SSC TE pH 7.5 Multichannel pipet 96-Well polypropylene, V-bottom Microμate 3M Scotch aluminum tape μastic wrap Ziμoc μastic bags At the conclusion of the PCR reaction, pipette the reaction products (100 μl) into the 96 well Millipore Multiscreen PCR μate. μace the Multiscreen μate on top of the Multiscreen Vacuum Manifold and apμy vacuum at 20 inches of Hg for 10 min until no liquid remains in the wells. Wash by adding 120 μl water to each well and apμy vacuum again for 13 min. To resuspend PCR products, add 35 μl of 3X SSC to each well. Cover μate with cover provided and shake vigorously on a μate shaker for 10 min. Remove as much as possible of the resuspended products (recovery about 30 μΓ) from each well using a multichannel pipette and μace in a new, 96 well μate for storage. Seal μate with 3M Scotch aluminum tape, wrap in moist paper towels and μastic wrap, then μace it in a Ziμoc μastic bag. Store at -20°C (non-defrosting freezer). Quality Control (QC) of PCR Products QC involves two parts Agarose gel analysis is used to examine the quality of each PCR. Failed PCR or PCR with multiμe products will be recorded for flagging purpose. The concentration of the DNA is determined by printing solution. Preparing to Print Day 0 -

Array the oligonucleotides into 384-well μates with identical volumes per well (typically 10-20 μl per well). If the oligonucleotide solutions were frozen, thaw the master μates overnight at 4oC. If the oligonucleotides were dried, resuspend them in 3x SSC, and let them incubate overnight at 4oC. Day 1 μace slides gently onto the arrayer μatter. Blow dust off the slides with compressed nitrogen. If the arrayer has a sonicator water bath for cleaning the pins, fill it with fresh water. Transfer four 384-well oligonucleotide-containing print μates from 4oC, and let them come to room temperature for at least 1 h. Centrifuge the μates at 1000 rpm for 2 min to remove condensation from the μate covers. Place a plate into the spotting robot's μate holder. Printing the Microarrays Start the print run. Keep careful notes about μate order and orientation. When a μate is finished printing and the print head has come to a comμete stop, either let the μate evaporate in a hood if the μates are stored dry, or cover the μate with foil and its lid and store the μate at 80°C. Insert the next print μate into the μate holder. When the print run is done, let the slides dry overnight, unless you are performing back-to-back print runs. Day 2 Transfer all slides from the arrayer into a μastic slide box. Store them in a desiccator. Power down the arrayer (Maier et al, 1994). RNA Extraction Protocol Snap Freezing in Liquid Nitrogen The tissue should be kept in the liquid nitrogen until further processing. Immersion in RNA Later

Upon extraction from the animal, immediately slice the tissue into pieces no wider than 0.5 cm and drop into RNA Later. (The volume of RNA Later should be at least ten times the volume of tissue) Homogenization For tissues that are snap frozen or slightly in excess, the homogenization of the tissue should be done by mortar and pestle (cooled to temp in a liquid nitrogen bath) with the simultaneous transfer at least 1ml TRIzol/100mg tissue to be homogenized into a falcon tube Transfer the tissue to the pestle and grind until a layer of very fine dust is left. Once homogenized, aliquot the solution to eppendorf tubes. Phase Separation Add 200 μl chloroform/1 mL TRIzol, vortex for 15 seconds, and leave at room temp for 2-3 minutes. Centrifuge samμes at 12,000g for 15 minutes at 2-80C. RNA Precipitation Following centrifugation, there will be three phases visible within the tube. Transfer the aqueous phase (top) to a fresh tube, being careful not to contaminate the solution with the other phases. Add 500 μl isopropanol/1mL TRIzol to the new tube and incubate at room temp for 10 minutes. Centrifuge samμes at 12,000g for 10 minutes at 2-8°C. RNA Wash and Re-suspension Following centrifugation, remove the supernatant. Wash RNA pellet with 80% EtOH/1ml TRIzol and vortex. Centrifuge samμes at 7,500g for 5 minutes at 2-80C. Remove supernatant. Allow remaining EtOH to air dry for 2-3 minutes. Transfer tubes to 700C heat block and let sit for 2-3 minutes. Dissolve the pellet in 81 μl of DEPC water.

DNase Treatment Add 8 μl of 10X DNase I Buffer. Add 2 μl of DNase I Enzyme. Vortex, quickly spin and incubate at 420C for 25 minutes. RNeasy Column Purification (Using Qiagen’s RNeasy Protocol) Add 350 μl Buffer RLT (with BME-10 μ/ml Buffer RLT). Add 250 μ 100% EtOH. Apμy entire volume to RNeasy column and spin full speed for 1 minute. Reapμy entire volume to RNeasy column and spin full speed for 1 minute. Transfer column to new 2ml collection tube. Add 750 μ Buffer RPE and spin full speed for 1 minute. Discard flow-through and add 750 μ Buffer RPE and spin full speed for 1 minute. Discard flow-through and spin full speed for 1 minute. Transfer column to new labeled 1.5 ml Eppendorf tube. Add 56 μ DEPC H2O and let sit for 2 minutes. Spin at full speed for 2 minutes. Discard column and transfer tube to ice. Alternate method for DNase treatment of Total RNA 1. Reaction solution 60 pg Total RNA 3 μ RNase inhibitor 10 μ 10X PCR buffer 6 μ MgCl2 (25mM) 20 μ DNase (Promega, 10U/μ) DEPC treated water upto 100 μ 100 μ Total reaction volume 2. Incubate at 370C for 30 minutes 3. Add 100 μ Phenol: Chloroform: Iso-amyl Alcohol (25:24:1) pH 4.5-5.2 4. Vortex for 30 sec 5. Spin for 5 minutes at room temperature at 14,000 RPM 6. Transfer upper phase to a new centrifuge tube

7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

Add 10 μ of 3M NaoAc (pH 4.5-5.2) and 200 μ 100% ethanol Mix by inverting several times Put in Dry ICE/ ethanol bath for 30 minutes or -80oC for 1 hour Spin for 15 minutes at 4oC Remove supertnatant with a pipette very carefully Resuspend pelleted RNA using pipette in 500 μ of 70% Ethanol Spin for 10 minute at 400C at 14000 RPM Remove supernatant with a pipette Aspirate remaining drops Air-dry pellet for 5-10 minute at room temperature Resuspend RNA pellet in 40 μ DEPC treated water Specification at OD260-280 using 2 μ in 198 μ of water.

Quantification and Quality Control Quantify each samμe using Nanodrop (or equivalent spectro¬photometer). Run 5 μ of each samμe on Agarose gel. cDNA Labeling Procedure Dilute the RNA samμe with DEPC-H2O to a final volume of 11. 5 μ. A typical labelling reaction require 5-8 pg total RNA for bacterial arrays and ~ 50 pg total RNA for Mouse Genome arrays. Add 1.5 μ random primers (stock concentration of 3pg/ μ). For the mouse genome arrays Oligo dT primers can be used in μace of random primers. Heat samμes to 70oC for 5 min., then snap-cool on ice. Make up master mix of the following reaction components, excluding the reverse transcriptase. 5X First-Strand Reaction Buffer

- 5.0 μl

100 mM DTT

- 2.5 μl

dNTP mix (5 mM each of dATP, dGTP, dCTP, 0.5 mM dTTP)

- 2.5 μ l

Cy3-dUTP (control) or Cy5-dUTP (samμe)

- 1.0 μl

Reverse Transcriptase (SUPERSCRIPT™ II)

- 1.0 μl

Add 12 μ of master mix including reverse transcriptase to each samμe. Mix, then centrifuge briefly to collect contents at the bottom of the tube. Incubate at 25oC for 10 min. and then 42oC for 90 min. Degrade RNA by addition of 5 μ 1M NaOH and incubate at 65-70°C for 15 minutes. Neutralize by adding 5 μ 1M HCl (can wait for samμes to cool before adding HCl, if necessary). Add 400 μ TE to a Microcon 30 and allow to stand for 10 minutes. Combine the appropriate Cy3- and Cy5-labelled samμes (If you will be performing a co-hybridization on the array). Add the combined probes to the Microcon 30 filter. Centrifuge at 12000 x g until nearly all of the volume is pushed through the filter. Repeat the buffer exchange once by adding a fresh 400 μ filtered TE and centrifuge as before. Check the volume periodically until there is only about 5-10 μ of samμe remaining in the filter unit. Recover the labelled cDNA by inverting the Microcon 30 filter into a new 1.5 ml collection tube and centrifuge at 10,000 x g for 2 minutes. Adjust volume to approx. 11 μ (De Risi et al., 1996). Genomic DNA Labeling Protocol Genomic DNA can be labeled with a simμe random-priming protocol based on Gibco/BRL's Bioprime DNA Labeling kit. Add 2 pg DNA of the samμe to be labeled to an eppindorf tube. Add ddH20 or TE 8.0 to bring the total volume to 21 μ. Then add 20 μ of 2.5X random primer/reaction buffer mix. Boil 5 min, then μace on ice. 2.5X random primer/reaction buffer mix: 125 mM Tris 6.8, 12.5 mM MgCl2, 25 mM 2-mercaptoethanol, 750 pg/ml random octamers. On ice, add 5 μ 10X dNTP mix. 1.2 mM each dATP, dGTP, and dTTP, 0.6 mM dCTP, 10 mM Tris 8.0, 1mM EDTA. Add 3 μ Cy5-dCTP or Cy3-dCTP. Add 1 μ Klenow Fragment. Incubate at 370C for 1 to 2 hours, then stop reaction by adding 5 μ of 0.5 M EDTA (pH 8.0).

As with RNA probes, purify the DNA probe using a Microcon 30 filter (Chee et al., 1996). Microarray hybridization (Amine coated slides) Pre-hybridization of arrays - The pre-hybridization of arrays takes μace during the Post Processing Procedure. Preparation of 2X Hybridization solution : Formamide 50 μ 20X SSC

50 μ

10% SDS

2 μ]

Again, add the 10 % SDS solution last to avoid precipitation Mix labelled targets with blocking reagents : Cy-3 and Cy-5 labelled cDNA/RNA 11 μ Yeast tRNA, 4 μg/ μ2

1 μ2

Hybridization arrays Add appropriate amount of 2X Hybridization Solution to nucleic acid mixture. Heat to 980C for 2 min. Snap cool on ice (briefly since SDS should not precipitate). Centrifuge at 12,000 x g briefly to collect samμe in bottom of tube. PLace new cover (lifter) slip over region of array on slide and apμy mixture under cover (lifter) slip. Apply 15 μ H2O to each well in the hybridization chamber to add humidity during incubation. Place array slide in microarray hybridization chamber, seal and incubate submerged under water or in hybridization oven at 420C overnight (minimum of 15 hours). Washing

Remove lifter slip while slide is submerged horizontally in the first wash solution. 1X SSC + 0.05% SDS 20X SSC

30 ml

10% SDS

3 ml

ddH2O

567 ml

Total

600 ml

Transfer slides to the second wash solution. Transfer slide rack to fresh 2nd wash and wash for an additional 2 minutes. Carry slides to centrifuge in final wash to prevent evaporation, and then transfer to the μate rack lined with a paper towel and spin @ 500-800 rpm for 3 minutes to dry. Scan slides as soon as possible. Image scanning After being hybridized and washed, the microarray is scanned by means of a dual-wavelength confocal laser scanner. The most common configuration of focal scanner utilizes laser excitation of small region of glass substrate, such that the entire image is gathered by moving the substrate or the confocal lens. Light emitted from the fluorescent samμe at each location is separated from unwanted light using a series of mirrors, filter and lenses, and the light is converted to an electrical signal with a photomultiμier tube (PMT) or an equivalent detector. For fluorescent signals to be detected, wavelengths of 532 nm and 635 nm are required for Cy3 and Cy5, respectively. Scanning of the hybridized microarray should be carried out immediately after the washing, because the fluorescent dyes lose signal intensity with time. Repeated scanning of the microarray also causes a decrease in fluorescent signal intensity, particularly for Cy5. For an accurate comparison of two samμes, the scanned signal intensities of Cy3 and Cy5 (van Hal et al., 2000) should be at the same level. Because the starting RNA volumes of the two samμes may not be exactly the same in most cases, the signal intensities of Cy3 and Cy5 must be adjusted to

be as close as possible, with the use of sets of positive control genes. Data interpretation Once the fluorescent emission from the microarrays is converted into digital output by the detection system, the data files are quantitated and interpreted. Quantitation is usually accomμished by superimposing a grid over microarray image and computing an average intensity value for each microarray element with automated software. Intensity values can be converted into relevant biological output such as number of mRNA per cell, by comparing the experimental and control elements present in a given microarray. Quantitative gene expression genotyping and other outputs are then correlated with the gene sequences represented in the microarray and higher older relationships such as coregulation and gene regulatory network can be identified (Fambrough et al., 1999; Feng et al., 1999; Zhao et al., 2000). Application of Microarray Technology 1. Patterns of expression analysis The idea that the global transcriptional response constitutes molecular phenotypes has recently received attention (Fathallah- Shaykh et al., 2003; Marton et al., 1998). The phenotypes are created by molecular systems in which single genes or molecules belong to rich networks of dynamic molecular interactions that include transcriptional regulation, signaling pathways, protein-protein, and protein-nucleic acid interactions (Hood, 2003). Theoretically, one could apμy microarrays to discover new molecular classifications of diseases, to study and define the molecular systems that create each individual phenotype, and to perturb the network to find the best targets that transition the whole system between phenotypes. The classic gene expression techniques, such as Northern or Southern blot analysis, are limited in their ability to evaluate the expression patterns of multiμe genes. However, microarray provides a better promise for comprehensive monitoring of gene expression in different cell or tissue. 2. Single Nucleotide Polymorphisms (SNPs) identification Due to advances in genomic studies, more and more single nucleotide

polymorphisms (SNPs) are found to be contributory factors for different diseases and can be used as genetic markers for molecular diagnostics. The apμication of microarray technologies to identify specific targets of defined genes that have clearly been imμicated in progression of diseases or development of particular phenotypes have been conventionally used.

3. Veterinary diagnostics

DNA microarrays can be used to detect multiμe pathogens based on differences in 16S rDNA sequences (Greisen et al., 1994). For examμe, nucleic acids can be extracted from a samμe and 16S rDNA sequences amμified by PCR using universal 16S primers. The resulting PCR products can be hybridised to an array consisting of many oligonucleotide probes, which can be designed to detect and characterize pathogens by taxonomy if sufficient discriminatory sequences are available (Klausegger et al., 1999). 4. Pharmacological industry and microarrays There are three major tasks with which the pharmaceutical industry deals on a regular basis: (a) to discover a drug for an already defined target, (b) to assess drug toxicity, and (c) to monitor drug safety and effectiveness. Relief was found with the increase in the knowledge of total genomic sequences of many prokaryotes and eukaryotes. These represent gold mines for drug targeting and discovery. Accordingly, there was an urgent need to develop high throughput monitoring technologies that are meant to identify targets and provide lead candidate optimization. Accordingly, there was a call for developing a technology that can help in basic research, drug discovery and evaluation. Gene expression microarray allows the simultaneous monitoring of the expression levels of thousands of genes (Wodicka et al., 1997; Fodor et al., 1993). This is an advantage that makes microarrays suitable for measuring the expression patterns of thousands of genes in parallel and generating clues to gene function that can help to identify appropriate targets for therapeutic intervention. Microarray also can be used to monitor changes in gene expression in response to drug treatments (Ivanov et al., 2000; Yeakley et al., 2002; Debouck et al., 1999). This makes gene chips the current frontrunner technology in the drug industry. Concluding remarks Microarray technology is becoming a corner stone in the development and integration of knowledge obtained from molecular targets. There are many microarray techniques with clinical apμications. Arrays have added an important dimension to the breadth and depth of knowledge that is obtained. The future of microarrays seems even more promising to the life sciences than PCR technology. Couμed with advanced Informatics, microarray diagnostics will furnish individualized investigation of the physiology and disease patterns of individuals and may be able to dispense the need for large,

costly and often difficult to interpret clinical trials. Despite these phenomenal accomμishments and further potential there was a major drawback to defeat; microarrays are entirely dependent on the state of knowledge of the genome under investigation. But this is fading as a principal problem due to comμetion whole genome projects of different species. Another criticism that has yet to be resolved is the concern that the expression levels achieved in these artificial systems are not physiologically relevant. Several methodological challenges to the practicality of microarrays do exists;but there seems little concern that these problems will yield in the foreseeable future. Looking at the larger picture it can be predicted that microarrays are on their way to revolutionize Bioscience and to allow us to understand how life really works. Troubleshooting Problem

Possible cause(s)

Suggested solution

Hybridization Follow the manufacturer's instructions system carefully Degraded reagents

Use high-quality reagents when performing hybridization experiments

Improper protocol

Follow the latest version of the

High Background

User Guide Improper pre- Use the proper amount of BSA hybridization during pre-hybridization. The BSA should buffer be at least molecular biology grade, as electrophoresis grade leads to insufficient blocking

Spot Mishandling Heterogeneity Poor signal

Follow the latest version of the User Guide

Slide scanned Take care to load the slide into

Poor differential signal

incorrectly

the scanner with the proper orientation

Non-specific binding

Ensure proper temperature for hybridization

References 1. Chee M, Yang R, Hubbell E, Berno A, Huang XC, Stern D, Winkler J, Lockhart DJ, Morris MS, Fodor SPA (1996). Accessing genetic information with high- density DNA arrays. Science 274: 610614. 2. De Risi J, Penland L, Brown PO, Bittner ML, Meltzer PS, Ray M, Chen Y, Su YA, Trent JM (1996). Use of a cDNA microarray to analyze gene expression patterns in human cancer. Nature Genetics 14: 457-460. 3. De Saizieu A, Certa U, Warrington J, Gray C, Keck W, Mous J (1988). Bacterial transcript imaging by hybridization of total RNA to oligonucleotide arrays. Nature Biotech 16: 45-48. 4. Debouck, C. and Goodfellow, P.N. (1999). DNA microarrays in drug discovery and development. Nature Genetics 21(1): 48-50. 5. Fambrough D, McClure K, Kazlauskas A, Lander ES. (1999). Gene expression profiling by DNA microarray Technology. Cell 97: 727-741. 6. Fathallah-Shaykh, H.M., He, B., Zhao, L.J., Engelhard, H.H., Cerullo, L., Lichtor, T., Byrne, R., Munoz, L., Von, Roenn, K., Rosseau, G.L., Glick, R., Sherman, C. and Farooq, K. (2003). Genomic expression discovery predicts pathways and opposing functions behind phenotypes. Journal of Biological Chemistry 278: 2383023833. 7. Fodor, S.P., Rava, R.P., Huang, X.C., Pease, A.C., Holmes, C.P. and Adams, C.L. (1993). Multiμexed biochemical assays with biological chips. Nature 364: 555-556. 8. Greisen, K., Loeffelholz, M., Purohit, A. and Leong, D. (1994). PCR primers and probes for the 16S rRNA gene of most species of

pathogenic bacteria, including bacteria found in cerebrospinal fluid. Journal of Clinical Microbiology 32:335-351. 9. Hood L. (2003). Systems biology: integrating technology, biology, and computation. Mechanisms of Ageing and Development 124: 916. 10. Ivanov, I., Schaab, C., μanitzer, S., Teichmann, U., Machl, A., Theml, S., Meier-Ewert, S., Seizinger, B. and Loferer, H. (2000). DNA microarray technology and antimicrobial drug discovery. Pharmacogenomics 1(2): 169-178 11. Khrapko KR, Khorlin AA, Ivanov IB, Chernov BK, Lysov Yu P, Vasilenko SK, Florent'ev VL, Mirzabekov AD (1991). Hybridization of DNA with oligonucleotides immobilized in gel: a convenient method for detecting single base substitutions. Molecular Biology 25: 581-591. 12. Klausegger, A., Hell, M., Berger, A., Zinober, K., Baier, S., Jones, N., Sperl, W. and Kofler, B. (1999). Gram type-specific broad-range PCR amμification for rapid detection of 62 pathogenic bacteria. Journal of Clinical Microbiology 37: 464-466. 13. Lashkari DA, DeRisi JL, McCusker, JH, Namath AF, Gentile C, Hwang SY, Brown PO, Davis RW (1997). Yeast microarrays for genome wide parallel genetic and gene expression analysis. Proceedings of National Academy of Science USA 94: 1305713062. 14. Maier E, Meier-Ewert S, Ahmadi AR, Curtis J, Lehrach H (1994). Apμication of robotic technology to automated sequence ?nger print analysis by oligonucleotide hybridisation. Journal of Biotechnology 35: 191-203. 15. Marton, M.J., DeRisi, J.L., Iyer, V.R., Meyer, M.R., Roberts, C.J., Stoughton, R., Burchard, J., Slade, D., Dai, H., Bassett, D.E. Jr, Hartwell, L.H., Brown, P.O. and Friend, S.H. (1998). Drug target validation and identification of secondary drug target effects using DNA microarrays. Nature Medicine 4: 1293-1301. 16. Schena M, Heller RA, Theriault TP, Konrad K, Lachenmeier E, Davis RW: Microarrays (1998). Biotech's discovery μatform for functional genomics. Trends in Biotechnology 16: 301-306.

17. Schena M, Shalon D, Heller R, Chai A, Brown PO, Davis RW (1996). Parallel human genome analysis: microarray-based expression monitoring of 1000 genes. Proceedings of National Academy of Science USA 93: 10614-10619. 18. Van Hal NL, Vorst O, van Houwelingen AM, Kok EJ, Peijnenberg A, Aharoni A, van Tunen AJ, Keijper J. (2000). The apμication of DNA microarrays in gene expression analysis. Journal of Biotechnology 78: 271-280. 19. Wodicka, L., Dong, H., Mittmann, M., Ho, M.H. and Lockhar, D.J. (1997). Genome-wide expression monitoring in Saccharomyces cerevisiae. Nature Biotechnology 15(13): 1359-1367. 20. Yeakley, J.M., Fan, J.B., Doucet, D., Luo, L., Wickham, E., Ye, Z., Chee, M.S. and Fu, X.D. (2002). Profiling alternative sμicing on fiber-optic arrays. Nature Biotechnology 20(4): 353-358. 21. Ying Ma, Changqun Cai, Lin Luo, Jiaqi Xie, Xiaoming Chen (2013). A resonance Rayleigh scattering detection of DNA hybridization based on interaction between DNA and surfactants. Analytical Methods 5 (11): 2688¬2693.

Chapter 18 In situ Hybridization In situ hybridization, also referred to a hybridization histochemistry, was introduced in 1969 by Gall and Pardue (Brady et al., 1990; Chan 1990). It is a powerful technique that allows to study the macroscopic distribution and cellular localization of DNA and RNA sequences in a heterogeneous cell population whereas normal hybridization requires the isolation of DNA or RNA, separating it on a gel, blotting it onto nitrocellulose and probing it with a comμementary sequence. During earlier times radioisotopes were the only labels available for nucleic acids, and autoradiography was the only means of detecting hybridized sequences. The basic technique of in situ hybridization utilizes the fact that DNA and RNA will undergo hydrogen bonding to comμimentary sequences of DNA or RNA remains the same. By labeling sequences of DNA or RNA of sufficient length (approximately 50-300 base pairs), selective probes can be made to detect particular sequences of DNA or RNA. In the past 40 years many improvements have been made on the basic in situ hybridization technique and its apμication has been carried out in all different tissues. A major advance in the method was achieved with the description in 1984 of in situ hybridization using single stranded RNA probe (Cox et al., 1984) otherwise known as riboprobes. Using in situ hybridization, it is possible to localize gene expression to specific cell types in specific regions and observe how changes in this distribution occur throughout development and correlate with behavioral manipulations. The sensitivity of the technique is such that threshold levels of detection are in the region of 10-20 copies of mRNA per cell. In spite of the high sensitivity and wide apμicability of in situ hybridization techniques, their use has been limited to research laboratories. This is probably due to the problems associated with radioactive probes, such as the safety measures required,

limited shelf life, and extensive time required for autoradiography. Other problems are the sequence to be detected will be at a lower concentration, be masked because of associated protein, or protected within a cell or cellular structure. Therefore, in order to probe the tissue or cells of interest one has to increase the permeability of the cell and the visibility of the nucleotide sequence to the probe without destroying the structural integrity of the cell or tissue. In situ Hybridization Preparation of RNase free slides 1. Soak the slides overnight in Dichrol at room temperature in a fume hood. 2. Wash the slides thoroughly to remove any residual Dichrol (Initially in tap water, then with distilled water). 3. Dry slides at 1500C for 20 minutes. 4. Dip slides in a 2% solution of APES (3-aminopropyltri- ethoxysilane) in dry acetone for 5 minutes. 5. Wash in 2 changes of acetone and three changes of DEPC-water. Dry overnight at 420C and store dry. Collection of Tissue 1. Collect tissue sections on RNAse - free slides coated with APES, dried in air for two hours and then stored at -200C. 2. Slides may be washed in DEPC-PBS following DEPC-water three times before storing at -200C. Preparation and sectioning of tissue 1. Prepare thin sections of 8-10 mm fixed in 4% paraformaldehyde or 10% phosphate buffered formalin and keep it room temp overnight. 2. Dehydrate the tissues after fixation in paraffin. 3. Make sections of 5-8 μm from paraffin embedded tissue blocks using rotary microtome onto precoated RNase free slides. 4. Dry slides at room temperature for 1 hour and incubate at 600C for 45 min.

5. For frozen tissue sectioning, bring the frozen tissue to -20oC from -800C. 6. Make sections of 5-8 μg using microtome onto precoated RNase free slides. 7. Dry the slides at room temperature for one hour followed by fixation with acetone for half an hour. Digoxigenin (DIG) labeling The DIG labeling method is based on a steroid isolated from digitalis μants (Digitalis purpurea and Digitalis lanata). Digoxigenin is linked to the C-5 position of uridine nucleotides via a spacer arm containing eleven carbon atoms. The DIG-labeled nucleotides may be incorporated, at a defined density, into nucleic acid probes by DNA polymerases (such as E.coli DNA Polymerase I, T4 DNA Polymerase, T7 DNA Polymerase, Reverse Transcriptase, and TaqI DNA Polymerase) as well as RNA Polymerases (SP6, T3, or T7 RNA Polymerase), and Terminal Transferase. DIG label may be added by random primed labeling, nick translation, PCR, 3'-end labeling/tailing, or in vitro transcription. Hybridized DIG-labeled probes may be detected with high affinity anti-digoxigenin (antiDIG) antibodies that are conjugated to alkaline phosphatase, peroxidase, fluorescein, rhodamine, or colloidal gold. Biotin labeling of nucleic acids Biotin can be used in the same way as digoxigenin; it can be detected by anti-biotin antibodies. However, streptavidin or avidin is more frequently used because these molecules have a high binding capacity for biotin. Generation of Digoxigenin probe 1. Synthesize the DNA probes labeled with DIG-UTP using probe synthesis kit 2. Set up the following reaction in 50 μl total volume: 5x Stratagene synthesis buffer 10 μl 1M DTT (RNAase free) 10mM NTPs/digoxygenin-UTP

0.5 μl 2.5 μ?

RNAsin 10 units RNA polymerase (T3, T7 or Sp6) 90 μ DNA temμate

2.5

DEPC-water

to 50 μ?

μg

Incubate at 37°C for 2 hours. 3. Remove 2 μl for mini-gel samμe. Add 20 units of RNAse-free DNAse and continue incubation for a further 10 minutes at 370C. Remove a second 2 μl samμe and check that DNA has been degraded on a 1% TBE mini-gel. 4. Add 52 μl of 'Stop' buffer to the reaction. 5. Separate unincorporated ribonucleotides on a Sephadex G50 spin column by spinning for 2 minutes on setting on the Lab benchtop centrifuge. 6. Transfer the purified probe to a clean tube. Add 1/9 volume of 3M NaOAc, pH 4.8 and 2 volumes of ethanol. Precipitate at - 80°C for 10 minutes. Spin down at 4°C for 15 minutes, wash the pellet in 95% EtOH/5% DEPC-water and spin again for 5 minutes. 7. Resuspend the pellet in 50 μl of an RNAse-free solution of 40mM NaHCO3/60 mM Na2CO3. Remove 1 μl samμe for OD260 measurement. Incubate at 600C for 35 minutes to hydrolyse the probe into small fragments (between 200-300 bp). 8. Precipitate hydrolysed probe again as in above. Resuspend the probe in hybridisation buffer to a final concentration of 10 μg/ ml. 9. Store probe at -200C until required. In situ Hybridisation on frozen tissue samμes 1. Prior to use warm the slides to room temperature for 15 min, dry at 500C for 15 min 2. Fixed it in 4% paraformaldehyde and rinse twice with DEPC treated PBS at room temperature for 5 min 3. Treat slides with 20 μg/ml Proteinase K in DEPC treated PBS at room temperature for 8-15 minutes

4. Stop digestion by rinsing twice with 0.2% glycine in PBS for 5 min each. 5. Wash once in DEPC-PBS at room temperature for five minutes. 6. Fix in 4% paraformaldehyde in DEPC-PBS for 15 minutes. 7. Rinse once in DEPC-water. 8. μace slides in an RNAse-free glass trough with a stir bar. 9. Add 250 ml 0.1 M RNAse-free triethanolamine-HCl pH 8.0. Add 0.625 ml acetic anhydride with constant stirring. Turn off stirrer when the acetic anhydride is dispersed and leave for a further 10 minutes. 10. Wash slides in DEPC-PBS at room temperature for five minutes. 11. Carry out prehybridisation in hybridization buffer at 600C for 90 min in a hybridization oven under moist conditions. 12. Drain prehybridisation buffer from the slides and reμace each section with sufficient hybridization buffer containing 2 μ^^ of gene specific digoxigenin-labeled DNA probe. 13. Heat the hybridization buffer containing probe to 950C for 10 min and quenched immediately on ice before apμying to tissue section. 14. Apμy cover slips. 15. Continue hybridization at 570C in hybridization oven. Washing Steps 1. Wash in 4 x SSC with 10mM DTT at room temperature for 10 minutes twice. 2. Wash in 2 x SSC with 10mM DTT at 500C for 15 minutes twice 3. Wash in 0.2 x SSC with 10mM DTT at room temperature for 10 minutes twice. 4. Wash in 0.1 Maleic acid buffer for 5 min at room temperature. 5. Incubate slides in 20% heat-inactivated sheep serum in PBT for 1-5 hours at room temperature. Enzymes Commonly peroxidase and alkaline phosphatase enzymes are used in in situ hybridization. With peroxidase (POD), use the diaminobenzidine (DAB)/imidazole reaction (Graham and Karnovsky,1966). With alkaline phosphatase (AP), use the 5-Bromo-4-chloro-3- indolylphosphate/Nitro-blue tetrazolium (BCIP/NBT) reaction.

Antibody visualization of Digoxygenin 1. Incubate with pre-absorbed anti-digoxygenin antibody diluted to a final concentration of 1:2000 at 40C overnight. 2. Wash three times with TBST for one hour each. 3. Wash twice with Alkaline Phosphatase buffer for one hour each. 4. For every ml of Alkaline Phosphatase buffer, add 4.5 μl of NBT and 3.5 μl of BCIP, and develop in the dark for 2-20 hours, depending on the abundance of the DNA/RNA. The product should usually be visible in an hour or two. 5. Wash twice in alkaline phosphatase buffer for five minutes each, and then wash at least three times in PTw buffered to pH 5.5 for 1 hour each in the dark, and then fix in MEMFA for an hour. Microscopy Bright field microscopy In brightfield microscopy the image is obtained by the direct transmission of light through the samμe. Evaluation of in situ hybridization results by brightfield microscopy is preferred for most routine apμications because the preparations are permanent. However, the sensitivity demanded by many apμications (e.g., single copy gene localization requires the detection of a few nanograms of DNA) may require more sophisticated microscopy. Dark field microscopy In the dark field microscope, the illuminating rays of light are directed from the side, so that only scattered light enters the microscope lenses. Consequently, the material appears as an illuminated object against a black background. Phase contrast microscopy Phase contrast microscopy exμoits the interference effects produced when two sets of waves combine. This is the case when light passing e.g., through a relatively thick or dense part of the cell (such as the nucleus) is retarded and its phase consequently shifted relative to light that has passed through an adjacent (thinner) region of the cytoμasm.

Digital imaging microscopy Digital imaging microscopes can detect signals that cannot be seen with conventional microscopes. Also, image processing technology provides enhancement of signal-to-noise ratios as well as measurement of quantitative data. Electron microscopy The resolution of microscopy is enhanced when electrons instead of light are used, since electrons have a much shorter wavelength (0.004 nm). The practical resolving power of most modern electron microscopes is 0.1 nm. Troubleshooting Problem Desired sequence is not identified

Possible cause(s) Probe unable to bind to target due to lack of sequence specificity

Faint signal Inefficient labeling of probe Damaged microscope High Probe binds background to other noise regions along with target

Suggested solution Improper tissue sectioning Optimize hybridization temperature Use alternate sequence or sense (rather than anti-sense) probes to determine whether the observed labeling pattern is specific to the target sequence Use multiple probes for the same gene sequence Follow appropriate procedure for labeling

Check microscope Pre-treat sections with non-labeled probe before treating with labeled probe Pre-absorb the tissue with specific non-labeled material like ficoll, bovine serum albumin, and polyvinyl pyrrolidone Apμy RNase to the tissue after hybridization to reduce background The probe concentration needs to be optimized as an excess of probe can yield a

nonspecific signal Increasing the hybridization and washing temperatures can improve signal specificity References 1. Wilson M.C. and Higgins G.A. (1990). In Situ Hybridization. Molecular Neurobiological Techniques Neuromethods 16: 239284. 2. Godard C.M. and Jones K.W. (1980). Improved method for detection of cellular transcripts by in situ hybridization Detection of poly (A) sequences in individual cells. Histocheestry 65: 291-300. 3. John H.A., Birnstiel M.L. and Jones K.W (1969). RNA-DNA hybrids at the cytological level. Nature 223: 582-587. 4. Lawrence J.B. and Singer R.H. (1985). Quantitative analysis of in situ hybridization methods for the detection of actin gene expression. Nucleic Acids Research 13: 1777-1799. 5. Rigby P.W.J., Dieckmann M., Rhodes C. and Berg P. (1977). Labeling deoxyribonucleic acid to high specific activity in vitro by nicktranslation with DNA polymerase I. Journal of Molecular Biology 113: 237-251. 6. Wallace R.B., Johnson M.J., Hirose T., Miyake T., Kawashima E.H., and Itakura K. (1981). The use of synthetic oligonucleotides as hybridization probes. Nucleic Acids Research 9: 879-894. 7. Wetmur J.G. and Davidson N. (1968). Kinetics of renaturation of DNA. Journal of Molecular Biology 31: 349-370. 8. Schwarzacher, T. and Heslop-Harrison, P. (2000). Practical in situ hybridization. 9. Brammer, S.P., Vasconcelos, S., Poersch, L.B., Oliveira, A.R. and Brasileiro- Vidal, A.C. (2013). Genomic in situ Hybridization in Triticeae: A Methodological Approach. ISBN 978-953-51-10903. 10. Langdale, J.A. (1994). In situ Hybridization. Springer Lab Manuals, 165¬180. ISBN 978-1-4612-2694-9. 11. Brady, M.A.W. and Finlan, M.F. (1990). Radioactive labels:

autoradiography and choice of emulsions for in situ hybridization: In Situ Hybridization: Princiμe and Practice (J.M. Polak & J.O'D. McGee eds.) Oxford: Oxford University Press. 12. Chan, VT-W and McGee, JO'D (1990). Non-radioactive probes: preparation characterization, and detection: In Situ Hybridization: Princiμe and Practice (J. M. Polak & J. O'D. McGee eds.) Oxford: Oxford University Press. 13. Cox, K.H., DeLeon, D.V., Angerer, L.M. and Angerer, R.C. (1984). Detection of mRNAs in sea urchin embryos by in situ hybridization using asymetric RNA probes. Developmental Biology 101:485-502. 14. Graham, R.C. and Karnovsky, M.J. (1966.) The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. Journal of Histochemistry and Cytochemistry 14: 291-30.2.

Chapter 19 Preparation of Required Buffers and Solutions RBC lysis buffer (IX) Ammonium chloride (NH4CI)

8.3 gm

Potassium bicarbonate (KHCO3)

1.0 gm

0.5 M EDTA (pH 8.0)

299 μl

Autoclaved and store at room temperature DNA extraction buffer 1 M Tris buffer (pH 8.00)

5 ml

5 M NaCl

40 ml

0.5 M EDTA buffer (pH 8.00) 2 ml Double distilled water

(up to) 500 ml

Autoclaved in batches of 100 ml and stored at room temperature Tris saturated phenol preparation 1. Measure the required volume of phenol, add 8-hydroxyquinoline to a final concentration of 0.1% (it is an anti-oxidant gives yellow colour to phenol) 2. Extract phenol once/twice with equal volume of 0.5 M Tris base (pH 10.5). 3. Then with equal volume of Tris base pH 8.0 until the pH of the phenol phase is >7.8

4. Add 0.2% β-mercaptoethanol and mix (antioxidant and cleaves disulfide bond) 5. Finally, 0.1 M Tris base (pH 8.0) is added to about l/3rd volume of phenol and store in amber coloured bottle at 4°C. Chloroform : isoamyl alcohol preparation (24:1) Chloroform

24 ml

Isoamyl alcohol

1 ml

Mix thoroughly and store in amber coloured bottle at 4°C. Phenol : chloroform: isoamyl alcohol preparation (25:24:1) Chloroform: Isoamyl alcohol (24:1) 25 ml Tris saturated phenol

25 ml

Mix thoroughly and store in amber coloured bottle at 4°C. DEPC treated water Add 0.05-0.1% (v/v) DEPC to double distilled water Mix and incubate on orbital shaker at room temperature overnight Then autoclave the water 2.7% EDTA solutions (pH 8.0) EDTA disodium salt

2.7 g

Double distilled water (up to)

100 ml

Adjust pH 8.0 using NaOH pellets Sterilize by autoclaving and store at room temperature 0.5 M EDTA solution (pH 8.0) EDTA disodium salt

186.1 g

Double distilled water (up to)

100 ml

Adjust pH 8.0 using NaOH pellets Autoclave and store at room temperature 5 M NaCl solution

Sodium chloride

29.22 g

Double distilled water (up to)

100 ml

Autoclaved and stored at room temperature 3 M sodium acetate Sodium acetate (anhydrous)

24.6 g

Double distilled water (up to)

100 ml

(Adjust pH 5.5 using glacial acetic acid) Autoclaved in batches of 20 ml 70% ethanol Ethanol

70 ml

Autoclaved double distilled water

30 ml

Mix thoroughly and store in amber coloured bottle at 4°C 1 M Tris HCI (pH 8.0) Tris HCl

157.6 g

Doubled distilled water (up to) 1000 ml Adjust pH 8 using NaOH pellets Autoclave and store at 4°C 10X TBE Buffer (Tris-Borate-EDTA) Tris Base

108.0 g

Boric Acid

55.0 g

EDTA (pH 8.0)

7.5 g

Autoclaved distilled water (up to) 1000 ml 6X Gel loading dye Sucrose Based Bromophenol blue

0.25%

Xylene cyanole FF

0.25%

Sucrose in water

40% (w/v)

Mix and store at 40C

Glycerol Based Glycerol in distilled water

30%

Bromophenol Blue

0.25%

Xylene cyanol FF

0.25%

Mix and store at 40C Ficoll® Based Ficoll® polymer in distilled water

15%

Bromophenol Blue

0.25%

Mix and store at room temperature Ethidium bromide Ethidium bromide

10 mg

Autoclaved distilled water (up to) 1 ml Wrap in aluminium foil and store in dark μace at ambient temperature Alkaline Lysis Solution I 50 mM glucose 25 mM Tris-Cl (pH 8.0) 10 mM EDTA (pH 8.0) Prepare Solution I from standard stocks in batches of approx. 100 ml, sterilize by autoclaving and store at 4°C. Alkaline Lysis Solution II 0.2 N NaOH (freshly diluted from a 10 N stock) 1% (w/v) SDS Prepare Solution II fresh and use at room temperature Alkaline Lysis Solution III 5 M potassium acetate 60.0 ml

Glacial acetic acid,

11.5 ml

H2O

28.5 ml

The resulting solution is 3 M with respect to potassium and 5 M with respect to acetate. Store the solution at 4°C and transfer it to an ice bucket just before use LB Media NaCl

10 g

Tryptone

10 g

Yeast extract

5

g

Deionized H2O to 950 ml To prepare LB (Luria-Bertani) medium, shake the ingredients, mentioned above with Distilled water until the solutes have dissolved. Adjust pH to 7.0 with 5 N NaOH and make up the final volume of the solution to 1 litre with deionized H2O. Then sterilize it for 20 minutes by autoclaving at 15 psi. SOC medium 2% tryptone 0.5% yeast extract 10 mM NaCl 2 mM KC1 10 mM MgCl2 10 mM MgSO4 20 mM glucose (adjust pH to 6.8 to 7.0) Ampicillin 10 mg/ml. Filter sterilise (do not autoclave). Store at -20oC for up to 1 year. Use ampicillin at a final concentration of 0.1 mg/ ml (i.e. 1 ml of 10 mg/ ml stock solution per 100ml of media). TE 10mM Tris pH 8.0 with HCl, 1mM EDTA For 1 liter: Dissolve 1.21g Tris base and 0.37g EDTA 2H2O in 800ml of ddH2O. Adjust the pH to 8.0 with HCl. Adjust the volume to 1 liter with ddH2O.

Stop Buffer (in situ hybridization) 1% SDS 20mM EDTA 20mM Tris pH7.5 100mM NaCl 1M Triethanolamine, pH 8.0 Add 66.5 Triethanolamine and 20 ml conc. HCl to 413.5 ml DEPCwater in an RNAse-free bottle. PTw 1x PBS 0.1% TWEEN-20 PBT 1x PBS 2 mg/ml BSA 0.1% Triton X-100 Hybridization Solution For 100 ml 50% Formamide

50ml

5x SSC

25ml

1 mg/ml Yeast tRNA in DEPC-H2O 2ml 100 μg/ml Heparin

10mg

1x Denhardt's Solution

1ml

0.1% Tween 20

0.1ml

0.1% CHAPS

0.1g

5mM EDTA

2.5ml

NBT 75 mg/ml Nitro blue tetrazolium in 70% dimethyl formamide and 30% water. BCIP 50 mg/ml 5-bromo-4-chloro-3-indoyl phosphate in 100% dimethyl formamide

TBS 500mM NaCl 20mM Tris, pH 7.5 TBST 500 mM NaCl 20 mM Tris, pH 7.5 1% Tween 20 MEMFA 0.1 M MOPS pH 7.5 2 mM EGTA 1 mM MgSO4 3.7% Formaldehyde Make a 10x stock of the salts and add fresh formaldehyde each time Alkaline Phosphatase Buffer 100 mM Tris, pH 9.5 50 mM MgCl2 100 mM NaCl 0.1% TWEEN 20 5 mM Levamisole - add fresh each time

Contents 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.

Cover Title Copyright Contents Foreword Preface 1 Laboratory Safety and Precautions 2 Isolation of Genomic DNA 3 Isolation of Plasmid DNA 4 Isolation of Total RNA 5 Quantification of Nucleic Acids 6 Polymerase Chain Reaction 7 Variants of Polymerase Chain Reaction 8 Reverse Transcription-Polymerase Chain Reaction (RT-PCR) 9 Agarose Gel Electrophoresis 10 Single Strand Conformation Polymorphism (SSCP) 11 Restriction Digestion 12 Real Time PCR 13 Chromatin Immunoprecipitation Assay 14 Microsatellite Analysis 15 DNA Ligation, Competent Cells Preparation and Transformation 16 Retrieving QTL & SNP Information from Databases 17 DNA Microarray 18 In situ Hybridization 19 Preparation of Required Buffers and Solutions