Training Manual On Molecular Marker Techniques For Genotype Identification (!!!) PDF

Training manual on
Molecular Marker Techniques for Genotype Identification
Compiled and edited by Mukesh Kumar Rana Sunil Archak Rakesh Singh
National Bureau of Plant Genetic Resources (NBPGR) Pusa Campus, New Delhi 110 012, India
Disclaimer
The contents of the manual are based on the modified protocols established in the laboratories of NRC on DNA Fingerprinting, NBPGR, New Delhi 110 012, India. No claims are made on the ownership of the protocols and appropriate citations are mentioned to credit the original author. The contents are not to be quoted. For original protocols, cited references may be consulted.
Protocols for laboratory use only (in the field of plant genetic research). Editors hold no responsibility if emplyoed for any other use.
Rana, MK, Archak S and Singh R (2012) An E-Manual on Molecular marker techniques for genotype identification. NBPGR, Pusa Campus, New Delhi 110 012. Pp. 49 NBPGR, New Delhi 110 012
Table of contents
1. 2. 3. 4. 5. DNA isolation Polymerase chain reaction Agarose gel electrophoresis Molecular data analysis Appendix 6-13 14-32 33-36 37-45
Safety in laboratories
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Laboratory safety may appear at first sight to be rather a dull subject and the temptation may be to read this section only superficially. Laboratories can be dangerous places in which to work and all users need to be aware of the potential hazards and to know what to do in cases of emergency. When working in a new laboratory it is important to become familiar with the layout of the room and the location of the safety equipment. Laboratory workers must also know the meaning of safety signs. Some of these safety signs are in plain English while others are in the form of pictograms. The list below describes protective measures and some particular hazards which may occur in molecular biology laboratories. This list is not exhaustive, but just an attempt to highlight precautions that should be employed with some materials.
Mandatory saftey rules

Laboratory coats must be worn to protect the wearer from chemical splashes and infectious material. Cotton is a better material for a labcoat than nylon as it has a greater absorptive capacity and is generally more resistant to chemical splashes. All chemicals should be considered as potentially dangerous and handled accordingly. Contact with skin and clothing should be avoided and even if a chemical is thought to be harmless it should not be tasted or smelt. Hazard warning symbols which are black in orange background are present on reagent bottles to warn of specific dangers and must be heeded. Deal with spills immediately as and when they occur. In general, large spills of powders should be treated in a fashion that avoids raising a dust. Where a spillage involves personal contamination remove contaminated clothing, wash with sufficient amount of cold water; and seek first-aid and/ or medical advice immediately. The biological materials, including antibodies, sera, hormones and biological substances, in general, pose a significant biological hazard. All such materials, whatever their origin, may harbour human pathogens and should be handled as potentially infectious material in accordance with local guidelines. Any recombinant DNA work associated with protocols is likely to require permission from the relevant regulatory body and one should consult ones local safety officer before embarking upon this work. The use of radioisotopes is subject to legislation and requires permission in most countries. Furthermore, national guidelines for their use and disposal must be rigorously adhered to. The procedures and protocols that use radioisotopes must only be carried
out by individuals who have received training in the use of such material using the appropriate facilities, protection and personal monitoring. Ultraviolet radiation is dangerous, particularly to the eyes. To minimize exposure, make sure that the ultraviolet light source is adequately shielded and wear protective goggles or a full safety mask that efficiently blocks ultraviolet light. Acrylamide is a neurotoxin and carcinogen. Wear a dusk mask. Treat unpolymerized acrylamide with extreme caution. Formamide is teratogenic and extreme care should be taken in its handling. Ethidium bromide is a potent mutagen. It must not be inhaled therefore wear a dust mask when handling a solid or opening a bottle containing ethidium bromide solution. Decontaminate the solutions before disposal as described below or use commercial disposal system. Keep readily availabe the important telephone numbers such as electrician, plumber, fire station, police station, hospital etc. for any eventuality.
Cautionary safety rules

Poisoning often arises from the accidental transfer of a compound to the mouth and this risk can be reduced by always keeping three simple rules in the laboratory: no smoking, no eating and drinking and no mouth pipetting. Eyes are especially vulnerable to splashes from reagents and safety spectacles or goggles should always be worn when undertaking procedures where there is a risk. Heavy duty gloves must be worn when handling corrosive substances such as strong acids and alkalis. The hazardous nature of these substances is obvious but the dangers inherent in skin contact with other chemicals are not always clear. Lightweight disposable gloves should, therefore, be worn during weighing and handling of chemicals to avoid the risk of absorption through skin.
Know the location and proper use of fire extinguishers, eyewash stations, and safety showers. Know the potential hazards of the materials, facilities, and equipment with which you will work. Place bags, lab coats, books etc. in specified locations (Never on the bench tops). Do not use plastic or polycarbonate containers, test tubes, pipettes etc. with phenol and or chloroform. Instead use polypropylene or glass with these organic compounds. Make sure to use gloves, goggles and lab coats when handling these chemicals.
Do not touch broken glassware with your hands. Use a broom and dustpan to clean it up. Dispose off broken glass in appropriate receptacles. Do not toss out into regular trash. Do not dispose of hazardous or noxious chemicals in laboratory sinks. Use proper containers in fume hood. Report all accidents to the instructor immediately.
Safety tips for handling some equipments

Always read instuctions carefully in the manuals and do not run instrumetns unless you know the operation. Always expel wet tips immediately after use. Never lay them down with the tip still attached as remaining liquid can go into the barrel, necessitating immediate cleaning. Always put the pipette back onto the stands when finished. Use fumehood whenever possible. Keep all noxious and volatile compounds in the fume hood. Remember that DNA is negatively charged and Runs to Red. Because most gels are run with Ethidium Bromide, the buffer in the tank will likely be contaminated. Therefore exercise caution when placing gels and removing them from the tank.. Always wear gloves when handling gel apparatus. Verify that the correct colored leads are connected to the appropriate terminals both at the tank end and the power pack before switching on current. Ensure that centrifuge is correctly balanced. And never leave the centrifuge operating and unattended. Melting agarose in the microwave results in superheated liquids, so always heat for short time periods and make sure the liquid is mixed between heating to ensure distribution of heat within. Never reheat agarose containing ethidium bromide in the microwave oven, as it could boil over, contaminating the interior of the oven. Remember to turn off always electicity switches before finishing for the day.
Decontamination of ethidium bromide solutions

Ethidium bromide is a powerful mutagen and is moderately toxic. Gloves should be worn when working with solutions that contain this dye. After use, these solutions should be decontaminated by one of the following methods described below. Decontamination of concentrated solutions of ethidium bromide (i.e. solutions containing >0.5mg/ml) Method 1 Add sufficient water to reduce the concentration of ethidium bromide to <0.5 mg/ml. To the resultant solution add 0.2 volume of fresh 5% hypophosphorous acid and 0.12 volume of fresh 0.5 M sodium nitrite. Mix carefully. (Check that the pH of the solution is <3.0). After incubation for 24 hours at room temperature, add a large excess of1M sodium bicarbonate. The solution may now be discarded. Method 2 Add sufficient water to reduce the concentration of ethidium bromide to <0.5mg/ml. Add 1volume of 0.5 M KMnO4. Mix carefully, and then add 1volume of 2.5N HCl. Mix carefully, and allow the solution to stand at room temperature for several hours. Add 1 volume of 2.5 N NaOH. Mix carefully, and then discard the solution. Decontamination of dilute solutions of ethidium bromide (e.g., electrophoresis buffer containing 0.5g/ml ethidium bromide) Method 1 The following method is from Lunn and Sansone (1987): Add 2.9 g of Amberlite XAD-16 for each 100ml of solution. Store the solution for 12 hours at room temperature, shaking it intermittently. Filter the solution through a Whatman No.1 filter, and discard the filtrate. Seal the filter and Amberlite resin in a plastic bag, and dispose of the bag in the hazardous waste. Method 2 Add 100 mg of powdered activated charcoal for each 100 ml of solution. Store the solution for 1 hour at room temperature, shaking it intermittently. Filter the solution through a Whatman No.1 filter, and discard the filtrate. Seal the filter and activated charcoal in a plastic bag, and dispose of the bag in the hazardous waste.
Selected references Karp A, Issac PG and Ingram DS. 1998. Safety in the molecular laboratory. In: Molecular tools for Screening Biodiversity (ed.) Chapman & Hall, London. 487490. Lunn G and Sansone EB. 1987. Ethidium bromide: destruction and decontamination of solutions. Anal. Biochem. 162: 453. Quillardet P and Hofnung M. 1988. Ethidium bromide and safety- Readers suggest alternative solutions. Letter to editor. Trends Genet.4: 89. Sambrook J, Fritsch EF and Maniatis T.1989. Molecular cloning - A Laboratory Manual, 2nd ed. Cold Spring Harbour Laboratory Press, New York.
1. DNA isolation and purification in plants ___________________________________________

Access to high quality genomic DNA for downstream analyses requires isolation of DNA from plant tissue. Numerous protocols have been published and the subtle and large changes in the protocols can be partly attributed to the species-level differences and between the type and concentration of secondary metabolites. The most commonly used method for DNA isolation is by Saghai-Maroof et al. (1984) which will be used in the practical demonstration with minor modifications to suit the material under consideration. DNA of good quality can be isolated from various parts of a fresh plant, herbarium specimens or even from ancient plant sources. Fresh leaf is the preferred tissue for DNA extraction as they contain low concentration of metabolites and plysaccharides. Although there are many published DNA isolation and purification techniques these methods have common features including disruption of the tissues, DNA release into extraction buffer and purification of the DNA molecule (Figure 1). The disruption of the tissue is the first step in the process of DNA isolation. Plant cells contain a rigid cell wall which needs shear forces to break it down and to release DNA in the extraction buffer. In order to avoid degradation from photolytic activity, tissue disruption is usually done at low temperatures. Usually tissue disruption by mortar and pestle is done in the presence of liquid nitrogen which minimizes oxidation of polyphenols, inhibits nuclease activity and aids the grinding process. After disrupting the plant cell wall, cell membrane also needs to be degraged in order to allow the release of the cellular constituents including DNA in the extraction buffer. The extraction buffer contains, in general, the following components: Detergent Detergents allow the release of membrane-bound DNA. These assist with the disruption of tissues by removing the lipid molecule from the cell membrane and allowing DNA and other cellular material to be released into the solution. The most commonly used detergents are cetyltrimethyl ammonium bromide (CTAB), sodium dodecyl sulphate (SDS), Triton X-100 and comercial biological laundary detergent. Detergents not only releases DNA from cellular membranes and proteins but also separates polysaccharides from the extracted DNA molecule. At low salt concentration, the positively charged CTAb solubilizes the cell membrane and forma a complex with negatively charged DNA, later the DNA can be precipitated from this complex mixture by increasing the salt concentration. Higher concentrations of CTAB are recommended for polysaccharide-rich plant tissues, but at the cost of little yield of the DNA. Buffering agent This is needed to control the pH of the extraction solution. Usually 100mM Tris HCl (pH 7.0 -8.0) is used as a buffering agent in most of the DNA extraction protocols.
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Chelating agent Chilating agent is required to inactivate the nuclease activity. Nuclease activity can cause shearing of DNA molecules. Keeping tissue frozen during or prior to homogenization reduces the risk of nuclease-activated degradation of DNA molecules. Activities of endogenous nuclease are dependent on the pH of the extraction buffer. The pH of the extraction buffer needs to be monitored carefully, especially after the addition of the plant material. Magnesium (Mg2++) ions are a cofactor for most of the nuclease activity, therefore addition of bivalent ion chelator EDTA in extraction buffer prevents DNA degradation. Salts are added to solubilize DNA and other molecules. Reducing agents, e.g. 2-mercaptoethanol (2-ME) to prevent peroxidase or polyphenoloxidase. Polyphenols form complexes with DNA very quickly and we need to include polyphenol absorbents, reductants and oxidants. Maintaining the freezing temperatures during tissue homogenization minimizes the polyphenol oxidation. 2-ME prevents DNA from quinones, disulphides, peroxidases and polyphenol oxidases and therefore retards polyphenol oxidation. Activated charcoal has also been reported as an effective endogenous polyphenol absorbent. DNA purification is usually conducted a two-step process, the first step involves treating the cell extracts with an organic agent like phenol or chloroform or mixtures of phenol, chlorofrom and isoamylalcohol (IAA). The second step involves centrifugation of the lysis mixture with the purpose of separating the DNA-containg aqueous phase from the organic phase, containing denatured protein and lipid contaminants. Phenol denatures proteins and eliminates cellular debris; chloroform also denatures proteins, and isoamylalcohol facilitates the separation from aqueous phase to organic phase and reduces foaming during centrifugation. Following the organic extraction step, DNA is precipitated by ethanol or isopropanol in the presence of sodium chloride, sodium acetate, or ammonium acetate. When the yield is high the precipitated DNA can be spooled out from the solution using a glass rod. Centrifugation is the preferred method when small amounts of DNA are precipitated. The DNA is usually rinsed with 70% ethanol to remove traces of salt and then dried. Polysaccharides in plants are diversified compounds which vary with species and with different developmental stages and cause the majority of problems in regard to purity of extracted DNA. Carbohydrates in plants are clear and sticky in nature and can easily be identified by their viscous consistency in the solution. Younger tissues are preferred because they contain fewer polysaccharides than older or mature tissues. Exclusion of polysaccharides by using high concentrations of NaCl is an easy, quick and inexpensive method for plant DNA isolation.
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During the DNA isolation a considerable amont of RNA is often extracted. RNA contamination can cause overestimation of the amount of DNA extracted when quantified. It usually appears as low molecular weight DNA in agarose gel electrophoresis. As RNA may cause suppression of PCR amplification and lead to mispriming of DNA templates during thermal cycling, its removal from the samples is essential. For this the DNA samples are generally dissolved in low concentration TE buffer and then treated with RNAse at 37oC from 30 min to 1 h.
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1.1 Equipments needed High speed centrifuge Microcentrifuge Auto-pippets (2-20l, 20-200l, 200-1000l) Waterbath Deep freezer (-20oC) Refrigerator Flourometer Liquid nitrogen container Gel documentations systems or UV transilluminator Gel electrophoresis appratus Power supply Pestle and mortar Vortexer
1.2 Supplies and solutions 50mL centrifuge tubes Liquid nitrogen 0.5 and 1.5mL microcentrifuge tubes Tips (2-20 L, 20-200 L, 200-1000 L) CTAB Buffer Isopropanol Saturated phenol Chloroform : isoamylalcohol (24:1) mixture 10:1 TE R-Nase A (10mg / ml) 70% ethanol 50x TAE buffer, pH 8.0
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1.3 Protocol (CTAB method by Saghai-Maroof et al. 1984) Weigh 5 g of clean young leaf tissue. Freeze the tissue rapidly in liquid nitrogen and grind to a powder with a pestle and mortar as the liquid nitrogen boils off. Add a little more liquid nitrogen, if necessary, to keep the powder from thawing while grinding. It is important not to let the tissue thaw once frozen, until it is added to the buffer. Transfer the frozen powder to 50 ml centrifuge tube with 20 ml CTAB buffer maintained at 60o C in a water bath. Mix vigorously or vortex. Incubate at 60o C for one hour. Mix intermittently. Fill the tube with chloroform: isoamyl alcohol. Mix gently by inverting for 5 min. Spin at 17,000 rpm for 10 min with SS34 rotor in Sorval RC-5C centrifuge at 25o C. Balance the tubes by adding chloroform: isoamyl alcohol before spinning in the Sorval. Transfer aqueous phase to a fresh centrifuge tube. Add equal amount of isopropanol and let the DNA to settle down for 20 min. Spool out the DNA. Drain out the excess chemicals with a pipette. Add 0.5 ml of 70% ethanol. Mix gently and incubate for 30 min. Decant and repeat the 70% ethanol treatment. Decant off and dry the pellet under vaccum. Dissolve DNA in minimum volume of 10: 1 TE or distilled deionized water. Add RNAse (0.2 ml) and incubate at 37o C for one hour. Add pronase (0.2 ml) and incubate at 37o C for one hour. Add equal volume of phenol: chloroform (1: 1), mix properly for at least 2 min and spin for 5 min. Take out the DNA supernatant and after this perform two chloroform: isoamyl alcohol extractions as before. Spin after each extraction. Precipitate DNA by adding 1/10 volume of 3M NaOAc and 2.5 times of the total volume chilled ethanol. Mix and spool out the DNA. Remove extra salts by two washings with 70% ethanol. Dry under vaccum. Add minimum volume of TE (10:1). Dissolve at room temperature. Store frozen at 20o C. 1.4 Quantity assessment of DNA
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DNA quantification is an important step in many procedures where it is necessary to know the amount of DNA that is present when carrying out restriction digests or performing different techniques such as PCR for RAPD or AFLPs or SSRs etc. There are several methods for quantifying DNA , the most widespread being: i) the comparison of an aliquot of the extracted sample with standard DNAs of known concentration using gel electrophoresis; ii) spectrophotometric determination, and iii) flourimetric determination 1.4.1 Electrophoresis of a DNA sample of unknown concentration with a known standard Set the electrophoresis apparatus with combs in such a way that 2 mm gap is maintained between the bottom of the gel and the comb tip. Prepare a 0.8% (w/ V) agarose gel suspension by heating in 1x TAE buffer. Allow the gel to cool and then pour it onto the gel tray and allow it to solidify for at least 30 min. Remove the combs. Place the solidified gel into the electrophoresis tank and pour 1x TAE until the gel is completely covered. Mix 4 l distilled deionized water, 1l loading dye and 1l DNA and load onto the gel. Load 2l of DNA standard of known concentration (25ng, 50ng, 100ng) into the side wells. Connect the apparatus to power supply and run the gel at 60-70 V for 1 h or until the dye moves 3-4 cm from the wells. Stain the gel with ethidium bromide (stock of 10mg/ mL) solution for 30-45 min. Alterntively, ethidium bromide to a concentration of 0.5g/ mL can be added in step 2 above. View the gel under UV light and photograph under UV light. Compare the intensity of the DNA bands of the samples with the intensity of the standard DNA bands. As the amount of DNA present in each standard DNA bands is known, the amount of DNA of each sample can be calculated by comparing the fluorescent yield of the sample with that of the standard. 1.4.2 DNA quantification by UV spectroscopy Take 5 l of the DNA sample in a quartz cuvette. Make up the volume to 1ml with distilled water. Measure absorbance of the solution at wavelengths 230, 260, 280 and, 300nm. Calculate the ratios A230 / A260 and A280 / A260. A good DNA preparation exhibits the following spectral properties A300 0.1 O.D. units A230 / A260 0.45 O.D. units A280 / A260 0.55 O.D.units). Calculate DNA concentration using the relationships for soluble stranded DNA, 1 O.D. at260 nm = 50g/ ml. This estimate is influenced by the contaminating substances like RNA and very low molecular weight DNA in the solution.
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Prepare a working stock of samples of about 100l with concentrations of 10 ng/ l. 1.4.3 DNA quantification using flourimeter The DNA extraction procedures do not eliminate RNA. Therefore, DNA concentration estimation by UV spectrophotometry as explained earlier will be highly inaccurate. RNase treatment may help in reducing the errors. However, fluorimetric estimations are more reliable as it measures the fluorescence emitted by the double stranded DNA- Hoechst 33258 dye complex, which is directly proportional to the amount of DNA in the sample. Since Hoechst 33258 dye does not bind to single stranded DNA and very small fragments of DNA, this procedure gives more reliable estimates of the DNA concentrations in the sample.
Reagents required 10x TNE ( 1000 ml buffer stock solution): 100 mM Tris 1M Na Cl 10 mM EDTA (Dissolve in 800 ml distilled water. Adjust pH to 7.4 with HCL. Add distilled water to 1000 ml. Filter and autoclave before use. Store at 4 o C for up to 3 months). Hoechst 33258 dye stock: Hoechst 33258 : I mg /ml in distilled water. (Add 10 ml distilled water to 10 mg H33258. Do not filter. Store at 4 o C for up to 6 months in an amber bottle.) Procedure Prepare the assay and DNA standard solutions as described below: DNA assay solution Low range (A) (10-500 ng/ml final DNA concentration) H33258 stock solution : 10.0 l 10x TNE buffer Distilled filtered water : : 10.0 ml 90.0 ml
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High range (B) (100-5000 ng/ml final DNA concentration) H33258 stock solution 10x TNE buffer Distilled filtered water : DNA standard 1:10 dilution (100 mg / ml) of 1mg / ml DNA standard stock solution. Mix the following: 1 mg / ml DNA standard stock 10x TNE buffer Distilled water : 100 l : 100 l : 800 l : 100.0 l : 10.0 ml 90.0 ml
Turn on the fluorimeter (Hoefer) at least 15 min before using. Zero the instrument: Prepare an assay blank using 2 ml of appropriate Assay solution (A or B for high DNA concentration). Dry the sides of a cuvette. Insert the cuvette into the well, close the lid, and press ZERO. After 0 displays, remove the cuvette. Calibrate the instrument: Deliver 2 l of the appropriate DNA standard solution (low or high range) to 2 ml of Assay solution in the cuvette. Mix by pipetting several times into a disposble transfer pipette. Place cuvette in well, close the lid and press CALIB. Enter 100 for the low range assay, 1000 for the high range assay and press ENTER. After the entered value displays, remove the cuvette. Zero the instrument: Empty and rinse the cuvette. Dry by draining cuvette and blotting upside down on a paper towel. Add 2 ml of the same Assay Solution used in step 3, insert the cuvette into the well, close the lid, and press ZERO. After 0 displays remove the cuvette. Measure the sample and mix well. Place the cuvette in the well, close the lid, and record the measurement. Measure subsequent samples. Repeat steps 5 and 6 for each sample.
Selected references
Saghai-Maroof MA Soliman KM, Jorgensen RA and Allard RW. 1984. Ribosomal DNA spacer-length polymorphism in barley: Mendelian inheritance, chromosomal location, and population dynamics, Proc Natl Acad Sci USA 81: 8014-8018.
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2. Polymerase Chain Reaction

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The advent of Polymerase Chain Reaction (PCR) by Kary B. Mullis in the mid1980s revolutionized molecular biology as we know it.The polymerase chain reaction (PCR) is a rapid procedure for in vitro enzymatic amplication of a specic segment of DNA. PCR has infinite applications which include direct cloning from genomic DNA or cDNA, in vitro mutagenesis and engineering of DNA, genetic ngerprinting of forensic samples, genotype identification, assays for the presence of infectious agents, prenatal diagnosis of genetic diseases, analysis of allelic sequence variations, analysis of RNA transcript structure, genomic footprinting, and direct nucleotide sequencing of genomic DNA and cDNA. There are three nucleic acid segments: the segment of double-stranded DNA to be amplied and two single-stranded oligonucleotide primers anking it (Figure 2). Additionally, there is a protein component (a DNA polymerase), appropriate deoxyribonucleoside triphosphates (dNTPs), a buffer, and salts. The primers are added in vast excess compared to the DNA to be amplied. They hybridize to opposite strands of the DNA and are oriented with their 3 ends facing each other so that synthesis by DNA polymerase (which catalyzes growth of new strands 53) extends across the segment of DNA between them. One round of synthesis results in new strands of indeterminate length which, like the parental strands, can hybridize to the primers upon denaturation and annealing. These products accumulate only arithmetically with each subsequent cycle of denaturation, annealing to primers, and synthesis.
Figure 2. The polymerase chain reaction
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However, the second cycle of denaturation, annealing, and synthesis produces two single-stranded products that together compose a discrete double-stranded product which is exactly the length between the primer ends. Each strand of this discrete product is complementary to one of the two primers and can therefore participate as a template in subsequent cycles. The amount of this product doubles with every subsequent cycle of synthesis, denaturation, and annealing; accumulating exponentially so that 30 cycles should result in a 2 -fold (270 millionfold) amplication of the discrete product.
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2.1
Composition of the PCR Reaction Mixture
A typical PCR reaction mixture contains template DNA, primers, MgCl2, dNTPs and Taq DNA polymerase each of which is described below.
2.1.1 Template DNA

Usually the template DNA amount is in the range of 0.1-1g for genomic DNA, for a total reaction mixture of 50l. Higher template DNA amounts usually increase the yield of nonspecific PCR products, but if the fidelity of synthesis is crucial, maximal allowable template DNA quantities in conjunction with limiting number of PCR cycles should be used to increase the percentage of "correct" PCR products. Nearly all routine methods are suitable for template DNA purification. Although even trace amounts of agents used in DNA purification procedures strongly inhibit Taq DNA polymerase, ethanol precipitation of DNA and repetitive treatments of DNA pellets with 70% ethanol is usually effective in removing traces of contaminants from the DNA sample.
2.1.2 Primers
PCR primers are usually 15-30 nucleotides in length. Longer primers provide sufficient specificity. The GC content should be 40-60%. The C and G nucleotides should be distributed uniformly within the full length of the primer. More than three G or C nucleotides at the 3'-end of the primer should be avoided, as nonspecific priming may occur. The primer should not be self-complementary or complementary to any other primer in the reaction mixture, in order to avoid primer-dimer and hairpin formation. The melting temperature of flanking primers should not differ by more than 5C, so the GC content and length must be chosen accordingly. All possible sites of complementarity between primers and the template DNA should be noted. If primers are degenerate, at least 3 conservative nucleotides must be located at the primer's 3'-end.
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Estimation of the melting and annealing temperatures of primer: If the primer is shorter than 25 nucleotides, the approx. melting temperature ( Tm) is calculated using the following formula: Tm = 4 (G + C) + 2 (A + T) G, C, A, T - number of respective nucleotides in the primer. Annealing temperature should be approx. 5C lower than the melting temperature. If the primer is longer than 25 nucleotides, the melting temperature should be calculated using specialized computer programs where the interactions of adjacent bases, the influence of salt concentration, etc. are evaluated.
2.1.3 Magnesium chloride (MgCl2) concentration

Since Mg2+ ions form complexes with dNTPs, primers and DNA templates, the optimal concentration of MgCl2 has to be selected for each experiment. Too few Mg 2+ ions result in a low yield of PCR product, and too many increase the yield of non-specific products and promote misincorporation. Lower Mg2+ concentrations are desirable when fidelity of DNA synthesis is critical. The recommended range of MgCl2 concentration is 14mM, under the standard reaction conditions specified. If the DNA samples contain EDTA or other chelators, the MgCl2 concentration in the reaction mixture should be raised proportionally.
2.1.4 dNTPs
The concentration of each dNTP in the reaction mixture is usually 200M. It is very important to have equal concentrations of each dNTP (dATP, dCTP, dGTP, dTTP), as inaccuracy in the concentration of even single dNTP dramatically increases the misincorporation level. When maximum fidelity of the PCR process is crucial, the final dNTP concentration should be 10-50M, since the fidelity of DNA synthesis is maximal in this concentration range. In addition, the concentration of MgCl2 should be selected empirically, starting from 1mM and increasing in 0.1mM steps, until a sufficient yield of PCR product is obtained.
2.1.5 Taq DNA polymerase

Usually 1-1.5 units of Taq DNA polymerase are used in 50l of reaction mix. Higher Taq DNA polymerase concentrations may cause synthesis of nonspecific products. However, if inhibitors are present in the reaction mix (e.g., if the template DNA used is not highly purified), higher amounts of Taq DNA polymerase (2-3 units) are helpful in obtaining a better yield of amplification products.
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2.2
Temperature Cycling
2.2.1 Initial denaturation step
The complete denaturation of the DNA template at the start of the PCR reaction is of key importance. Incomplete denaturation of DNA results in the inefficient utilization of template in the first amplification cycle and in a poor yield of PCR product. The initial denaturation should be performed over an interval of 1-5 min at 95C if the GC content is 50% or less. This interval should be extended up to 10 min for GC-rich templates. If the initial denaturation is no longer than 3 min at 95C, Taq DNA polymerase can be added into the initial reaction mixture. If longer initial denaturation or a higher temperature is necessary, Taq DNA polymerase should be added only after the initial denaturation, as the stability of the enzyme dramatically decreases at temperatures over 95C. 2.2.2 Denaturation step Usually 0.5-2 min denaturation at 94-95C is sufficient, since the PCR product synthesized in the first amplification cycle is significantly shorter than the template DNA and is completely denatured under these conditions. If the amplified DNA has a very high GC content, denaturation time may be increased up to 3-4 min. Alternatively, additives facilitating DNA denaturation - glycerol (up to 10-15 vol.%), DMSO (up to 10%) or formamide (up to 5%) - should be used. In the presence of such additives, the annealing temperature should be adjusted experimentally, since the melting temperature of the primer-template DNA duplex decreases significantly. If additives are used, the amount of Taq DNA polymerase in the reaction mix should be increased, because DMSO and formamide, at the suggested concentrations, inhibit the enzyme approximately 50%. Alternatively, a common way to decrease the melting temperature of the PCR product is to substitute dGTP with 7-deaza-dGTP in the reaction mix.
2.2.3 Primer annealing step

Usually the optimal annealing temperature is 5C lower than the melting temperature of primer-template DNA duplex. Incubation for 0.5-2 min is usually sufficient. However, if nonspecific PCR products are obtained in addition to the expected product, the annealing temperature should be optimized by increasing it stepwise by 12C.
2.2.4 Extension step

Usually the extension step is performed at 70-75C. The rate of DNA synthesis by Taq DNA polymerase is highest at this temperature (2-4 kb/min), and a 1min extending time is sufficient for the synthesis of PCR fragments up to 2 kb. When larger
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DNA fragments are amplified, the extension time is usually increased by 1min for each 1000 bp.
2.2.5 Number of cycles

The number of PCR cycles depends on the amount of template DNA in the reaction mix and on the expected yield of the PCR product. For less than 10 copies of template DNA, 40 cycles should be performed. If the initial quantity of template DNA is higher, 25-35 cycles are usually sufficient.
2.2.6 Final Extension Step

After the last cycle, the samples are usually incubated at 72C for 5-15min to fillin the protruding ends of newly synthesized PCR products. Also, during this step, the terminal transferase activity of Taq DNA polymerase adds extra A nucleotides to the 3ends of PCR products. Therefore, if PCR fragments are to be cloned into T/A vectors, this step can be extended to up to 30 min.
2.3
Some variants of PCR

RT-PCR : Reverse-Transcriptase- PCR is one of the most sensitive methods for
detection and analysis of rare mRNA transcripts or other RNA present in low abundance. RNA can not serve as template for PCR, so it is first transcribed into dDNA with reverse transcriptase and the cDNA copy is then amplified.
2.3.1
2.3.2 Nested PCR: Nested PCR is a variation of the PCR in that two pairs (instead of one pair of PCR primers) are used to amplify a fragment. The first pair of primers amplifies a fragment similar to a standard PCR. However, a second pair of primers called nested primers bind inside the first PCR product fragment to allow amplification of a second PCR product which is shorter than the first one. 2.3.3 Hot start PCR: It is carried out to reduce non-specific amplification. It is done by separating the DNA mixtures from enzymes by a layer of wax which melts when heated in a cycling reaction. A number of companies also produce hot start PCR products. 2.3.4 Multiplex PCR: Here two or more unique targets of DNA sequences in the same specimen are amplified simultaneously. One can be used as control to verify the integrity of PCR. It can be used for mutational analysis and identification of pathogens. 2.3.5 Q/C-PCR (Quantitative/ Comparative: It uses an internal control DNA sequence (but of different size) which compete with the target DNA (competitive PCR) for the same set of primers. It is used to determine the amount of target template in reaction.
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2.4
PCR-based molecular marker techniques
This section describes some of the basic molecular marker techniques like RAPD, ISSR, SSR and gene specific amplification that make use of PCR. After PCR one needs to separate the amplified DNA fragments using gel electrophoresis which is described separtely in next chapter.
2.4.1 Random Amplified Polymorphic DNA (RAPD)

Three main techniques fall within the category of PCR-based markers using arbitrary primers: RAPD, DAF and AP-PCR. MAAP is the acronym proposed, but not commonly used to encompass all of these closely related techniques. The random amplified polymorphic DNA (RAPD) technique is a PCR-based method that uses a short primer (usually 10 bases) to amplify anonymous stretches of DNA. With this technique, there is no specific target DNA, so each particular primer will adhere to the template DNA randomly. As a result, the nature of the obtained products will be unknown. The DNA fragments generated are then separated and detected by gel electrophoresis. Polymorphism of amplified fragments are caused by: (1) base substitutions or deletions in the priming sites, (2) Insertions that render priming sites too distant to support amplification, or (3) insertions or deletions that change the size of the amplified fragment. Equipments Refrigerator (4oC) Deep freezer (-20oC) Microwave or hot plate Laminar air flow Fume hood pH meter Centrifuge Standard balance Thermocycler Gel electrophoresis units Power supply units UV transilluminator/ gel documentation system Vortexer Pipetman (2-20 L, 20-200 L, 200-1000 L)
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Supplies and solutions 10X Taq poymerase buffer, 25 mM magnesium chloride, 2 mM dNTPs (mixture of dATP, dCTP, dGTP and dTTP), Taq poymerase (5 Units/ L), 10 M RAPD primers, template or genomic DNA (10ng /L), sterile double distilled water, TAE buffer, agarose, gel loading dye, DNA size standard, ethidium bromide, tips, 0.5mL and 1.5 mL microcentrifuge tubes, 0.2 mL PCR tubes, staining trays Protocol Take out MgCl2, dNTPs, primers and buffer from the deep freezer and let these thaw at room temperature. Taq DNA polymerase needs to taken out only when it is to be added and return immediately to into the deep freezer after use. For setting up PCR reaction, prepare master mix in 1.5 L centrifuge tube of the following components except template DNA: Sr. No. Component Stock Required Amount concentration concentration required for one reaction 25mM 5M 25mM 5U/ L 10ng/ L 1x 3mM 0.5M 0.2mM 1U 50ng 2.5L 3.0L 2.5L 0.2L 0.2L 5.0L 11.6L
1 2 3 4 5 6 7
Taq polymerase buffer MgCl2 Primer dNTPs Taq DNA polymerase Template DNA
Sterile double-distilled water (to make 25 L volume) Total volume
25.0 L
Dispense 20L of above master mix in each of the 0.2mL PCR tube. Add 5 L of template DNA and mix thoroughly and gently by pipetting 3-4 times.
24
Turn on the thermocyler and place samples in it. The following general thermocycling steps are followed: Step1: Initial denaturation at 94C for 5.0min Step2: Denaturation at 94 C for 1.0min Step3: Primer annealing at 45-55 C for 1.0min Step4: Primer extension at 72 C for 2.0min Step5: Go to 2, 39 times Step6: Final extension at 72 C for 10min Step7: 4 C for ever
After the run is completed turn off the machine and remove the samples. Add 2.5 L gel loading dye in each sample and carry out electrophoresis or store samples at 4oC until electrophoresis.
Agarose gel electrophoresis
To prepare 100mL of a 1.5% agarose solution, measure 1.5g agarose into a glass beaker or flask and add 100ml 1X TAE.
Microwave or stir on a hot plate until agarose is dissolved and solution is clear. Allow solution to cool to about 55C before pouring. (ethidium bromide can be added at this point to a concentration of 0.5 g/ ml)
Place the comb in gel tray. Pour 50 C gel solution into tray to a depth of about 5mm. Allow the gel to solidify for about 20 min at room temperature.
To run, gently remove the comb, place the tray in electrophoresis chamber, and cover (just until wells are submerged) with electrophoresis buffer (the same buffer used to prepare the agarose).
To each of the RAPD sample from Step II above, add 2.5 L of 6% gel loading dye solution. Mix well. Load 25 l of DNA per well. Load also the DNA size standards along side RAPD reactions.
Connect the electrodes to the power pack and carry out electrophoresis at 50-150 Volts until the bromophenol blue dye has reached three fourth of the gel length.
Stain the gel with ethidium bromide.

25
Examine the gel under UV light in the gel documentation system (or transilluminator) and obtain a photograph.
Note: Ethidium bromide is a mutagen and a probable carcinogen. Wear gloves when working with ethidium bromide solutions. Also use care not to contaminate the work area with the solution. UV light is damaging and must be used with caution. UV light causes burns and can damage the eyes.
Selected references
Caetano-Anolls G, Bassam BJ and Gresshoff PM. 1991b. DNA amplification using very short arbitrary oligonucleotide primers. Bio/Technology 9:553-557. Welsh J and McClelland M. 1990. Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res. 18:7213-7218. Williams JGK, Kubelik AR, Livak KJ, Rafalski JA and Tingey V. 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res. 18:6531-6535.
26
2.4.2
Inter-Simple Sequence Repeats (ISSR)
ISSRs are semiarbitrary markers amplified by PCR in the presence of one primer complementary to a target microsatellite. Amplification in the presence of non-anchored primers also has been called microsatellite-primed PCR, or MP-PCR. Such amplification does not require genome sequence information and leads to multilocus and highly polymorphous patterns. Each band corresponds to a DNA sequence delimited by two inverted microsatellites (Figure 3). Like RAPDs, ISSRs markers are quick and easy to handle, but they seem to have the reproducibility of SSR markers because of the longer length of their primers. ISSR technique differs from the RAPD in the sense that here longer primer sequences are used and hence the annealing temperature is also higher which together provide more stringency to the technique. Consequent to higher stingent conditions, ISSR markers are more reproducible as comapared to the RAPD markers.
Figure 3. Generation of ISSR markers
Equipments
Refrigerator (4oC) Deep freezer (-20oC) Microwave or hot plate Laminar air flow Fume hood pH meter Centrifuge Standard balance Thermocycler Gel electrophoresis units Power supply units UV transilluminator/ gel documentation system Vortexer Pipetman (2-20 L, 20-200 L, 200-1000 L)
27
Supplies and solutions

10X Taq poymerase buffer, 25 mM magnesium chloride, 2 mM dNTPs (mixture of dATP, dCTP, dGTP and dTTP), Taq poymerase (5 Units/ L), 10 M RAPD primers, template or genomic DNA (10ng /L), sterile double distilled water, TAE buffer, agarose, gel loading dye, DNA size standard, ethidium bromide, tips, 0.5mL and 1.5 mL microcentrifuge tubes, 0.2 mL PCR tubes, staining trays
Protocol
Take out MgCl2, dNTPs, primers and buffer from the deep freezer and let these thaw at room temperature. Taq DNA polymerase needs to taken out only when it is to be added and return immediately to into the deep freezer after use. For setting up PCR reaction, prepare master mix in 1.5 L centrifuge tube of the following components except template DNA: Sr. No. Component Stock Required Amount concentration concentration required for one reaction 10x 25mM 5M 25mM 5U/ L 10ng/ L 1x 3mM 0.5M 0.2mM 1U 50ng 2.5L 3.0L 2.5L 0.2L 0.2L 5.0L 11.6L
1 2 3 4 5 6 7
Taq polymerase buffer MgCl2 Primer dNTPs Taq DNA polymerase Template DNA
25.0 L
Dispense 20L of above master mix in each of the 0.2mL PCR tube. Add 5 L of template DNA and mix thoroughly and gently by pipetting 3-4 times. Turn on the thermocyler and place samples in it. The following general thermocycling steps are followed:
28
Step1: Initial denaturation at 95C for 5.0 min Step2: Denaturation at 94 C for 1.0 min Step3:Primer annealing at 45-55 C for 1.0 min Step4: Primer extension at 72 C for 2.0 min Step5: Go to 2, 39 times Step6: Final extension at 72 C for 8 min Step7: 4 C for ever After the run is completed turn off the machine and remove the samples. Add 2.5 L gel loading dye in each sample and carry out electrophoresis or store samples at 4oC until electrophoresis.

To prepare 100mL of a 1.5% agarose solution, measure 1.5g agarose into a glass beaker or flask and add 100ml 1X TAE. Microwave or stir on a hot plate until agarose is dissolved and solution is clear. Allow solution to cool to about 55C before pouring. (ethidium bromide can be added at this point to concentration of 0.5 g/ mL) Place the comb in gel tray. Pour 50 C gel solution into tray to a depth of about 5mm. Allow the gel to solidify for about 20 min at room temperature. To run, gently remove the comb, place the tray in electrophoresis chamber, and cover (just until wells are submerged) with electrophoresis buffer (the same buffer used to prepare the agarose). Load 25 L of DNA per well. Load also the DNA size standards along side ISSR reactions. Connect the electrodes to the power pack and carry out electrophoresis at 50-150 Volts until the bromophenol blue dye has reached three fourth of the gel length. Stain the gel with ethidium bromide solution. Examine the gel under UV light in the gel documentation system (or transilluminator) and obtain a photograph.
Selected references
Zietkiewicz E, Rafalski A and Labuda D. 1994. Genome fingerprinting by simple sequence repeat (SSR)-anchored polymerase chain reaction amplification. Genomics 20: 176-183.
29
2.4.3
Simple Sequence Repeats (SSR)
PCR-based assays for SSRs have become the most popular and powerful of the current methods for genotype identification as they reveal highly polymorphic Mendelian markers. A SSR locus also known as STR (Short Tandem Repeat) or microsatellite, consists of reiterated short sequences (usually di-, tri-, or tetranucleotides) that are tandemly arrayed at a particular choromosomal location with variation in repeat copy number. A microsatellite array can be considered a reminiscent of a minisatellite array, except that each of its repeat units is much shorter. Therefore alleles at SSR loci can be distinguished in acrylamide (rather than agarose) gels. Microsatellite loci were discovered in the late 1980s and soon were shown to be characteristic features scattered abundantly throughout the nuclear genomes of most plants. The first and most laborious step in SSR assay is primer development, which requires construction of a genomic library for the target species, screening that library for clones that contain SSRs, sequencing the inserts from those positive clones, and using the information from unique sequences flanking each repeat region to synthesize PCR primers. With the availability of huge number of ESTs (Expressed Sequence Tags) and BAC clones in the public domain database NCBI for many crop plant species, generation of EST-deived SSRs has become and economic alternative to hunt for SSRs. Once primers are available, large number of individuals can be readily screened for Menedelian genotypes at specific SSR loci displaying codominant alleles.
Figure 4: PCR amplification of SSR markers
30
Equipments Refrigerator (4oC) Deep freezer (-20oC) Microwave or hot plate Laminar air flow Fume hood pH meter Centrifuge Standard balance Thermocycler Gel electrophoresis units Power supply units UV transilluminator/ gel documentation system Vortexer Pipetman (2-20 L, 20-200 L, 200-1000 L)
Supplies and solutions 10X Taq poymerase buffer, 25 mM magnesium chloride, 2 mM dNTPs (mixture of dATP, dCTP, dGTP and dTTP), Taq poymerase (5 Units/ L), 10 M RAPD primers, template or genomic DNA (10ng /L), sterile double distilled water, TAE buffer, agarose, gel loading dye, DNA size standard, ethidium bromide, tips, 0.5mL and 1.5 mL microcentrifuge tubes, 0.2 mL PCR tubes, staining trays Protocol Take out MgCl2, dNTPs, primers and buffer from the deep freezer and let these thaw at room temperature. Taq DNA polymerase needs to taken out only when it is to be added and return immediately to into the deep freezer after use. For setting up PCR reaction, prepare master mix in 1.5 L centrifuge tube of the following components except template DNA:
31
Sr. No.
Component
Stock Required Amount concentration concentration required for one reaction 10x 25 mM + 5 M 1x 3 mM 0.1 M 2.5 L 3.0 L 1.0 L
1 2 3
Taq polymerase buffer MgCl2 Primer (Forward Reverse) dNTPs Taq DNA polymerase Template DNA
4 5 6 7
25 mM 5U/ L 10 ng/ L
0.2 mM 0.5 U 20 ng -
0.2 L 0.1 L 2.0 L 16.2 L
25.0 L
Dispense 23 L of above master mix in each of the 0.2 mL PCR tube. Add 2.0 L of template DNA and mix thoroughly and gently by pipetting 3-4 times. Turn on the thermocyler and place samples in it. The following general thermocycling steps are followed: Step1: Initial denaturation at 95C for 5.0 min Step2: Denaturation at 94 C for 1.0min Step3: Primer annealing at suitable annealing temp. for 1.0 min Step4: Primer extension at 72 C for 2.0 min Step5: Go to 2, 39 times Step6: Final extension at 72 C for 8 min Step7: 4 C for ever
After the run is completed turn off the machine and remove the samples. Add 2.5 L gel loading dye in each sample and carry out electrophoresis or store samples at 4oC until electrophoresis.
32
Prepare 3% metaphore agarose solution (usually we add metaphor agarose and normal agarose in a ratio of 3:1; measure 2.25 g metaphor agarose and add 0.75 g normal agarose for 100 mL of gel solution in 1X TAE buffer). Microwave or stir on a hot plate until agarose is dissolved and solution is clear. Allow the solution to cool to about 55C before pouring. (ethidium bromide can be added at this point to concentration of 0.5g/ mL) Place the combs in gel tray. Pour the gel solution into the casting tray and allow to solidify for about 30 min at 4 C. To run, gently remove the comb, place the tray in electrophoresis chamber, and cover (just until wells are submerged) with electrophoresis buffer (the same buffer used to prepare the agarose). Load 25l of DNA per well. Load also the DNA size standards along side the SSR reactions. Connect the electrodes to the power pack and carry out electrophoresis at 100 Volts for 2:30 h or until the bromophenol blue dye has reached three fourth of the gel length. Stain the gel with ethidium bromide (if not already included in the gel). Examine the gel under UV light in the gel documentation system (or transilluminator) and obtain a photograph.
Selected references
Tautz D.1989. Hypervariablity of simple sequences as a general source of polymorphic DNA markers. Nucleic Acids Res. 17: 6463-6471. Weber JL and May PE. 1989. Abundant class of human DNA polymorhisms which can be typed using the polymerare chain reaction. Am. J. Hum. Genet. 44: 388-396. Litt M and Luty JA. 1989. A hypervariable microsatellite revealed by in vitro amplification of a dinucleotide repeat within the cardiac muscle actin gene. Am. J. Hum. Genet. 44:397-401.
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2.4.4
Functional markers for genotype identification
Genetic markers were originally used in genetic mapping to determine the order of genes along chromosomes. In 1913, Alfred H. Sturtevant generated the first genetic map using six morphological traits (termed factors) in the fruit fly ( Drosophila melanogaster) and, soon after, Karl Sax produced evidence for genetic linkage between a qualitative and a quantitative trait loci (seed color and seed size) in the common bean (Phaseolus vulgaris). Since these pioneer studies, genetic markers have evolved from morphological markers through isozyme markers to DNA markers. Today, genetic markers are used in both basic plant research and plant breeding to characterize plant germplasm, for gene isolation, for marker-assisted introgression of favorable alleles and for variety protection. Different approaches (including association studies) have recently been adopted for the functional characterization of allelic variation in plants and to identify sequence motifs affecting phenotypic variation. The term 'functional markers' for DNA markers derived from such functionally characterized sequence motifs. Functional markers are superior to random DNA markers such as RFLPs, ISSRs and AFLPs owing to complete linkage with trait locus alleles. Functional marker development requires allele sequences of functionally characterized genes from which polymorphic, functional motifs affecting plant phenotype can be identified.
Equipments
Refrigerator (40C) Deep freezer (-200C) Microwave Oven or Hotplate Laminar Airflow Fume hood pH Meter Vortex Centrifuge Analytical Balance PCR Thermal cycler Gel electrophoresis unit Power supply unit UV transilluminator/ gel documentation system Micro Pipettes (2-20 ul, 20-200ul, 200-1000 ul)
Supplies and solutions
34
10X Taq Ploymerase buffer, 25 mM Magnesium Chloride, 10mM dNTPs (mixture of dATP, dCTP, dTTP, dGTP), Taq Polymerase (5unit/ul), sterile double distilled water, TAE buffer, Meataphore Agarose, gel loading dye, DNA size markers, ethidium bromide, tips etc.
Protocol
I)
PCR Cocktail preparation

Set up PCR reaction with the following components: Sr. No. Component Stock Required Amount concentration concentration required for one reaction 10x 25 mM 10 M 10 M 10 mM 5U/ L 10 ng/ L 1x 3 mM 0.5 M 0.5 M 0.2 mM 1U 40 ng 1.0 L 1.2 L 0.5 L 0.5 L 0.2 L 0.2 L 4.0 L 2.4 L
1 2 3
Taq polymerase buffer MgCl2 Primer (reverse) Primer (forward)
4 5 6 7
dNTPs Taq DNA polymerase Template DNA
10.0 L
II)
Thermocycling conditions
The following general thermocycling steps are followed: Step1: Initial denaturation at 950C for 4.00 min Step2: Denaturation at 940C for 0.30 min Step3: Primer annealing at 59.10C for 0.30 min
35
Step4: Primer extension at 720C for 2.00 min Step5: Go to 2, 35 times Step6: Final extension at 720C for 7.00 min Step7: 40C for ever After PCR, amplified DNA fragment is analyzed through gel electrophoresis. III) Metaphor gel electrophoresis
To prepare gel (4%) first dissolve metaphor agarose in small pinches to avoid clotting in prechilled 0.5x TBE buffer and then heat it to get dissolved. Allow solution to cool at 55oC and add ethidium bromide stain at the rate of 2.5l /100ml. Pour the gel in to the casting tray with combs and allow it to polymerize at room temperature. Shift the gel along with tray and combs in to the electrophoresis tank and remove combs carefully. Prepare PCR samples for loading by mixing with 1x loading dye and load in the preformed wells. Carry out electrophoresis in 0.5x TBE buffer at 80 volts for 3.5 hours. Examine the gel under UV light in the gel documentation system (or transilluminator) and take photograph.
Selected references
Sturtevant AH. 1913. The linear arrangement of six sex-linked factors in Drosophila, as shown by their mode of association. J. Exp. Zool. 14: 4359. Sax K. 1923. The association of size differences with seed-coat pattern and pigmentationm in Phaseolus vulgaris. Genetics 8: 552560. Henry RJ (ed.). 2001. Plant Genotyping CABI Publishing.
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3.
Agarose Gel Electrophoresis
__________________________________________
Virtually all scientific investigations involving nucleic acids use agarose gel electrophoresis as a fundamental tool. Agarose gel electrophoresis is a simple and highly effective method for separating, identifying, and purifying 0.5-to 25-kb DNA fragments.
3.1
Equipments and supplies
The following equipments and supplies are needed for carrying out agarose gel electrophoresis:

An electrophoresis chamber and power supply Gel casting trays, which are available in a variety of sizes and composed of UVtransparent plastic. The open ends of the trays are closed with tape while the gel is being cast, then removed prior to electrophoresis. Sample combs, around which molten agarose is poured to form sample wells in the gel. Electrophoresis buffer, usually Tris-acetate-EDTA (TAE) or Tris-borate-EDTA (TBE). Loading buffer, which contains something dense (e.g. glycerol) to allow the sample to "fall" into the sample wells, and one or two tracking dyes, which migrate in the gel and allow visual monitoring or how far the electrophoresis has proceeded. Ethidium bromide, a fluorescent dye used for staining nucleic acids. NOTE: Ethidium bromide is a known mutagen and should be handled as a hazardous chemical - wear gloves while handling. Transilluminator (an ultraviolet lightbox), which is used to visualize ethidium bromidestained DNA in gels. NOTE: always wear protective eyewear when observing DNA on a transilluminator to prevent damage to the eyes from UV light. The protocol for agarose gel electrophoresis can be divided into three stages: gel preparation; running and staining each of which is detailed below.
3.2.1 Gel preparation

To prepare an agarose gel, agarose powder is mixed with electrophoresis buffer to the desired concentration, and then heated in a microwave oven until completely melted. Most commonly, ethidium bromide is added to the gel (final concentration 0.5 ug/ml) at this point to facilitate visualization of DNA after electrophoresis. After cooling the solution to about 60oC, it is poured into a casting tray containing a sample comb and allowed to solidify at room temperature or in a refrigerator.
37
3.2.2 Gel running

After the gel has solidified, the comb is removed, using care not to rip the bottom of the wells. The gel, still in its plastic tray, is inserted horizontally into the electrophoresis chamber and just covered with buffer. Samples containing DNA mixed with loading buffer are then pipeted into the sample wells, the lid and power leads are placed on the apparatus, and a current is applied. You can confirm that current is flowing by observing bubbles coming off the electrodes. DNA will migrate towards the positive electrode, which is usually colored red. The distance DNA has migrated in the gel can be judged by visually monitoring migration of the tracking dyes. Bromophenol blue and xylene cyanol dyes migrate through agarose gels at roughly the same rate as doublestranded DNA fragments of 300 and 4000 bp, respectively.
3.3.3 Gel staining

When adequate migration has occured, DNA fragments are visualized by staining with ethidium bromide. This fluorescent dye intercalates between bases of DNA and RNA. It is often incorporated into the gel so that staining occurs during electrophoresis, but the gel can also be stained after electrophoresis by soaking in a dilute solution of ethidium bromide. To visualize DNA or RNA, the gel is placed on a ultraviolet transilluminator. Be aware that DNA will diffuse within the gel over time, and examination or photography should take place shortly after cessation of electrophoresis.
3.4
Critical parameters for agarose gel electrophoresis
3.4.1 Agarose concentration

Molecules of linear, duplex DNA travel through gel matrices at a rate that is inversely proportional to the log10 of their molecular. The molecular weight of a fragment of interest can therefore be determined by comparing its mobility to the mobility of DNA standards of known molecular weight. This is the most valuable feature of agarose gel electrophoresis, as it provides a reproducible and accurate means of characterizing DNA fragments by size.
Figure 5: Effect of agraose concentration on migration

38
Agarose concentration plays an important role in electrophoretic separations, as it determines the size range of DNA molecules that can be adequately resolved. For most analyses, concentrations of 0.5% to 1.0% agarose are used to separate 0.5-to 30-kb fragments. However, low agarose concentrations (0.3 to 0.5%) are used to separate large DNA fragments (20 to 60 kb), and high agarose concentrations (1 to 1.5%) can resolve small DNA fragments (0.2 to 0.5 kb).
3.4.2 Applied voltage

In general, DNA fragments travel through agarose at a rate that is proportional to the applied voltage. With increasing voltages, however, large DNA molecules migrate at a rate proportionately faster than small DNA molecules. Consequently, higher voltages are significantly less effective in resolving large DNA fragments. For separating large DNA molecules, it is best to run gels at both low agarose concentrations and low applied voltages (1 V/cm, 0.5% agarose).
3.4.3 Electrophoresis buffers

The two most widely used electrophoresis buffers are Tris/ acetate (TAE) and Tris/borate (TBE). While these buffers have slightly different effects on DNA mobility, the predominant factor that should be considered in choosing between the two is their relative buffering capacity. Tris/acetate is the most commonly used buffer despite the fact that it is more easily exhausted during extended or high-voltage electrophoresis. Tris/borate has a significantly greater buffering capacity, but should be avoided for purification of DNA from gels (see gel purification protocols).
3.4.4 DNA conformation

Closed circular (form I), nicked circular (form II), and linear duplex (form III) DNA of the same molecular weight migrate at different rates through agarose gels. In the absence of ethidium bromide, closed circular supercoiled DNAs such as plasmids migrate faster than their linear counterpart DNAs. Supercoiling essentially winds the molecules up, giving them a smaller hydrodynamicradius and allowing them to pass more readily through the gel matrix. Nicked or relaxed circular molecules that have lost all of their superhelicity migrate appreciably slower than either supercoils or linear molecules. The intercalating dye (ethidium bromide) is commonly incorporated into the gel and running buffer. The dye reduces the mobility of linear duplexes and has a particularly pronounced effect on the mobility of closed circular DNA. Ethidium bromide changes the superhelical density of closed circular molecules by inducing positive supercoils.
39
Figure 6: Mobility of different forms of DNA With increasing concentrations of ethidium bromide, negative supercoils are gradually removed, causing a concomitant decrease in the mobility of the DNA molecule. This occurs until a critical free-dye concentration is reached where no more superhelical turns remain (usually between 0.1 to 0.5 g/ ml). As still more ethidium bromide is bound, positive superhelical turns are generated which, like negative supercoils, cause an increase in the electrophoretic mobility of the molecules. By running gels at different concentrations of ethidium bromide, therefore, form I DNAs can easily be distinguished from other topoisomers.
Selected references
Current Protocols in Molecular Biology. 2007. John Wiley & Sons, Inc.
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4. Molecular data analysis

_______________________________________________
A number of molecular marker techniques like RAPD, RFLP, AFLP, SSRs, ISSRs etc. are available at present and each of them differs in its informational contents. Multilocus approaches may be convenient but have some technical/ analytical drawbacks such as dominance. As a consequence of simultaneous visualization of many marker alleles, multilocus data are typically analysed as pair-wise comparison of complex patterns that only have meaning relative to others in the same study, thus results are to a limited extent comparable among studies. Single locus markers, on the other hand are characterised by codominance and thus are more flexible and supply more robust and comparable data.
4.1
Data collection
Once gel images are in hand, entry of band information into computer can be done manually or it can be read from gel directly by a computer installed software. The band (alleles) can be scored as presence/ absence (1 or 0) in case of dominant markers (e.g. RAPD, ISSR, AFLP) which can not distinguish homozygous and heterozygous individuals. Alternatively, alleles can be simply coded as their allele size in base pairs in case of codominant markers where heterozygote yields two bands and homozygote one band (e.g. SSR or microsatellite, RFLP). However, SSR and RFLP markers can be scored as presence/ absence but some information will be lost.
4.2
Data analysis
Data can be broadly analysed for two purposes: intra-population analysis, e.g. F coefficient and other differentiation measuers and inter-population for genetic distance and analyses of molecular variance. There are many software packages with different analytical methods that can be either downloaded from internet or purchased. Genotype identification may be used for either protection of breeders rights or for quality control of cultivar production or for identification of duplicates in genebanks. The easiet approach is to identify genotypes with unique bands/ markers. However, some statistics can also be used in genotype identification particularly for the efficacy of either specific primers or DNA marker techniques. These functions include polymorphism information content, marker index, resolving power, discriminating power, probability of idenitcal match by chance etc.
41
Some of the softwares commonly used for molecular data analysis are listed below and use of one such software i.e. NTSYS is described.
NTSYS
http://www.exetersoftware.com/cat/ntsyspc/ntsyspc.html (paid software)
Arlequin Phylip PAUP POPGENE
http://cmpg.unibe.ch/software/arlequin3 http://evolution.genetics.washington.edu/phylip.html http://paup.csit.fsu.edu/ (paid software) http://www.ualberta.ca/~fyeh/download.htm
4.3
Commonly used statistical indices 4.3.1 For genotype identification

Resolving power (Rp) Resolving power (Rp)= sum of Band Informativeness of all bands produced by a primer. Band Informativeness (Ib) = 1-(2 x | 0.5 p|), where p is the proportion of accessions containing the band. So Resolving power of a primer (Rp) = Ib Marker Index (MI) Marker index is product of expected heterozygosity/ gene diversity and effective multiplex ratio. Expected heterozygosity/ gene diversity = (1- pi2)/ n, where Pi is the frequency of the ith allele and n is the number of cultivars. Effective multiplex ration = number of polymorphic products amplified per reaction.
42
Minimal number of markers needed to identify a set of genotypes (n) n = In X / [In (1/1-D)], where X is the number of unique genotypes, D is the genetic diversity over the loci and n is the number of loci analyzed. Number of genotypes that can be identified (X) X = [1/(1-D)]n , where X is the unique genotypes, D is the genetic diversity over loci. Probability of chance identity (CIp) CIp = [(XD]n, where XD is average similarity index among genotypes and n is the average number of amplified products per genotype.
4.3.2 At species level diversity

a)
Polymorphism information content (PIC) PIC = 1 [pi2 + (1-pi)2] Where, pi = frequency of ith marker in the data set. PIC = 2 pi (1-pi), in case of markers with null alleles where pi is the frequency of the ith null allele. b) Percent polymorphic loci (Ps) Ps = Number of polymorphic loci/ total number of loci compared. A loci is said to be polymorphic when Pi of most common allele is < 1.0. c) Mean number of alleles per locus (As) As = Average of allele frequency over all loci. d) Gene diversity (Hes) Hes = 1- pi2, where pi is mean frequency of ith allele. Mean diversity He is calculated by averaging the Hes values over all loci which ranges from 0 to 1. He is also defined as the probability that two alleles randomly chosen
43
from a population will be different. He is the expected proportion of heterozygous loci in a randomly chosen individual. He is the average proportion of heterozygotes in a random mating population.
e)
Effective number of alleles (Aes) Aes = 1/ (1- Hes), which is equal to the number of alleles when all alleles
have the same frequency.
e)
Observed heterozygosity (Ho) Ho = Proportion of heterozygous individuals in a sample. Ho = He under random mating and no selection.
4.3.3
f)
At population level diversity

Percent polymorphis loci (Pp) Pp = Proportion of loci polymorphic in each population averaged over all populations
g)
Number of alleles per locus (Ap) Ap = Number of alleles for each population/ number of populations
h)
Genetic diversity for each locus and population (Hep) Hep = 1- 1- pi2, where pi is the frequency of ith allele in each population. Mean Hep is got for each locus by averaging over all populations and an overall mean (Hep) is obtained by averaging over all loci.
i)
The effective number of alleles (Aep) Aep = 1/ (1- Hep)
j)
Shannon-Weaver Information index (SWI or H)
44
SWI or H = pi In pi, where pi is frequency of ith allele at a locus. This parameter is not bound by the 0 to 1 limit. No genetic interpretation and statistical properties partly known for it.
4.3
Use of NTSYS-pc software for molecular data analysis

4.3.1 Conversion of DNA fragment data into binary data
First we need first to convert the fragment data into binary data (1 = present, 0 = absent). To do this conversion, DNA fragments should be labeled based on their migration in the gel, starting from position 1 and so (usually starting from the top to the bottom), with a rule that fragments with same mobility in the gel (considered having same molecular weight) is given same label.
4.3.2
Data preparation in MS Excel Sheet
Prepare rectangular data matrix in Excel, following this format:

1 8 10 0 ABCDEFGHIJ X1 0 0 0 1 1 1 1 0 0 0 X2 1 1 1 0 1 1 1 1 1 0 X3 1 1 1 0 1 1 1 1 1 0 X4 1 0 1 0 1 1 0 1 1 0 X5 0 1 0 1 1 0 1 0 0 0 X6 0 0 0 1 1 1 0 0 0 0 X7 0 0 0 1 1 1 0 0 0 0 X8 1 1 1 0 0 0 1 1 1 1 Description: Number 1 on first row first column is a code for matrix type, in this case is rectangular matrix data (There are 8 different matrix types). Number 8 on first row second column is the number of row in the matrix, in this case is the number of marker used. Number 10 on first row third column is the number of column in the matrix, in this case is the number of taxa used. Number 0 on first row fourth column is explanation that there is no missing data in the matrix. If we have missing data, we should change 0 into 1, and give the code for missing data in the next column, for example -999. Letter A J on second row second column and so are taxa codes. Code X1 X8 on first column are marker codes.
45
Save the rectangular data matrix in MS Excel Workbook file type (extension .XLS) with a suitable name, for example EXERCISE.
4.3.3 Data editing in NT-Edit

Open program NT-Edit in folder NTSYS-pc 2.1. On menu File, click Open file in grid. Browse file EXERCISE.XLS on the corresponding folder, then click Open. Then the rectangular data matrix will be shown in the monitor. On menu File, click Save file as. Give name EXERCISE using NTSYS file type (*.NTS)
4.3.4 Calculation of similarity coefficient in NTSYS-pc Open program NTSYS-pc 2.10x in folder NTSYS-pc 2.1
On the main menu option in the left side of monitor, choose menu Similarity Then click option Qualitative data It will be shown some parameters that we need to fill in their arguments. On parameter Input file, click twice on the empty argument. Browse file EXERCISE.NTS in the corresponding folder. On the parameter Coefficient, choose Dice by clicking on the argument. On the parameter Output file, click twice on the empty argument. Choose folder that you want to save the output file, and give a suitable name for the output file, for example SIMILARITY The file will automatically be saved in NTSYS file type (*.NTS) For the other parameters, let them in default (no need to change) Click Compute
4.3.5 Cluster analysis in NTSYS-pc On the main menu option in the left side of monitor, choose menu Clustering
Then click option SAHN On the parameter Input file, click twice on the empty argument. Browse file SIMILARITY.NTS in the corresponding folder. On the parameter Output file, click twice on the empty argument. Choose the folder that you want to save the output file, and give a suitable name for the output file, for example UPGMA The file will automatically be saved in NTSYS file type (*.NTS) On parameter In case of ties, choose FIND by clicking the argument. For the other parameters, let them in default (no need to change) Click Compute
4.3.6 Visualisation of phylogeny tree in NTSYS-pc On the main menu option in the left side of monitor, choose menu Clustering
Then click option Tree plot
46
On parameter Input file, click twice on the empty argument. Browse file UPGMA.NTS in the corresponding folder. Click Compute
Selected references
Rohlf, FJ. 1998. NTSYSpc Version 2.0: User Guide. Applied Biostatistics Inc. Hernan Laurentin, 2009. Data analysis for molecular characterization of plant genetic resources. Genetic Resources and Crop Evolution 56: 277-292. Powell et al. 1997. Comparison of PCR-based marker systems for the analysis of genetic relationships in cultivated potato. Molecular Breeding 3: 127-136.
47
Appendix
_______________________________________________________
Chloroform : Isoamyl alcohol (24:1)
Remove 160 mL chloroform from a full 4 liter bottle and add 160 mL isoamyl alchol to the bottle containing chloroform. This will be 24:1 chloroform: isoamylalcohol mixture. Mix it well.
CTAB Buffer (200mL)

4M NaCl (70mL) 1M Tris (20 ml) 0.5 M EDTA, pH 8.0 (8 ml) 2% 2-Mercaptoethanol (4 ml) 10% CTAB (40 ml) dH2O (58 ml) Adjust pH to 8.0 with HCl, autoclave before use
EDTA 0.5 M
Add 186.1 g EDTA (disodium, dihydrate, Sigma # E-5134) to 800 ml of ddH20. Add about 20g of NaOH pellets while stirring to bring the pH to 8.0. Add the last few grams slowly to avoid overshooting the pH. Note that the EDTA won't completely dissolve until the pH is around 8. Filter with 0.5 micron filter and autoclave.
Ethanol (70 %)
Add 740 mL Ethanol, 95 %, to 260 ml ddH2O.
Ethidium bromide
Drop 1 g ethidium bromide per 100 mL ddH2O. Stir for several hours to dissolve ethidium bromide.
NaCl (5 M)
Add ddH2O to 292.2 g NaCl to a total volume of 1 liter. Autoclave to sterilize.
NaOH
Add ddH2O to 160.04g NaOH to a total volume of 1 liter and stir.
48
Proteinase K, stock
20 mg/ mL H2O Store at 20oC. Conc. in Xn = 50 g/ ul: Stock 400X Recommended aliquotting 100 L into 0.5 mL tubes. Store at 20 C.
RNAse (10mg/ mL)

Dissolve RNase A in 10mM Tris-Cl, pH 7.5, 15 mM NaCl. Heat at 100oC for 15 min. Cool to room temperature and store as aliquots at -20 oC.
TE pH 8.0 (Standard TE)

Tris 1M pH 8.0 EDTA 0.5 M pH 8.0 H2O Aliquot and autoclave : 40 mL : 8 mL : 3952 mL
50X TAE (Tris acetate)

242 g Tris base 57.1 ml glacial acetic acid 100.0 ml 0.5 M EDTA (pH 8.0) Bring to 1 liter with d H2O Dilute 1/50 to final conc of 0.04 M Tris-acetate, 0.001 M EDTA
5X TBE (Tris borate)

Tris base (MW = 121.14) NA2EDTA (pH 8.0) Boric acid Verify final pH. Note: For EDTA (free acid) use 1.46 g/ L) 54 g 4.0 g 27.5 g
10:1 TE
10 mM Tris, 1 mM EDTA, adjust pH to 8.0 with HCl, autoclave before use.
49

Training Manual On Molecular Marker Techniques For Genotype Identification (!!!) PDF

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Training manual on

Molecular Marker Techniques for Genotype Identification

Molecular Marker Techniques for Genotype Identification

Molecular Marker Techniques for Genotype Identification

Molecular Marker Techniques for Genotype Identification

Molecular Marker Techniques for Genotype Identification

Mandatory saftey rules

Molecular Marker Techniques for Genotype Identification

Cautionary safety rules

Molecular Marker Techniques for Genotype Identification

Safety tips for handling some equipments

Decontamination of ethidium bromide solutions

Molecular Marker Techniques for Genotype Identification

Molecular Marker Techniques for Genotype Identification

1. DNA isolation and purification in plants ___________________________________________

Molecular Marker Techniques for Genotype Identification

Molecular Marker Techniques for Genotype Identification

Molecular Marker Techniques for Genotype Identification

Molecular Marker Techniques for Genotype Identification

Molecular Marker Techniques for Genotype Identification

2. Polymerase Chain Reaction

Figure 2. The polymerase chain reaction

Molecular Marker Techniques for Genotype Identification

Composition of the PCR Reaction Mixture

2.1.1 Template DNA

Molecular Marker Techniques for Genotype Identification

2.1.3 Magnesium chloride (MgCl2) concentration

2.1.5 Taq DNA polymerase

Molecular Marker Techniques for Genotype Identification

2.2.3 Primer annealing step

2.2.4 Extension step

2.2.5 Number of cycles

2.2.6 Final Extension Step

Some variants of PCR

PCR-based molecular marker techniques

2.4.1 Random Amplified Polymorphic DNA (RAPD)

Molecular Marker Techniques for Genotype Identification

Sterile double-distilled water (to make 25 L volume) Total volume

Molecular Marker Techniques for Genotype Identification

Agarose gel electrophoresis

Stain the gel with ethidium bromide.

Molecular Marker Techniques for Genotype Identification

Inter-Simple Sequence Repeats (ISSR)

Figure 3. Generation of ISSR markers

Supplies and solutions

Sterile double-distilled water (to make 25 L volume) Total volume

Agarose gel electrophoresis

Simple Sequence Repeats (SSR)

Figure 4: PCR amplification of SSR markers

Molecular Marker Techniques for Genotype Identification

0.2 L 0.1 L 2.0 L 16.2 L

Sterile double-distilled water (to make 25 L volume) Total volume

Molecular Marker Techniques for Genotype Identification

Agarose gel electrophoresis

Molecular Marker Techniques for Genotype Identification

Functional markers for genotype identification

Supplies and solutions

Molecular Marker Techniques for Genotype Identification

PCR Cocktail preparation

Taq polymerase buffer MgCl2 Primer (reverse) Primer (forward)

dNTPs Taq DNA polymerase Template DNA

Sterile double-distilled water (to make 25 L volume) Total volume