Training manual on Molecular Marker Techniques for Genotype Identification (!!!).pdf

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Molecular Marker Techniques for Genotype Identification

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

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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

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Table of contents

1. DNA isolation 6-13

2. Polymerase chain reaction 14-32

3. Agarose gel electrophoresis 33-36

4. Molecular data analysis 37-45

5. Appendix

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Safety in laboratories

___________________________________________

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 one’s 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

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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.

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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

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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.

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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. 487-

490.

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.

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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 mis-

priming 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 µL, 20-200 µL, 200-1000 µL)

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 0.5µg/ 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 : 10.0 ml

Distilled filtered water : 90.0 ml

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High range (B)

(100-5000 ng/ml final DNA concentration)

H33258 stock solution : 100.0 l

10x TNE buffer : 10.0 ml

Distilled filtered water : 90.0 ml

DNA standard

1:10 dilution (100 mg / ml) of 1mg / ml DNA standard stock solution.

Mix the following:

1 mg / ml DNA standard stock : 100 l

10x TNE buffer : 100 l

Distilled water : 800 l

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

___________________________________________

The advent of Polymerase Chain Reaction (PCR) by Kary B. Mullis in the mid-

1980s revolutionized molecular biology as we know it.The polymerase chain reaction

(PCR) is a rapid procedure for in vitro enzymatic amplification of a specific segment of

DNA. PCR has infinite applications which include direct cloning from genomic DNA or

cDNA, in vitro mutagenesis and engineering of DNA, genetic fingerprinting 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 amplified and two single-stranded oligonucleotide primers flanking 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 amplified. 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 5→3) 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 228

-fold (270 million–fold) amplification of the discrete product.

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 0.1-1µg for genomic DNA,

for a total reaction mixture of 50µl. 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 5°C, 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. 5°C 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 Mg2+

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 1-

4mM, 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 200µM. 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 10-50µM, 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 50µl 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 95°C 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 95°C, 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 95°C.

2.2.2 Denaturation step

Usually 0.5-2 min denaturation at 94-95°C 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 5°C 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 1-

2°C.

2.2.4 Extension step

Usually the extension step is performed at 70-75°C. 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

22


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 72°C for 5-15min to fill-

in 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 3’-

ends 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

2.3.1 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.

2.3.2 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.

23


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)


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 µL, 20-200 µL, 200-1000 µL)

24


Supplies and solutions

10X Taq poymerase buffer, 25 mM magnesium chloride, 2 mM dNTP’s (mixture

of dATP, dCTP, dGTP and dTTP), Taq poymerase (5 Units/ µL), 10 µM RAPD primers,

template or genomic DNA (10ng /µL), sterile double distilled water, TAE buffer, agarose,

gel loading dye, DNA size standard, ethidium bromide, tips, 0.5mL and 1.5 mL micro-

centrifuge 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 µL centrifuge tube of the

following components except template DNA:

Sr. No. Component Stock

concentration

Required

concentration

Amount

required

for one

reaction

1 Taq polymerase buffer - 1x 2.5µL

2 MgCl2 25mM 3mM 3.0µL

3 Primer 5µM 0.5µM 2.5µL

4 dNTPs 25mM 0.2mM 0.2µL

5 Taq DNA polymerase 5U/ µL 1U 0.2µL

6 Template DNA 10ng/ µL 50ng 5.0µL

7 Sterile double-distilled

water (to make 25 µL

volume)

- - 11.6µL

Total volume 25.0 µL

Dispense 20µL of above master mix in each of the 0.2mL PCR tube.

Add 5 µL of template DNA and mix thoroughly and gently by pipetting 3-4 times.

25


Turn on the thermocyler and place samples in it.

The following general thermocycling steps are followed:

Step1: Initial denaturation at 94ºC for 5.0min

Step2: Denaturation at 94º C for 1.0min

Step3: Primer annealing at 45-55º C for 1.0min

Step4: Primer extension at 72º C for 2.0min

Step5: Go to 2, 39 times

Step6: Final extension at 72º C for 10min

Step7: 4º C for ever

After the run is completed turn off the machine and remove the samples.

Add 2.5 µL 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 55ºC before pouring. (ethidium bromide can be added

at this point to a concentration of 0.5 µg/ ml)

Place the comb in gel tray.

Pour 50º 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 µL of 6% gel loading dye

solution. Mix well. Load 25 µl 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.

26


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-Anollés 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.

27


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)



Laminar air flow

Fume hood

pH meter

Centrifuge

Standard balance

Thermocycler


Power supply units


Vortexer

Pipetman (2-20 µL, 20-200 µL, 200-1000 µL)

28








Protocol







concentration

Required

concentration

Amount

required

for one

reaction

1 Taq polymerase buffer 10x 1x 2.5µL

2 MgCl2 25mM 3mM 3.0µL

3 Primer 5µM 0.5µM 2.5µL

4 dNTPs 25mM 0.2mM 0.2µL

5 Taq DNA polymerase 5U/ µL 1U 0.2µL

6 Template DNA 10ng/ µL 50ng 5.0µL



volume)

- - 11.6µL


Dispense 20µL of above master mix in each of the 0.2mL PCR tube.

Add 5 µL of template DNA and mix thoroughly and gently by pipetting 3-4 times.



29


Step1: Initial denaturation at 95ºC for 5.0 min

Step2: Denaturation at 94º C for 1.0 min

Step3:Primer annealing at 45-55º C for 1.0 min

Step4: Primer extension at 72º C for 2.0 min


Step6: Final extension at 72º C for 8 min






To prepare 100mL of a 1.5% agarose solution, measure 1.5g agarose into a glass

beaker or flask and add 100ml 1X TAE.


Allow solution to cool to about 55ºC before pouring. (ethidium bromide can be

added at this point to concentration of 0.5 µg/ mL)

Place the comb in gel tray.

Pour 50º C gel solution into tray to a depth of about 5mm. Allow the gel to solidify

for about 20 min at room temperature.




Load 25 µL 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.

30


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

31


Equipments

Refrigerator (4oC)



Laminar air flow

Fume hood

pH meter

Centrifuge

Standard balance

Thermocycler


Power supply units


Vortexer

Pipetman (2-20 µL, 20-200 µL, 200-1000 µL)







Protocol






32


Sr.

No.

Component Stock

concentration

Required

concentration

Amount

required

for one

reaction

1 Taq polymerase buffer 10x 1x 2.5 µL

2 MgCl2 25 mM 3 mM 3.0 µL

3 Primer (Forward +

Reverse)

5 µM 0.1 µM 1.0 µL

4 dNTPs 25 mM 0.2 mM 0.2 µL

5 Taq DNA polymerase 5U/ µL 0.5 U 0.1 µL

6 Template DNA 10 ng/ µL 20 ng 2.0 µL



volume)

- - 16.2 µL


Dispense 23 µL of above master mix in each of the 0.2 mL PCR tube.

Add 2.0 µL of template DNA and mix thoroughly and gently by pipetting 3-4 times.



Step1: Initial denaturation at 95ºC for 5.0 min

Step2: Denaturation at 94º C for 1.0min

Step3: Primer annealing at suitable annealing temp. for 1.0

min

Step4: Primer extension at 72º C for 2.0 min


Step6: Final extension at 72º C for 8 min





33



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).


Allow the solution to cool to about 55ºC before pouring. (ethidium bromide can be

added at this point to concentration of 0.5µg/ 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

ºC.




Load 25µl 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).


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.

34


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


Micro Pipettes (2-20 ul, 20-200ul, 200-1000 ul)


35


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:


concentration

Required

concentration

Amount

required

for one

reaction

1 Taq polymerase buffer 10x 1x 1.0 µL

2 MgCl2 25 mM 3 mM 1.2 µL

3 Primer (reverse) 10 µM 0.5 µM 0.5 µL

Primer (forward) 10 µM 0.5 µM 0.5 µL

4 dNTPs 10 mM 0.2 mM 0.2 µL

5 Taq DNA polymerase 5U/ µL 1 U 0.2 µL

6 Template DNA 10 ng/ µL 40 ng 4.0 µL



volume)

- - 2.4 µL


II) Thermocycling conditions


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

36


Step4: Primer extension at 720C for 2.00 min


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.


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: 43–59.

Sax K. 1923. The association of size differences with seed-coat pattern and

pigmentationm in Phaseolus vulgaris. Genetics 8: 552–560.

Henry RJ (ed.). 2001. Plant Genotyping CABI Publishing.

37


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 UV-

transparent 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 bromide-

stained 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.

38


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 double-

stranded 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

39


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.

40


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 µg/ 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.

41


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 breeder’s 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.

42


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 http://cmpg.unibe.ch/software/arlequin3

Phylip http://evolution.genetics.washington.edu/phylip.html

PAUP http://paup.csit.fsu.edu/ (paid software)

POPGENE 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.

http://www.exetersoftware.com/cat/ntsyspc/ntsyspc.html

http://cmpg.unibe.ch/software/arlequin3

http://evolution.genetics.washington.edu/phylip.html

http://paup.csit.fsu.edu/

http://www.ualberta.ca/~fyeh/download.htm

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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

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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 At population level diversity

f) 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)

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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

A B C D E F G H I J

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.

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• 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)


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”

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• On parameter Input file, click twice on the empty argument.

• Browse file “UPGMA.NTS” in the corresponding folder.


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.

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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.

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Proteinase K, stock

20 mg/ mL H2O

Store at –20oC. Conc. in Xn = 50 µg/ ul: Stock 400X

Recommended aliquotting 100 µL 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 : 40 mL

EDTA 0.5 M pH 8.0 : 8 mL

H2O : 3952 mL

Aliquot and autoclave

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) 54 g

NA2EDTA (pH 8.0) 4.0 g

Boric acid 27.5 g

Verify final pH.

Note: For EDTA (free acid) use 1.46 g/ L)

10:1 TE 10 mM Tris, 1 mM EDTA, adjust pH to 8.0 with HCl, autoclave before use.

Training manual on Molecular Marker Techniques for Genotype Identification (!!!).pdf

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