Compiled and Edited by Sunil Archak Ambika B Gaikwad KV Bhat

Compiled and Edited by

Sunil Archak Ambika B Gaikwad

KV Bhat

PCR Techniques for

Characterization of Germplasm

National Bureau of Plant Genetic Resources New Delhi

Table of Contents

1. Preparation of PCR ready DNA 04

2. Polymerase Chain Reaction 11

3. Random Amplified Polymorphic DNA (RAPD) 25

4. Inter-Simple Sequence Repeats (ISSR) 31

5. Sequence Tagged Microsatellite DNA (STMS) 39

6. Sequence Characterized Amplified Regions (SCARs) 45

7. Appendix 52

Archak S, Gaikwad AB and Bhat KV (2013). PCR Techniques for Characterization of Germplasm. NBPGR, New Delhi. Pp. 55

© NBPGR, New Delhi 110 012

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 authors.

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 employed for any other use.

2

General Safety

1. Be serious and alert when working in the lab

2. Be sure you understand the procedure to be employed in the experiment and the hazards

associated with it

3. Read all the directions for the investigation for the experiment several times and follow the

exactly as written; when in doubt, ask your trainer for assistance

4. Never perform activities that are not authorized by your trainer

5. Never handle equipment unless you have permission

6. Take extreme care not to spill any chemicals in the lab. If a spill occurs, immediately follow proper

clean-up procedure

7. Never just pour chemicals in the sink or trash can

8. Never eat or taste anything in the lab

9. Wash your hands before and after every experiment

10. Keep the work area clean and uncluttered, especially, free of back packs, books or papers

Dress Properly

1. Wear laboratory apron always

2. Tie back long hair to keep your hair out of chemicals, equipment, or flames

3. Remove or tie back any article of clothing or jewelry that can hang down and touch chemicals or

flames

Using Chemicals Safely

1. Never mix chemicals for the “fun” of it; they may react in a dangerous way

2. Never directly inhale fumes from a reaction

3. Dispose of chemicals as directed by your trainer

4. Be extra careful when working with acids or bases; rinse any acids off your skin with water. Notify

your trainer immediately of an acid spill; Immediately report all injuries or accidents

5. Ethidium Bromide is a carcinogenic agent; never allow skin contact

6. UV rays are harmful to naked eyes; look only through safe cover

Using Glassware Safely

1. If glassware breaks, notify your trainer and dispose of the glassware immediately

2. Never eat or drink from lab glassware; it may look clean but it has been used for a variety of

purposes

End-of-Investigation

1. When lab time is over or when you have completed the experiment, clean up your work area and

return all equipment to its proper place

2. Wash your hands after every investigation

3. Turn off all equipment before leaving the room

3

Notes

4

1. Preparation of PCR ready DNA

Good quality DNA is a prerequisite for reproducible results

A number of published methods are available for the extraction of genomic DNA. Most

routinely followed protocols in plant DNA isolation are essentially based on Saghai-Maroof et

al. (1984) with minor modifications to suit the material under consideration.

Principle

For plant cells with a rigid cell wall, the disruption of cells usually requires the tissue to be

ground using a pestle and mortar in a pool of liquid nitrogen. The powdered plant tissue is

then transferred to an extraction buffer that contains detergent to disrupt the membranes.

Cetyl trimethyl ammonium bromide (CTAB) is commonly used for this purpose. The

extraction buffer also contains a reducing agent (2-mercaptoethanol) and a chelating agent

(ethylene di-amine tetra-acetic acid, EDTA). This helps to inactivate nucleases that are

released from the plant cell and can cause serious degradation of the genomic DNA. Their

effects can be minimized by keeping the reactions cold, when possible. Phenolic compounds

may also be released on disruption of plant tissues and these may interfere with subsequent

uses of the DNA (e.g. if it is to be used in the PCR). Polyvinyl pyrolidone (PVP) can be

added to the extraction buffer to remove phenolic compounds.

Phenol extraction can be used to remove any traces of proteins and the genomic DNA can

be precipitated using either ethanol or isopropanol. Precipitated DNA can be hooked out of

the solution or collected by centrifugation. It is important that DNA is not sheared, for this

reason the DNA should not be vortexed or pipetted repeatedly and all manipulations should

be as gentle as possible.

For additional details on the current DNA extraction protocols, refer to Appendix.

All DNA extractions have three steps:

Lysis of cell walls and membranes to free DNA into solution.

Purification of DNA by precipitating proteins and polysaccharides.

Precipitation of DNA and resuspension in a buffer.

5

Instruments and materials

Cryocan for Liquid Nitrogen

Liquid Nitrogen

Pestle and mortar

Water bath

Vortexer

High speed centrifuge

Micro centrifuge

Auto-pipettes 2-20l, 20-200l, 200-1000l

Deep freezer (-20oC)

Refrigerator

Horizontal gel electrophoresis unit with accessories

UV trans-illuminator and gel documentation system

Fluorometer

Glassware and Plasticware

250ml flask

Reagent bottles

Oakridge tube (30/50ml)

Tube stands

0.5 and 1.5ml microcentrifuge tubes

Tips for pipettes (10l, 200l, 1ml)

Reagents

CTAB Buffer

o 1.4 M NaCl, 100 mM Tris, 20 mM EDTA, 2% 2-Mercaptoethanol, 1.5% CTAB;

Adjust pH to 8.0 with HCl, autoclave before use

Isopropanol

Saturated phenol pH 8.0

Chloroform : isoamylalcohol ( 24:1) mixture

10:1 TE (10 mM Tris, 1 mM EDTA, Adjust pH to 8.0 with HCl, autoclave before use)

RNase A, 10mg/ml (Dissolve RNase A in 10mM Tris, pH 7.5, 15 mM NaCl. Heat at

100 oC for 15 min. Cool to room temperature. Store as aliquots at -20 oC)

70% ethanol

50xTAE/20x TBE, pH 8.0 (2 M tris-acetate/tris-borate, pH 8.0; 0.05 M EDTA, pH 8.0)

10x Loading Buffer (0.25% Bromophenol Blue; 0.25% Xylene Cyanol FF; 50% Glycerol;

1x TAE/TBE)

Ethidium bromide stock (10 mg/ml ethidium bromide in double distilled water)

Ethidium bromide is hazardous! A potent carcinogenic and teratogenic

6

Extraction

Weigh 1-2g of clean young leaf tissue and grind to fine powder with a pestle and

mortar after freezing in liquid nitrogen.

Transfer to 30 ml centrifuge tube with 10 ml CTAB buffer maintained at 60o C in a

water bath. Mix vigorously or vortex.

Incubate at 60o C for one hour. Mix intermittently.

Add 10ml of chloroform : isoamyl alcohol. Mix gently by inverting for 5 min.

Spin at 15,000rpm (~12000 g) for 10 min in a SS34 rotor in Sorval RC-5C centrifuge

at 25o C.

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 solution with a pipette (Alternatively, spin at

15,000rpm for 5 min in a SS34 rotor in Sorval RC-5C centrifuge at 25o C.)

Add 0.5 ml of 70% ethanol. Mix gently and incubate for 30 min. Decant and repeat

the 70% ethanol wash. Decant off and dry the pellet under vaccum.

Dissolve DNA in minimum volume of 10: 1 TE.

Add RNAse (0.2 ml) and incubate at 37o C for one hour.

Add equal volume of phenol : chloroform: isoamyl alcohol (25:24:1), mix properly for

at least 2 min and spin for 5 min. Pipette out the aqueous phase and perform two

chloroform: isoamyl alcohol extractions.

Precipitate DNA by adding 1/10 volume of 3M sodium acetate (pH 5.2) 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 (alternatively, air dry at room

temperature).

Dissolve in TE; aliquot into 1.5ml tubes; store working DNA samples at 4 °C and the

rest at -20 °C.

Quantification of DNA

DNA quantification is a prerequisite step for downstream enzymatic procedures including PCR

amplification and restriction digestion. There are several methods for quantifying DNA, the most

common being: i) electrophoretic comparison with standard DNA samples of known

concentration; ii) spectrophotometric determination, and iii) fluorometric determination.

7

Electrophoresis of a DNA sample of unknown concentration with known

standards

Seal the open ends of the gel tray with tape. Place the well-forming comb in the

appropriate slot. Ensure that the gel tray is leveled.

Prepare a 0.8% agarose gel in 1x TAE/TBE (not water). Pour warm agarose onto the

gel tray and allow it to set for at least 30 min (Optionally, EtBr may be added to the

gel).

Remove the comb and tape. Place the gel into the electrophoresis tank and pour 1x

TAE/TBE 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 (DNA ladder or digested DNA)

into the extreme wells.

Electrophorese at 60-70V, until the dye moves down about 3-4 cm from the wells.

Stain the gel with ethidium bromide for 30-45 min (Not if EtBr is added into the gel).

Illuminate the gel with UV light and photograph under UV light (UV is hazardous!).

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.

DNA quantification by UV spectroscopy

Take 1l 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; 1OD

at 260nm = 50g/ml. This estimate is influenced by the contaminating substances

like RNA and very low molecular weight DNA in the solution.

Prepare a working stock of samples of 100l with concentrations of 10 ng/l.

DNA quantification by fluorometry

The DNA extraction procedures do not always eliminate RNA completely. Therefore,

DNA concentration estimation by UV spectrophotometry might be inaccurate depending

upon the RNA contamination. RNase treatment may help in reducing the errors.

However, fluorometric estimations are more reliable as it measures the fluorescence

8

emitted by the double stranded DNA only, with 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 ( 1000ml, buffer stock solution):

o 100mM Tris; 1M NaCl; 10mM 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 oC for up to 3 months)

Calf thymus DNA (standard)

Hoechst 33258 dye stock: Hoechst 33258: 1mg/ml in distilled water (Add 10ml

distilled water to 10mg H33258. Do not filter. Store at 4 °C for up to 6 months in an

amber bottle)

Procedure:

Prepare the assay and DNA standard solutions as described below:

Low range (A)

(10-500ng/ml final DNA concentration)

High range (B)

(100-5000ng/ml final DNA concentration)

H33258 stock solution : 10.0l

10x TNE buffer : 10.0ml

Distilled filtered water : 90.0ml

H33258 stock solution : 100.0l

10x TNE buffer : 10.0ml

Distilled filtered water : 90.0ml

Turn on the fluorometer at least 15 min before using

Calibrate the instrument to Zero: Prepare an assay blank using 2 ml of appropriate

Assay solution (A or B for high DNA concentration). Wipe and 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 2, 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.

Reference

Saghai-Maroof MA, Soliman KM, Jorgensen RA and Allard RW (1994) Ribosomal DNA spacer

length polymorphism in barley: Mendelian inheritance, chromosomal location and population

dynamics. Proc. Natl. Acad. Sci. USA 81: 8014-8018.

9

DNA Extraction Notes

10

DNA Extraction Notes

11

2. Polymerase Chain Reaction (PCR)

PCR has provided seamless novelties to molecular biology protocols

Principle

Polymerase Chain Reaction (PCR), invented by Kary B. Mullis (1993 Nobel laureate), is a

technique for in vitro exponential amplification of a small quantity of a specific nucleotide

sequence. It requires the presence of template sequence, two oligonucleotide primers that

hybridize to opposite strands and flank the region of interest in the target DNA, nucleotides and a

thermostable DNA polymerase. The reaction is cycled for template denaturation, primer

annealing, and the extension of the primers by DNA polymerase until enough copies are made

for further analysis.

PCR allows the production of more than 10 million copies of a target DNA sequence from

only a few molecules (template). The sensitivity of this technique means that the sample

should not be contaminated with any other DNA or previously amplified products

(amplicons) that may reside in the laboratory environment.

The reagents for PCR should be prepared separately and used solely for this purpose.

Autoclaving of all solutions, except dNTPs, primers and Taq DNA Polymerase is

recommended. Solutions should be aliquoted in small portions and stored in designated

PCR areas. Aliquots should be stored separately from other DNA samples.

A control reaction, omitting template DNA, should always be performed, to confirm

the absence of contamination.

12

PCR variety

1. Hot-start PCR - to reduce non-specific amplification. Can also be done by separating the DNA mixtures from enzyme by a layer of wax which melts when heated in cycling reaction. A number of companies also produce hot start PCR products.

2. Touch-down PCR - start at high annealing temperature, then decrease annealing temperature in steps to reduce non-specific PCR product. Can also be used to determine DNA sequence of known protein sequence.

3. Nested PCR - use to synthesize more reliable product - PCR using a outer set of primers and the product of this PCR is used for further PCR reaction using an inner set of primers.

4. Inverse PCR - for amplification of regions flanking a known sequence. DNA is digested; the desired fragment is circularized by ligation, then PCR using primer complementary to the known sequence extending outwards.

5. AP-PCR (arbitrary primed)/RAPD (random amplified polymorphic DNA) - methods for creating genomic fingerprints from species with little-known target sequences by amplifying using arbitrary oligonucleotides. It is normally done at low and then high stringency to determine the relatedness of species or for analysis of Restriction Fragment Length Polymorphisms (RFLP).

6. RT-PCR (reverse transcriptase) - using RNA-directed DNA polymerase to synthesize cDNAs which is then used for PCR and is extremely sensitive for detecting the expression of a specific sequence in a tissue or cells. It may also be use to quantify mRNA transcripts. See also Quantitative RT-PCR, Competitive Quantitative RT-PCR, RT in situ PCR, and Nested RT-PCR.

7. RACE (rapid amplification of cDNA ends) - used where information about DNA/protein sequence is limited. Amplify 3' or 5' ends of cDNAs generating fragments of cDNA with only one specific primer each (+ one adaptor primer). Overlapping RACE products can then be combined to produce full cDNA.

8. DD-PCR (differential display) - used to identify differentially expressed genes in different tissues. First step involves RT-PCR, then amplification using short, intentionally nonspecific primers. Get series of band in a high-resolution gel and compare to that from other tissues, any bands unique to single samples are considered to be differentially expressed.

9. Multiplex-PCR - two or more unique targets of DNA sequences in the same specimen are amplified simultaneously. One can be use as control to verify the integrity of PCR. Can be used for mutational analysis and identification of pathogens.

10. Q/C-PCR (quantitative comparative) - uses an internal control DNA sequence (but of different size) which compete with the target DNA (competitive PCR) for the same set of primers. Used to determine the amount of target template in the reaction.

11. Recursive PCR - used to synthesize genes. Oligos used are complementary to stretches of a gene (>80 bases), alternately to the sense and to the antisense strands with ends overlapping (~20 bases). Design the oligo avoiding homologous sequence (>8) is crucial to the success of this method.

Preparation of Reaction Mixture

To perform several parallel reactions, prepare a master mix containing water, buffer, dNTPs,

primers, MgCl2 and Taq DNA polymerase in a single tube, which can then be aliquoted into

individual tubes. Template DNA is added subsequently. This method of setting reactions

minimizes the possibility of pipetting errors and saves time by reducing the number of

reagent transfers.

13

Reaction Mixture Set Up

1. Gently vortex and briefly centrifuge all solutions after thawing 2. Add, in a thin-walled PCR tube, on ice:

Reagent Final concn Template DNA 10pg-1 µg 10X Taq buffer 1X 2 mM dNTPs mix 0.2 mM of each Primer I 0.1-1 µM Primer II 0.1-1 µM Taq DNA Polymerase 1.25 u / 50 µl 25 mM MgCl2 1-4 mM Sterile de-ionized water -

* Selection of 25 mM MgCl2 solution volume:

3. Gently vortex the sample and briefly centrifuge to collect all drops from walls of tube. 4. Overlay the sample with half volume of mineral oil or add an appropriate amount of

wax. This step may be omitted if the thermal cycler is equipped with a heated lid. 5. Place samples in a thermocycler and start PCR.

Components of the Reaction Mixture

Template DNA

Usually the amount of template DNA is in the range of 0.01-1 ng for plasmid or phage DNA

and 0.1-1 µg for genomic DNA, for a total reaction mixture of 50 µl. Higher amounts of

template DNA usually increase the yield of nonspecific PCR products, but if the fidelity of

synthesis is crucial, maximal allowable template DNA quantities together with a limited

Final concn of MgCl2 in 50µl reaxn mix, mM 1.0 1.25 1.5 1.75 2.0 2.5 3.0 4.0

Volume of 25 mM MgCl2, µl 2 2.5 3 3.5 4 5 6 8

14

number of PCR cycles should be used to increase the fraction of "correct" PCR products.

Nearly all routine methods are suitable for template DNA purification. Although even trace

amounts of reagents used in DNA purification procedures (phenol, EDTA etc.) 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.

Primers

Guidelines for primer selection:

PCR primers are usually 15-30 nucleotides in length. Longer primers provide higher

specificity.

The GC content should be 40-60%. The C and G nucleotides should be distributed

uniformly throughout 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.

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 being number of

respective nucleotides in the primer. Annealing temperature may be approximately

5°C lower than the melting temperature.

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 1-4 mM, under the

standard reaction conditions specified. In our experiments, at a final dNTP concentration of

0.2 mM, MgCl2 concentration ranges of 1.5±0.25 mM (in Taq buffer with KCl) and of

2.0±0.5 mM (in Taq buffer with (NH4)2SO4) are suitable in most cases. If the DNA samples

contain EDTA or other chelators, the MgCl2 concentration in the reaction mixture should be

raised proportionally.

dNTPs

The concentration of each dNTP in the reaction mixture is usually 200 µM. It is very

important to have equal concentrations of each dNTP (dATP, dCTP, dGTP, dTTP), as

inaccuracy in the concentration of even a single dNTP dramatically increases the

misincorporation level.

15

When maximum fidelity of the PCR process is crucial, the final dNTP concentration should

be 10-50 µM, since the fidelity of DNA synthesis is maximal in this concentration range. In

addition, the concentration of MgCl2 should be selected empirically, starting from 1 mM

and increasing in 0.1 mM steps, until a sufficient yield of PCR product is obtained.

Taq DNA Polymerase

Usually 1-1.5 unit of Taq DNA polymerase is used in 50 µl 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 u) may be necessary to obtain a

better yield of amplification products.

Reaction Overlay

If necessary, the reaction mixture can be overlaid with mineral oil or paraffin (melting

temperature 50-60°C) of special PCR grade. One-half of the total reaction volume is usually

sufficient.

Cycling Conditions

Amplification parameters depend greatly on the template, primers and amplification

apparatus used.

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 1-3 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.

Denaturation Step

16

Usually denaturation for 0.5-2 min 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 10-15vol.%), 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 when these additives are used. The amount of enzyme

in the reaction mix should be increased since DMSO and formamide, at the suggested

concentrations, inhibit Taq DNA Polymerase by approx. 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.

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.

Extension Step

Usually the primer extension step is performed at 70-75°C. The rate of DNA synthesis by

Taq DNA polymerase is highest at this temperature. Recommended extending time is 1 min

for the synthesis of PCR fragments up to 2 kb. When larger DNA fragments are amplified,

the extending time is usually increased by 1 min for each 1000 bp.

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.

Final Extension Step

After the last cycle, the samples are usually incubated at 72°C for 5-15 min 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

prolonged to up to 30 min.

17

Calculating concentrations for PCR

Primers

i) Oligonucleotide primers are generally supplied as "so many OD units/ml" - but what does

this mean, in terms of mg/ml, or mmol/ml, etc?

If a primer is Y nucleotides (nt) long

If the MW of ssDNA is (330 daltons per nt) x (length in nt)

If the concentration of primer (=ssDNA) producing an OD of 1 at 254 nm in a 1 cm

cuvette, is 37 ug/ml

Then: the MW of the primer is 330*Y daltons

And: X OD/ml = 37 X ug/ml

= 37 X mg/l

= 37 X /330Y mM

= 37 X1000/330Y uM

For example: A 17-mer oligo-deoxynucleotide is supplied as 12.6 OD units/ml; we need to make a 5 uM

stock solution for PCR

MW: 17 x 330 = 5610 daltons

Concentration: 12.6 OD x 37 ug/ml = 466 ug/ml = 466 mg/l = 0.466 g/l

Molarity: 0.466/5610 = 0.000083 Molar = 83 uM

Therefore:

we need 5ul of oligo stock solution in 83ul (+78ul water) to make a 5uM solution (if

1ul in 83ul gives a 1uM soln.)

Calculation of amounts for PCR reactions: if we need a final concentration of 0.5 uM

oligo in the PCR reaction mix (final volume 50 ul), we add 5 ul of 5 uM stock to the

reaction mix (1/10 final dilution).

Nucleotides

Stocks of nucleotides for PCR (or other procedure) are nearly always dNTPs (de-

oxynucleotides), and concentrations are almost always given on EACH dNTP; that is, the

given concentration is each nucleotide in the mix, not the total concentration. This means

that a 2.5 mM dNTPs mix for PCR contains 2.5 mM of each dNTPs, and 10 mM total dNTPs.

Example: Make up a 2.5 mM stock solution of dNTPs from stock 100mM individual dNTPs,

FIRST mix equal volumes of each nucleotide (e.g. 50 ul); this gives you 200ul

of 25mM mixed dNTPs (Remember: concn. expressed is of each dNTPs).

THEN dilute this (or aliquot) 1/10 with WATER - aliquot into 100 ul amounts

and freeze.

18

To make master mix: multiply amount of dNTPs per reaction by number of reactions.

Procedure

1. Thaw 10x PCR Buffer, dNTP mix, primer solutions, and MgCl2 at room temperature. Keep the solutions on ice after complete thawing. Mix well before use to avoid localized differences in salt concentration.

2. Program the thermal cycler according to the manufacturer’s instructions. A

typical PCR cycling program is outlined as follows:

Step Time Temp

Initial denaturation: 3 min 94°C

3-step cycling

Denaturation: 0.5–1 min 94°C

Annealing: 0.5–1 min 50–68°C

Extension: 1 min 72°C

Number of cycles: 25–35

Final extension: 10 min 72°C

For maximum yield and specificity, temperatures and cycling times should be optimized for each new target or primer pair.

3. Prepare a master mix as shown earlier. The master mix typically contains all of the components needed for PCR except the template DNA. Prepare a volume of master mix 10% greater than that required for the total number of PCR assays to be performed. A negative control (without template DNA) should be included in every experiment. The optimal Mg2+ concentration should be determined empirically but in most cases a concentration of 1.5 to 2mM, will produce satisfactory results. Keep the master mix on ice.

4. Mix the master mix thoroughly, and dispense appropriate volumes into PCR tubes. Mix gently, for example, by pipetting the master mix up and down a few times. It is recommended that PCR tubes are kept on ice before placing in the thermal cycler. Add template DNA to the individual tubes containing the master mix.

5. When using a thermal cycler with a heated lid, do not use mineral oil. Proceed

directly to step 6. Otherwise, overlay with approximately 100 μl mineral oil. Place the PCR tubes in the thermal cycler and start the cycling program. Note: After amplification, samples can be stored overnight at 2–8°C, or at –20°C for longer storage.

6. Electrophorese amplification products. Attach tape to the ends of the gel tray. Position the well-forming comb and ensure that the gel tray is horizontal. Prepare a 1.5% agarose gel. Pour agarose onto the gel tray and allow it to set for at least 30 min. Remove comb and tape. Place the gel into the electrophoresis tank and pour 1x TAE until the gel is completely submerged.

7. Mix 4l amplification product, 4l water and 1l loading dye, and load onto the gel.

Load 2l of DNA standard into one of the wells. Electrophorese at 60-70 V until the

19

dye is 3-4 cm from the wells. Illuminate the gel with UV light and photograph under UV light. Compare the size of the DNA bands of the samples with the sizes of the standard DNA bands.

Tips

Reagent concentrations - "less is usually better" (more specific)

o primers: final concentration 0.1-1.0 M o MgCl2: final concentration 1.0-4.0 mM (depends on Taq used) o dNTPs: final concentration 0.2 mM each dNTP (depends on Taq used)

note: sensitive to repeated freeze/thaws Vortex or finger-flick reagents to mix well before use Primer design very important

o the higher the annealing temperature the better (TANN > 50°C) o primer pairs should have melting temps within 5°C of each other

Annealing temperature and step times are important Titrations are a good idea

o most commonly temperature and MgCl2 Hot starts improve reaction efficiency (fewer primer-dimers)

o manual: add Taq to tubes in thermocycler at 94oC (or MgCl2 or dNTPs) o TaqStart Antibody (Clontech) o Faststart Taq (Roche) o hotstart comparison gel

Always check program on thermalcycler Run negative control(s) to check for contamination Make a flow chart of what tried and in what order Run a positive control (a sample known to amplify well) Always run a ladder on gel (will indicate whether failed PCR or failed detection

system) Additives for fragments that are very long, G-C rich or prone to secondary structure

o glycerol, formamide, NMP - lower denaturing and annealing temperatures by a few degrees

o DMSO decreases incidence of secondary structure

Troubleshooting PCR

1. I get (many) longer unspecific products. What can I do?

Decrease annealing time Increase annealing temperature Decrease extension time Decrease extension temperature to 62-68º C Increase KCl (buffer) concentration to 1.2x-2x, but keep MgCl2 concentration at 1.5-

2mM. Increase MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration constant. Take less primer Take less DNA template Take less Taq polymerase If none of the above works: check the primer for repetitive sequences (BLAST align

the sequence with the databases) and change the primer(s) Combine some/all of the above

20

2. I get (many) shorter unspecific products. What can I do?

Increase annealing temperature Increase annealing time Increase extension time Increase extension temperature to 74-78º C Decrease KCl (buffer) concentration to 0.7-0.8x, but keep MgCl2 concentration at 1.5-

2mM Increase MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration constant Take less primer Take less DNA template Take less Taq polymerase If none of the above works: check the primer for repetitive sequences (BLAST align

the sequence with the databases) and change the primer(s) Combine some/all of the above

3. Reaction was working before, but now I can't get any product.

Make sure all PCR ingredients are taken in the reaction (buffer, template, Taq, etc) Change the dNTP solution (very sensitive to cycles of thawing and freezing,

especially in multiplex PCR) If you just bought new primers, check for their reliability (bad primer synthesis ?) Increase primer amount Increase template amount Decrease annealing temperature by 6-10º C and check if you get any product. If you

don't, check all your PCR ingredients. If you do get products (including unspecific ones) reaction conditions as described above.

Combine some/all of the above

4. My PCR product is weak. Is there a way to increase the yield?

Gradually decrease the annealing temperature to the lowest possible. Increase the amount of PCR primer Increase the amount of DNA template Increase the amount of Taq polymerase Change buffer (KCl) concentration (higher if product is lower than 1000bp or lower if

product is higher than 1000bp) Add adjuvants. Best, use BSA (0.1 to 0.8 µg/µL final concentration). You can also try

5% (v/v, final concentration) DMSO or glycerol. Check primer sequences for mismatches and/or increase the primer length by 5

nucleotides Combine some/all of the above

5. My two primers have very different melting temperatures (Tm) but I cannot change their locus. What can I do to improve PCR amplification?

An easy solution is to increase the length of the primer with low Tm. If you need to keep the size of the product constant, add a few bases at the 3' end. If size is not a concern, add a few bases at either the 3' or the 5' end of that primer.

6. I have a number of primer pairs I would like to use together. Can I run a multiplex PCR with them? How? Very likely, yes.

21

Try amplify all loci seaprately using the same PCR program. If one of the primer pairs yields unspecific products, keep the cycling conditions constant and change other parameters as mentioned above (#1 and #2).

Mix equimolar amounts of primers and run the multiplex reaction either in the same cycling conditions or by decreasing only the annealing temperature by 4º C.

If some of the loci are weak or not amplified, read below !!

7. How many loci can I amplify in multiplex PCR at the same time?

Literature describes up to 25 loci or so. Many commercial kits target 8-10 loci.

8. One or a few loci in my multiplex reaction are very weak or invisible. How can amplify them?

The first choice should be increasing the amount of primer for the "weak" loci at the same time with decreasing the amount of primer for all loci that can be amplified. The balance between these amounts is more important than the absolute values used!

Check primer sequences for primer-primer interactions

9. Short PCR products in my multiplex reaction are weak. How can I improve their yield?

Increase KCl (buffer) concentration to 1.2x-2x, but keep MgCl2 concentration at 1.5-2mM

Decrease denaturing time Decrease annealing time and temperature Decrease extension time and temperature Increase amount of primers for the "weak" loci while decreasing the amount for the

"strong" loci. Add adjuvants. Best, use BSA (0.1 to 0.8 µg/µL final concentration). You can also try

5% (v/v, final concentration) DMSO or glycerol Combine some/all of the above

10. Longer PCR products in my multiplex reaction are weak. How can I improve their yield?

Decrease KCl (buffer) concentration to 0.7-0.8x, but keep MgCl2 concentration at 1.5-2mM

Increase MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration constant. Increase denaturing time Increase annealing time Decrease annealing temperature Increase extension time and temperature Increase amount of primers for the "weak" loci while decreasing the amount for the

"strong" loci Add adjuvants. Best, use BSA (0.1 to 0.8 µg/µL final concentration). You can also try


11. All products in my multiplex reaction are weak. How can I improve the yield?

22

Decrease annealing temperature in small steps (2º C) Decrease extension temperature to 62-68º C Increase extension time Increase template concentration Increase overall primer concentration Adjust Taq polymerase concentration Change KCl (buffer) concentration, but keep MgCl2 concentration at 1.5-2mM Increase MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration constant. Add adjuvants. Best, use BSA (0.1 to 0.8 µg/µL final concentration). You can also try


12. Unspecific products appear in my multiplex reaction. Can I get rid of them somehow?

If long: increase buffer concentration to 1.2-2x, but keep MgCl2 concentration at 1.5-2mM If short: decrease buffer concentration to 0.7-0.9x, but keep MgCl2 concentration at 1.5-2mM Gradually increase the annealing temperature Decrease amount of template Decrease amount of primer Decrease amount of enzyme Increase MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration constant Add adjuvants. Best, use BSA (0.1 to 0.8 µg/µL final concentration). You can also try 5% (v/v, final concentration) DMSO or glycerol If nothing works: run PCR reactions for each (multiplexed) locus individually, using an annealing temperature lower than usual. Compare the unspecific products for each locus tested with the unspecific products seen when running the multiplex PCR. This may indicate which primer pair yields the unspecific products in the multiplex reaction. Combine some/all of the above

(Note: primer-primer interactions in multiplex PCR are usually translated into lack of some amplification products rather than the appearance of unspecific products)

Troubleshooting Shortcut

Weak or no amplification:

o increase # cycles

o increase time at step(s)

o add final extension of 10

min

o increase quantity of

template

o increase Taq concentration

by 2X

o try different Taq (e.g.

Faststart)

o increase MgCl2, primers,

dNTPs

o reamplify PCR product from

previous reaction

Non-targeted bands:

o high weight bands: decrease Taq

concentration

o low weight bands: decrease MgCl2

concentration

o decrease # cycles

o raise annealing

temperature

o decrease primer

concentration

o decrease dNTPs

Possible inhibition

o dilute template 5:1 with 10mM Tris-

HCl (pH 8.0) or ddH2O

o add BSA - final

concentration 1X

Smears above band

o decrease # cycles

Carpet

o decrease concentration of

reagent(s)

23

PCR Notes

24

PCR Notes

25

3. Random amplified polymorphic DNA

(RAPD)

RAPD is an inexpensive yet powerful typing method applicable in many species

Principle

Random amplified polymorphic DNA (RAPD) is a PCR based technique for identifying genetic

variation. It involves the use of a single arbitrary primer in a PCR reaction, resulting in the

amplification of many discrete DNA products. The technique was developed independently by

two different laboratories (Williams et. al., 1990; Welsh and McClelland, 1990) and called as

RAPD and AP-PCR (Arbitrary primed PCR) respectively. This procedure detects nucleotide

sequence polymorphisms in a DNA amplification-based assay using only a single primer of

arbitrary nucleotide sequence. In this reaction, a single species of primer binds to the genomic

DNA at two different sites on opposite strands of the DNA template. If these priming sites are

within an amplifiable distance of each other, a discrete DNA product is produced through

thermocylic amplification. These products are separated on agarose gels and visualized by

ethidium bromide staining. The polymorphisms between individuals result from sequence

differences in one or both of the primer binding sites, and are visible as the presence or absence

of a particular RAPD band. Such polymorphisms thus behave as dominant genetic markers.

RAPD related techniques

AP-PCR and DAF (DNA amplification fingerprinting) are the two other PCR based related

techniques for DNA genotyping using arbitrary primers. Whereas RAPD uses single arbitrary

primers of 9 or 10 nucleotides in length, low stringency annealing conditions, separation on

agarose gels and detection by ethidium bromide staining, DAF (Cateno-Anolles et al.,1991)

requires single/ multiple arbitrary primers greater than 4 nucleotides in length, separation by

polyacrylamide gel electrophoresis and detection by silver staining. AP-PCR uses single arbitrary

primers 20 to 34 nucleotides in length, low stringency followed by high stringency annealing,

separation by polyacrylamide gel electrophoresis and detection by autoradiography. The

resolution obtained is low (upto 10 products), moderate (3-20) and high (upto 100 products) with

RAPD, AP-PCR and DAF respectively.

26

Instruments and Reagents

1. Thermocycler

2. Vortexer

3. Refrigerators (4º C)

4. Deep freezer ( –20ºC)

5. Horizontal gel electrophoresis apparatus with accessories

6. Weighing balance

7. UV trans-illuminator and gel documentation system

8. Microwave oven

9. Micro centrifuge

10. Table-top mini-cooler

11. Auto-pipettes 2-20l, 20-200l, 200-1000l

PCR

1. Genomic DNA

2. Taq DNA polymerase

3. primers

4. dNTP mix

5. MgCl2

6. Buffer for DNA polymerase

7. Sterile milli-Q/distilled water

Agarose gel electrophoresis

1. Electrophoresis grade agarose

2. TBE buffer

3. DNA size marker

4. DNA Loading buffer

5. Ethidium bromide solution


1. 250ml flask

2. Reagent bottles

3. 0.5ml and 1.5ml micro-centrifuge tubes

4. Tips for pipettes (10l, 200l, 1ml)

5. 0.2 ml thin walled PCR tubes

6. Staining trays

27

Procedure

Switch on the thermocycler about 15 min earlier to setting up of the reaction. Pipette out

accurately into sterile 0.5 or 0.2 ml microtubes the reagents in the following order.

Since, the pipetting of small volumes is difficult and often inaccurate; a master mix is prepared

where constituents common to all the reactions are combined in one tube multiplying the

volume for one reaction with the total number of samples. Later, the appropriate amount of the

master mix is aliquoted to each tube and DNA template (or the variable constituent) is added

separately in each tube.

Component Final concentration

Volume per

reaction (in l)

Volume for master mix

(in l, for 10 reactions)

10x PCR buffer (with 15mM MgCl2)

1x 2.5 25

25 mM MgCl2 2 mM 0.5 5

dNTP mix (10 mM each) 200 M 2.5 25

Primer F (OPE 03) 0.5 M 2.5 25

Primer R --- 0 ---

Taq DNA polymerase

(5u/l)

1 unit 0.2 2

Water --- 14.3 143

Template DNA 25 ng 2.5 ---

Total Volume --- 25 225

Mix by repeated pipetting. Spin down the contents (2-5 sec) and place the tubes firmly in the

wells. Start thermocycler programmed as follows:

The temperature cycling conditions are:

Step Time (minutes) Temperature (° C)

Initial Denaturation 4 94

3-step cycling

Denaturation 1 94

Annealing 1 35

Extension 2 72

Number of cycles 40

Final extension 5 72

At the end of the run take out the tubes and add 2.5 μl of 10x loading dye. Spin for 2-5 sec.

Store at 4oC till electrophoresis.

28

Agarose Gel Electrophoresis:

The amplification products in RAPD analysis are usually smaller than 4 kb size. Hence, they

are separated by electrophoresis in 1.4 to 1.8 % agarose gels and visualized by staining with

ethidium bromide and viewing under UV light.

1. Prepare the gel tray by taping the open ends. Place the comb and level the tray.

2. Boil and prepare 1.4% agarose gel in 1xTAE buffer. Cool to 60oC. Pour into gel tray

avoiding air bubbles. Allow to set for 30-40 min.

3. Remove the adhesive tapes. Place in electrophoresis tank filled with 1xTAE buffer.

Remove the comb carefully. Pour buffer till the gel is fully immersed.

4. Load the samples carefully. Take care to load suitable DNA size markers (about 200

ng). Connect the leads and start electrophoresis run at constant 60 V. The voltage

varies with the gel strength and length.

5. Stop the run when bromophenol blue dye has traveled less than 2/3 the length of gel.

6. Stain in 0.5 to 1 μg/ ml ethidium bromide in distilled water for 30 - 40 min. Wash briefly

in distilled water.

7. View under UV light. Photograph the gel.

8. The amplification products are scored across the lanes comparing their respective

molecular weights. The data can be analyzed statistically using appropriate software

packages.

References

1. JGK Williams et al. (1990) DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res 18: 6531-6535.

2. J Welsh and M McClelland (1990) Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res 18: 7213-7218.

3. Cateno-Anolles et al. (1991) Bio/Technology 9: 553-557

29

RAPD Notes

30

RAPD Notes

31

4. Inter Simple Sequence Repeats (ISSR)

ISSR polymorphisms are not affected by repeat polymorphisms within the SSR

Principle

Discovery of hypervariable tandem repeats having a longer repeat unit in human genome and

demonstration of their applications heralded the birth of DNA fingerprinting. (Jeffreys et al, 1985).

The term minisatellites was invented to describe such sequences that were used in fingerprinting

studies leading to widespread application in individual identification, parentage testing and

genome mapping. Soon after, minisatellites were reported in plants in 1988 and by 1993, plant

minisatellites were demonstrated to be useful markers for variation between and within species.

Meanwhile, ubiquitous existence and variability of much simpler tandem repeats were discovered

(Tautz and Renz, 1984). These were simple sequences also known as microsatellites.

Microsatellites are a type of simple repeated sequences comprising tandem copies of, usually, di-

, tri- or tetra-nucleotide repeat units, for example (TA)6 and CGGCGACGA(CGG)8TGGCGG in

rice.

Microsatellites have repeat unit length of usually 1-10 bp and length of locus not greater than 150

bp. Among repeated DNA sequences, microsatellite copies in a genome are the highest and

distributed throughout the genome. Both mini and microsatellites were routinely used in

hybridisation-based protocols to generate classical DNA fingerprints. In 1989, synthetic

32

oligonucleotide probes that recognise simple repetitive DNA sequences were introduced to plant

DNA fingerprinting. These multilocus probes reveal hypervariable target regions. Since mini- and

microsatellites are characterised by highly variable copy numbers of identical or closely related

basic motifs, this class of DNA polymorphism was designated variable number of tandem repeats

(VNTR). Possible mechanisms of molecular basis of VNTR variability include replication slippage,

transposition, recombinational events and gene conversion. In vitro experiments have shown that

replication slippage may actually result in considerable amplification of a given repeat. The

oligonucleotide probes bind to hypervariable target regions, revealing polymorphism in the

microsatellite repeats. If the same oligonucleotides are used as primers in a PCR reaction, the

resulting amplification pattern is expected to exhibit polymorphism in the inter-microsatellite

region. Such differences are designated as Inter Simple Sequence Repeat Polymorphism (ISSR).

However, first studies employing ISSR markers were published only in 1994 (Zietkiewicz et al,

1994; Gupta et al, 1994). The initial studies focussed on cultivated species, and demonstrated the

hypervariable nature of ISSR markers. Since then several studies have demonstrated the

potential and utility of the technique in a wide range of applications and plant families. ISSR

polymorphism has been employed for deciphering genetic relationships and diversity facilitating

germplasm management; for generating DNA fingerprints and genetic linkage maps; for gene

tagging; for detecting somaclonal variation, somatic hybrids and microspore-derived plants; and

for the isolation of STMS markers that are considered as better DNA markers.

ISSR technique is a PCR-based multi-locus marker system that employs oligonucleotide primers

homologous to SSR sequences such as (GATA)n to amplify mainly the inter-SSR regions.

Amplification products are obtained only if SSRs in opposite orientation are found within a PCR-

able distance, with flanking sequences matching the oligonucleotides. ISSR markers are

multilocus, unmapped dominant markers. Various workers have developed techniques to use

microsatellite sequences as PCR primers with minor variations. This has resulted in many

synonyms of ISSR technique viz. SSR–anchored PCR; anchored SSR-PCR (ASSR-PCR);

microsatellite primed PCR (MP-PCR); anchored microsatellite primed PCR (AMP-PCR); single

primer amplification reaction (SPAR); randomly amplified microsatellites (RAM) and randomly

amplified multiple polymorphism (RAMP). Distinction is required to be understood between ISSR

and STMS. STMS (Sequence Tagged Microsatellite Site) is also a PCR-based marker, where

unique DNA sequences flanking microsatellites (GACA)n, (CT)n etc., are used to construct

oligonucleotide primers. Resulting amplification products spanning the microsatellite region show

rich allele divergence. However, ISSR polymorphisms are not influenced by repeat

polymorphisms within the SSR.

ISSR technique is nearly identical to RAPD technique except that ISSR primer sequences are

designed from microsatellite regions. Higher stringency of amplification in the form of longer

primers (16-25 bp) and elevated annealing temperatures (45-60º C) makes ISSR markers more

reproducible than RAPD markers. While the molecular basis of underlying polymorphism has not

been addressed yet, the use of these oligonucleotides as PCR primers instead of hybridisation

probes offers one important advantage. It combines the high degree of DNA polymorphism

detected by conventional multilocus probes in DNA fingerprinting experiments with the technical

simplicity and speed of the PCR method, facilitating large-scale experiments. Polymorphism

levels and extent of utility in ISSR experiments depend upon nature of primers used (unanchored,

3’-anchored or 5’-anchored), motif of the repeats targeted (di-, tri-, tetra-, penta-nucleotides or

higher), sequence of the primer (AT rich, GC rich or balanced), length of the primer, extent of

optimisation in the PCR (both reaction composition and reaction condition- mainly annealing

33

temperature), method of detection (agarose gel electrophoresis-ethidium bromide or PAGE-silver

staining/radioactive detection). Unanchored primers show a tendency to slip within the repeat

units during PCR, which may result in background smear. To prevent internal priming, slippage

and to avoid stutterings, ISSR primers can be anchored to unique genomic sequences flanking

the repeat by extending them at either of ends using degenerate nucleotides up to 5 bases. 3'-

anchoring is shown to give clearer banding pattern and 5'-anchoring is reported to produce more

bands and higher polymorphism. However, such anchoring allows only a subset of microsatellites

to function as priming sites.

Generation, polymorphism and usefulness of the bands ultimately depend upon the frequency

and the organisation (dispersed or clustered) of microsatellites in a species/genotype. ISSR

technique has some drawbacks also. Co-migrating bands may not be homologous, optimisation

may take little longer and neither exact molecular nature of bands is known nor the molecular

basis of the polymorphism is explained. In spite of this, ISSR technique has developed into a

successful molecular profiling technique for a variety of purposes in various taxa investigated.


1. Thermocycler

2. Vortexer




6. Weighing balance


8. Microwave oven

9. Micro centrifuge



PCR

1. Genomic DNA


3. primers

4. dNTP mix

5. MgCl2





2. TBE buffer

3. DNA size marker




34

1. 250ml flask

2. Reagent bottles




6. Staining trays

Procedure

A. Setting up PCR programme

PCR reaction conditions for routine ISSR is given below



3-step cycling

Denaturation 1 94

Annealing 1 55

Extension 3 72

Number of cycles 35


Annealing temperature (T) needs to be empirically optimised for number and clarity of bands with

melting temperature (Tm) of the oligonucleotide as the reference point. To calculate Tm following

thumb rule may be applied:

Tm = 81.5 + 16.6 (log [Na+]) + 0.41 (%G+C) – (675/n)

Where, n=number of bases in oligo and Na+ = molar salt concentration

A simpler and easy way of calculation is by the formula,

Tm (º C) = [2*(A+T) + 4*(G+C)]

A list of selected ISSR primers with their Tm and annealing temperatures is given below;

construct the programme accordingly in the thermocycler and deposit in the memory.

Primer sequence (C) Tm (C) Annealing Temperature

(ACTG)4 48 45

(GATA)4 40 43

(GAAGTGGG)2 52 50

(AT)5 (GT)5 50 52

(GTG)6 60 57

(TCC)5 50 52

B. Setting up PCR reaction

1. Thaw PCR reagents- MgCl2, primers and Buffer for Taq DNA polymerase

2. Vortex vigorously MgCl2 to remove gradients

35

3. Prepare PCR master-mix by adding components as given below

Component Final concentration Volume per reaction

(in l)



10x PCR buffer 1x 2.0 20

25 mM MgCl2 2 mM 1.6 16

dNTP mix (10 mM each)

200 M 2.0 20

Primer F (ISSR03) 0.5 M 1.0 10

Primer R --- --- ---

Taq DNA polymerase

(1u/l)

1 unit 1.0 10

Water --- 9.9 99



4. Dispense aliquots of master-mix into PCR tubes

5. Add template DNA and mix gently by pipetting 2-3 times

6. Place the reaction tubes on the thermocycler plate which is switched on earlier

7. Set off the designated program following the instruction of the machine’s menu

8. After the completion of the PCR, add loading dye and proceed with electrophoresis (The

tubes can be stored at 4º C if electrophoresis not planned immediately)

C. Electrophoresis

1. Prepare Agarose gel of final strength 1.5% in 0.5X TBE buffer; allow it to cool to room

temperature

2. Set the gel tray with combs appropriate for loading 25l volume and 12 samples and level it

3. Pour the lukewarm gel into the tray gently, avoiding bubbles; gel sets within 30 minutes

4. Transfer the gel to the buffer tank filled with fresh TBE buffer (0.5X)

5. Flush the wells with buffer; load the samples, along with DNA size marker in the extreme

wells.

6. Electrophorese the samples at 4 volts/cm of electrode distance

7. Prepare ethidium bromide staining solution and stain the gel for few minutes; destain till the

background fluorescence is removed (Alternatively, ethidium bromide can be added in the

gel before pouring to a final concentration of 0.5g/ml; samples loaded after brief pre-run)

8. View the DNA profile developed under ultraviolet light

9. Document the results

36

Trouble shooting

Problem Possible cause Remedy

No amplification Missing reaction components Repeat the amplification

Bad quality components Change the components, maintain them in aliquots

Low DNA quality Try with other primers, if problem persists purify DNA or isolate fresh DNA

Improper annealing temperature Further optimisation of annealing temperature required at lower values

All ISSR primers may not yield amplification products in all species

Change the primer

Inconsistent amplification


Improper dissolution of DNA or impurities in DNA or improper quantification

Dissolve completely before quantification; repeat clean up procedure; quantify DNA again

Improper reaction setting Practice for perfection; avoid setting more PCR reactions at a stretch (beyond 96)

Fuzzy/ indistinct bands

Poor amplification Repeat the amplification


Over used electrophoresis buffer Change to fresh buffer

Improper setting of comb Take care while repeating

Improper annealing temperature Further optimisation of annealing temperature required at higher values

High background Faulty staining procedure or bad water quality or over staining in case of PAGE

Use milli-Q water, good quality chemicals and optimise staining time

Foggy photographs High background (agarose gel) De-stain thoroughly before photography

Wrong filter used Use proper filters

Counting of bands difficult due to clutter

Inadequate separation of bands Use longer gels or higher strength gels

Reference

1. Jeffreys AJ, Wilson V and Thein SL. 1985. Hypervariable minisatellite regions in human

DNA. Nature 314: 67-73

2. Tautz D and Renz M, 1984. Simple sequences are ubiquitous repetitive components of

eukaryote genomes. Nucleic Acids Research 12: 4127-4138

3. Zietkiewicz E, Rafalski A and Labuda D (1994) Genome fingerprinting by simple

sequence repeat (SSR)-anchored polymerase chain reaction amplification. Genomics 20,

176-183

4. Gupta M, Chyi Y-S, Romero-Severson J and Owen JL (1994) Amplification of DNA

markers from evolutionarily diverse genomes using single primers of simple-sequence

repeats. Theor Appl Genet 89, 998-1006.

37

ISSR Notes

38

ISSR Notes

39

5. Sequence Tagged Microsatellite DNA

(STMS)

STMS markers are the most robust and informative of all the available DNA markers

Principle

Eukaryotic genome contains repeat regions of different lengths and motifs. The repeat

sequences that have less than six base long central motifs are called microsatellites or Simple

Sequence Repeats (SSRs). These simple repetitive DNA sequences of SSRs which are spread

throughout the genome of eukaryotes provide the basis of PCR based multiallelic, co-dominant

genetic marker system called Sequence Tagged Microsatellite Sites (STMS). The regions

flanking the microsatellites are generally conserved among the genotypes of the same species.

Polymerase Chain Reaction (PCR) primers to the flanking regions are used to amplify the SSR

containing DNA fragments. The length polymorphism is created when PCR products from the

different individuals vary in length as a result of variation in the number of repeat units in the

SSR.

Detection of microsatellites involves electrophoresis of the amplified fragments through high

resolution gels capable of separating fragments that differ by as much as 2 base pairs.

Polyacrylamide gels are most effective for separating small fragments of DNA. These have a

high resolving power and fragments of DNA that differ in size by as little as 1 bp can be

separated from each other. Although they can be run very rapidly and can accommodate

comparatively large quantities of DNA, polyacrylamide gels have the disadvantage of being more

difficult to prepare and handle than the routine agarose gels as they have to be run in a vertical

configuration. Metaphor Agarose is a high resolution agarose that challenges polyacrylamide in

terms of resolution of small sized fragments. It is an intermediate melting temperature (75°C)

agarose with twice the resolution capabilities of the finest-sieving agarose products. Hence it has

an advantage over polyacrylamide gels as by using the routine submarine gel electrophoresis,

fragments that differ in size by 2% can be separated.

40


1. Thermocycler

2. Vortexer




6. Weighing balance


8. Microwave oven

9. Micro centrifuge



PCR

1. Genomic DNA


3. primers

4. dNTP mix

5. MgCl2





2. TBE buffer

3. DNA size marker




1. 250ml flask

2. Reagent bottles




6. Staining trays

41

Procedure


Annealing temperature (T) needs to be empirically optimized for number and clarity of bands with


thumb rule may be applied: Tm = 81.5 + 16.6 (log [Na+]) + 0.41 (%G+C) – (675/n)


A simpler and easy way of calculation is by the formula, Tm (º C) = [2*(A+T) + 4*(G+C)]

PCR reaction conditions is given below



3-step cycling

Denaturation 1 94

Annealing 1 50

Extension 2 72

Number of cycles 35





6. Prepare PCR master-mix by adding components as given below:

PCR reaction composition (25l):


Volume per

reaction (in l)




25 mM MgCl2 2 mM 2.0 20

dNTP mix (2.5 mM each) 200 M 2.0 20

Primer F (122) 0.05 M 0.75 7.5

Primer R (123) 0.05 M 0.75 7.5

Taq DNA polymerase 1 unit 0.2 2

Water --- 13.8 138







42



C. Electrophoresis:

1. Choose a beaker that is 2-4 times the volume of the solution.

2. Add chilled 0.5X electrophoresis buffer and a stir bar to the beaker.

3. Slowly sprinkle in the agarose powder while the solution is rapidly stirred.

4. Remove the stir bar if not Teflon® coated.

5. Soak the agarose in the buffer for 15 minutes before heating. This reduces the

tendency of the agarose solution to foam during heating.

6. Weigh the beaker and solution before heating.

7. Cover the beaker with plastic wrap.

8. Pierce a small hole in the plastic wrap for ventilation.

9. For agarose concentrations >4%, the following additional steps will further help

prevent the agarose solution from foaming during melting/dissolution:

a. Heat the beaker in the microwave oven on Medium power for 1 minute.

b. Remove the solution from the microwave.

c. Allow the solution to sit on the bench for 15 minutes.

i. Heat the beaker in the microwave oven on Medium power for 2

minutes.

ii. Remove the beaker from the microwave oven.

10. Caution: Any microwaved solution may become superheated and foam over

when agitated. Gently swirl the beaker to resuspend settled powder and gel pieces.

11. Reheat the beaker on HIGH power until the solution comes to a boil.

12. Hold at boiling point for 1 minute or until all of the particles are dissolved.

13. Remove the beaker from the microwave oven.

14. GENTLY swirl the beaker to thoroughly mix the agarose solution.

15. After dissolution, and sufficient hot distilled water to obtain the initial weight.

16. Mix thoroughly.

17. Cool the solution to 50°C-60°C prior to casting. Adust the comb on the gel casting

tray. Pour the gel solution gently without introducing bubbles. Once the gel is cast,

allow the molten agarose to cool and gel at room temperature. The gel must then

be placed at 4°C for 20 minutes to obtain optimal resolution and gel handling

characteristics.

18. Transfer the gel to the buffer tank filled with fresh TBE buffer (0.5X). Flush the wells

with buffer; load the samples, along with DNA size marker in the extreme wells.

19. Electrophorese the samples at 4 volts/cm of electrode distance.

20. Prepare ethidium bromide or gel star staining solution and stain the gel for few

minutes; destain till the background fluorescence is removed (Alternatively, ethidium

bromide can be added in the gel before pouring to a final concentration of 0.5g/ml;

samples loaded after brief pre-run).

21. View the DNA profile developed under ultraviolet light and document the results.

43

STMS Notes

44

STMS Notes

45

6. Sequence Characterized Amplified

Regions (SCARs)

SCARs represent simple yet effective way to generate specific markers

Principle

In 1993, a new type of RAPD-derived DNA marker was introduced (Paran and Michelmore 1993), which got around several drawbacks inherent to RAPD technique. SCAR markers were generated by cloning and sequencing (at least ends) selected RAPD fragments, and designing longer (24-mer) primer pairs that are complementary to the original RAPD amplicons. Amplification with SCAR primers results in characterized loci and not random ones. These SCARs may retain the dominant behavior of the original RAPD fragment or may exhibit codominant segregation. Generation of SCARs from anonymous fragments is not restricted to RAPD but any arbitrary amplicons including AFLPs.

Essential steps in SCAR marker Development

1. Run RAPD reaction and electrophorese the amplicons. Identify the band of interest

(a private band or a polymorphic band)

2. Excising band from the agarose gel

Place the agarose gel on UV transilluminator and cut out band using a sterile

scalpel. Make sure that not to touch any of the other bands surrounding the band

of interest. Trim the band on the UV box with the scalpel if necessary.

Employ any of the commercially available kit (for instance QIAquick™ Gel

Extraction Kit from QIAGEN) to extract the DNA from the gel piece. Essentially,

the procedure involves melting the gel piece in a buffer, exclusion of the DNA,

and purification with the help of anion exchange columns.

46

3. Transformation/Cloning reaction:

Using the gel extracted DNA, run PCR with the original primer

Electrophorese an aliquot on an agarose gel to confirm amplicons size

If the band on the gel is clean and clear of the expected size, use the rest of the

PCR product carry out transformation.

Set-up the TA cloning reaction using any of the commercial kits (for instance

TOPO TA Cloning® Kit from Invitrogen) following manufacturers manual

4. Transformation

Prepare E. coli competent cells or obtain commercially available chemically

competent cells and thaw on ice.

Add 2 µl of the cloning reaction to the cells and carry out heat shock procedure

Meanwhile prepare the culture plates by warming LB plates in 37 °C for 30 min;

place 40 µl of X-gal and 40 µl of IPTG in the center of a plate; and spread evenly

until completely dry. Keep plates covered while working in the hood to avoid

photodegredation of X-gal.

Place the transformed bacterial cells in the center of the plate and spread evenly

until completely dry and incubate overnight at 37 °C.

Select white colonies, incubate in the culture medium overnight and extract

plasmid DNA. Sequence the insert

Design primers using any of the primer design software (for instance Primer 3;

http://biotools.umassmed.edu/bioapps/primer3_www.cgi)


1. Thermocycler

2. Vortexer




6. Weighing balance


8. Microwave oven

9. Micro centrifuge



PCR

1. Genomic DNA


3. primers

4. dNTP mix

5. MgCl2

47





2. TBE buffer

3. DNA size marker




1. 250ml flask

2. Reagent bottles




6. Staining trays

Procedure


PCR reaction conditions are given below



3-step cycling

Denaturation 1 94

Annealing 1 55

Extension 1 72

Number of cycles 35


Annealing temperature (T) needs to be empirically optimised for number and clarity of bands with


thumb rule may be applied: Tm = 81.5 + 16.6 (log [Na+]) + 0.41 (%G+C) – (675/n)


A simpler and easy way of calculation is by the formula, Tm (º C) = [2*(A+T) + 4*(G+C)]




3. Prepare PCR master-mix by adding components as given below:

PCR reaction composition (25l)

48


Volume per

reaction (in l)




25 mM MgCl2 2 mM 2.0 20

dNTP mix (2.5 mM each) 200 M 2.0 20

*Primer F (ROC11 F) 0.05 M 0.75 7.5

*Primer R (ROC11 R) 0.05 M 0.75 7.5

Taq DNA polymerase 1 unit 0.2 2

Water --- 13.8 138



* for bean common mosaic virus resistance gene







C. Electrophoresis:

1. Choose a beaker that is 2-4 times the volume of the solution.

2. Add chilled 0.5X electrophoresis buffer and a stir bar to the beaker.

3. Slowly sprinkle in the agarose powder while the solution is rapidly stirred.

4. Remove the stir bar if not Teflon® coated.

5. Soak the agarose in the buffer for 15 minutes before heating. This reduces the

tendency of the agarose solution to foam during heating.

6. Weigh the beaker and solution before heating.

7. Cover the beaker with plastic wrap.

8. Pierce a small hole in the plastic wrap for ventilation.

9. For agarose concentrations >4%, the following additional steps will further help

prevent the agarose solution from foaming during melting/dissolution:

a. Heat the beaker in the microwave oven on Medium power for 1 minute.

b. Remove the solution from the microwave.

c. Allow the solution to sit on the bench for 15 minutes.

i. Heat the beaker in the microwave oven on Medium power for 2

minutes.

ii. Remove the beaker from the microwave oven.

10. Caution: Any microwaved solution may become superheated and foam over

when agitated. Gently swirl the beaker to resuspend any settled powder and gel

pieces.

11. Reheat the beaker on HIGH power until the solution comes to a boil.

12. Hold at boiling point for 1 minute or until all of the particles are dissolved.

13. Remove the beaker from the microwave oven.

14. GENTLY swirl the beaker to thoroughly mix the agarose solution.

15. After dissolution, and sufficient hot distilled water to obtain the initial weight.

16. Mix thoroughly.

49

17. Cool the solution to 50°C-60°C prior to casting. Adust the comb on the gel casting

tray. Pour the gel solution gently without introducing bubbles. Once the gel is cast,

allow the molten agarose to cool and gel at room temperature. The gel must then

be placed at 4°C for 20 minutes to obtain optimal resolution and gel handling

characteristics.

18. Transfer the gel to the buffer tank filled with fresh TBE buffer (0.5X). Flush the wells

with buffer; load the samples, along with DNA size marker in the extreme wells.

19. Electrophorese the samples at 4 volts/cm of electrode distance.

20. Prepare ethidium bromide or gel star staining solution and stain the gel for few

minutes; destain till the background fluorescence is removed (Alternatively, ethidium

bromide can be added in the gel before pouring to a final concentration of 0.5g/ml;

samples loaded after brief pre-run).

21. View the DNA profile developed under ultraviolet light and document the results.

Reference

1. Paran I and Michelmore RW (1993). Development of reliable PCR-based markers linked

to downy mildew resistance genes in lettuce. Theor. Appl. Genet. 85:985–993.

50

SCAR Notes

51

SCAR Notes

52

Appendix

DNA extraction protocols (excerpts from www.qiagen.com )

Disruption using mortar and pestle

The most common disruption method involves freezing samples in liquid nitrogen and grinding with a

mortar and pestle:

1. Freeze tissue in liquid nitrogen immediately after harvesting .Do not let the sample to thaw at

any time during disruption.

2. Pre-cool mortar to –20°C and keep on dry ice.

3. Pour liquid nitrogen into the mortar, and pre-cool pestle by placing the grinding end in the

liquid nitrogen.

4. Place frozen tissue in mortar and grind until a fine, whitish powder results.

5. Add liquid nitrogen as necessary, being careful the sample does not spill out of the mortar.

6. Using a pre-cooled spatula, transfer the powder to pre-cooled containers of the appropriate

size. To avoid thawing, large samples may be transferred to several containers.

7. Ensure all liquid nitrogen has evaporated before closing the container. To prevent the sample

from thawing after evaporation, the container should be cooled by placing it in dry ice or

liquid nitrogen.

Nucleic acid content of plant tissues

The nucleic acid content can vary widely between different plant starting materials. For example, a

tissue sample comprised of small cells will have a higher cell density, and therefore is likely to contain

more nucleic acids than a sample of the same size which is comprised of larger cells. In addition, DNA

contents depend on the haploid genome size and the ploidy of the sample. Arabidopsis has a small

diploid genome and correspondingly lower DNA yields than wheat which has a large hexaploid

genome. RNA content varies less predictably than DNA content. Highly proliferating tissues, such as

meristems, typically contain more RNA than mature tissues. This variation in nucleic acid content should

be considered when purifying nucleic acids.

DNA isolation methods

Plant molecular biology studies often require a simple, rapid, and reproducible method for preparing

DNA from a wide variety of species. A number of factors that can affect the yield and purity of DNA

must be considered, including incomplete cell lysis and carryover contamination of carbohydrates,

polyphenolics, flavones, and other metabolites. DNA isolation methods must be scaleable for different

sample sizes and provide sufficient throughput to meet demanding project timelines.

DNeasy® Plant kits

53

With the DNeasy Plant procedure, plant cells or tissues are first mechanically disrupted and then lysed

by the addition of lysis buffer and incubation at 65°C. During this step, RNase contained in the lysis

buffer digests RNA in the lysate. After lysis, proteins and polysaccharides are removed by salt

precipitation. Precipitates and cellular debris are removed in a single step by a brief spin through a

QIAshredder unit. The cleared lysate is transferred to a new tube and a binding buffer containing

ethanol is added to promote binding of the DNA to the DNeasy membrane. The sample is then applied

to a DNeasy spin column or a 96-well plate and spun briefly in a centrifuge. Contaminants, such as

proteins and polysaccharides, are efficiently removed by two stringent wash steps. The highly specific

binding properties of the DNeasy membrane allows efficient purification and eliminates the need for

additional extraction or precipitation steps which are often required for traditional isolation methods.

Pure DNA is eluted in a small volume of low-salt buffer or water. DNeasy purified DNA typically has

A260/A280 ratios of 1.7–1.9, and absorbance scans show a symmetric peak at 260 nm, confirming

high purity. The DNA-binding DNeasy membrane is available in either spincolumn or 96-well formats.

The combination of easy, high-throughput disruption using the Mixer Mill MM 300 and reliable

purification using the DNeasy 96 Plant Kit provides convenient DNA isolation from plant tissues in 96-

well format. DNeasy Plant Kits have been used to isolate high-quality DNA from a wide range of plant

species and tissues, including troublesome sources rich in polysaccharides and polyphenolics.

1. Plant Nucleic Acid Purification Technical hints and Applications (2000). QIAGEN, 10/2000.

Available at www.qiagen.com

CTAB lysis

This “home-made” DNA isolation method uses the detergent cetyltrimethylammonium bromide (CTAB) to

lyse plant cells. After lysis, contaminants are removed by a chloroform extraction step. During

extraction, it is essential that the correct salt concentration is used to ensure that contaminants are

separated into the organic phase and DNA stays in the aqueous phase. DNA is recovered from the

aqueous phase with a subsequent precipitation either by adding alcohol or lowering the salt

concentration so that DNA forms insoluble complexes with the CTAB. DNA preparations isolated using

this method may contain enzyme-inhibiting contaminants and therefore may not be sufficiently pure for

sensitive downstream applications such as PCR.

1. Richards, E., Reichardt, M., and Rogers, S. (1994) Preparation of genomic DNA from plant tissue.

In: Ausubel, F.M. et al., eds. Current Protocols in Molecular Biology. New York: John Wiley &

Sons, Inc., p 2.3.1.

2. Doyle, J.J. and Doyle, J.L. (1987) A rapid isolation procedure for small quantities of fresh leaf

tissue. Phytochemical Bulletin 19, 11.

3. Murray, M.G., and Thompson, W.F. (1980) Rapid isolation of high-molecular-weight plant DNA.

Nucleic Acids Res. 8, 4321.

Dellaporta (salting-out) method

This method involves grinding plant tissue in an SDS-containing lysis buffer, filtering the lysate, and

precipitating proteins and other compounds contained in the lysate with high salt concentrations.

Removal of proteins and other contaminants using this salting-out method may be inefficient. RNase

treatment and repeated alcohol precipitation are typically necessary before the DNA can be used in

http://www.qiagen.com/

54

downstream applications. Difficult samples may require manual removal of precipitated DNA from the

alcohol suspension to reduce co-precipitation of contaminants by centrifugation. However, this step may

not sufficiently remove contaminants, and DNA preparations may contain enzyme-inhibiting compounds.

Yields and purity using this method are often variable.

1. Dellaporta, S.L., Wood, J., and Hicks, J.B. (1993) A plant DNA minipreparation: version II. Plant

Mol. Biol. Rep. 1, 19.

ROSE method

The ROSE method involves disruption and lysis of plant cells followed by incubation at high

temperatures (90°C for 20 minutes). The lysate is then used directly in downstream applications.

Considered a “quick-and-dirty” technique, this method may not be suitable for extremely sensitive

applications because isolated DNA often contains enzyme-inhibiting contaminants (see Table 5, next

page). Furthermore, high levels of contamination often result in DNA degradation during storage.

Therefore, the ROSE method is appropriate for a limited range of applications.

1. Steiner, J.J., Poklemba, C.J., Fjellstrom, R.G., and Elliot, L.F. (1995) A rapid one-tube genomic DNA

extraction process for PCR and RAPD analyses. Nucleic Acid Res. 23, 2569.

CsCl density gradient

Plant DNA can be isolated by centrifugation through a cesium chloride (CsCl) density gradient. After

plant cells are lysed with detergent and treated with protease, the cleared lysate is precipitated with

isopropanol. The resuspended DNA is then mixed with CsCl and ethidium bromide and centrifuged for

several hours. Although this method allows the isolation of high-quality DNA, it is time consuming, labor

intensive, and expensive, making it inappropriate for routine use.

1. Richards, E., Reichardt, M., and Rogers, S. (1994) Preparation of genomic DNA from plant tissue.

In: Ausubel, F.M. et al., eds. Current Protocols in Molecular Biology. New York: John Wiley &

Sons, Inc., p 2.3.1.

55

Comparison of plant DNA purification methods

Criterion DNeasy Plant kit

CTAB Dellaporta ROSE CsCl gradient

Sample source Plant cells and tissues

Plant cells and tissues

Fresh plant tissue

Plant tissues and cells

Plant tissues*

Can be used with a broad range of plant species?

Yes No† No† No† Yes

DNA quality High Medium Low Very low High

Alcohol precipitation required?

No Yes Yes No Yes

Preparation time for 24 samples

<1 h‡ 2–4 h (plus overnight re-suspension)

2–4 h (plus overnight re-suspension)

1 h 12 h (for 8 samples)§

Reproducibility High Variable Variable Poor High**

Method convenient? Yes Moderately No Yes No

DNA storage Long-term Long-term Short-term No Long-term

Performance in downstream applications

Excellent Moderate Poor Very poor Excellent

* It is recommended that plants are grown in the dark for 2 days before isolating DNA. **Protocol may need to be optimized for some species. †Using the DNeasy Plant Mini Kit. ‡Does not include the recommended 2 day dark treatment. §Depends on handling.

56

NBPGR

Nodal organization in India to

manage plant genetic

resources

Compiled and Edited by Sunil Archak Ambika B Gaikwad KV Bhat

Documents

Transcript of Compiled and Edited by Sunil Archak Ambika B Gaikwad KV Bhat