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Most molecular markers fall into one of three basic categories of techniques that use either hybridization or are based on the polymerase chain reaction (PCR):
Category # 1: Hybridization Based (non-PCR) Techniques:
Category I includes restriction fragment length polymorphism (RFLP) analysis, where probes are hybridized to filters containing DNA which has been digested with restriction enzymes.
The resultant fragments are separated by gel electrophoresis and transferred onto filters by Southern blotting. Hybridization can also be carried out with probes for mini-satellite or microsatellite sequences to give variable number of tandem repeats (VNTR) and oligonucleotide fingerprinting.
Category # 2: Arbitrarily-primed PCR and Other PCR-based Multi-Locus Profiling Techniques:
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In Category 2 we can group together all PCR-based techniques which use ‘arbitrary’ or semi-arbitrary primers for amplification of DNA products, including those derived from RNA. A common feature of these techniques is the lack of requirement for sequence information from the genome under investigation.
The range of different approaches in this category differs in the length and sequence of the primers used, the stringency of the PCR conditions and the method of fragment separation and detection.
In one group within this category, single arbitrarily chosen primers are used in PCR conditions, in which, the primer will initiate synthesis, even when the match with the template is imperfect. DNA techniques of this kind have been collectively termed multiple arbitrary amplicon profiling (MAAP) and arbitrarily amplified DNA (AAD).
In these cases, each amplified product will be derived from a region of the genome that contains two short segments, which share sequence similarity to the single primer and which are on opposite strands and sufficiently close together for the amplification to work.
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They include random amplified polymorphic DNA (RAPD) analysis in which the amplification products are separated on agarose gels in the presence of ethidium bromide and visualised under ultraviolet light, arbitrarily primed PCR (AP-PCR) and DNA amplification fingerprinting (DAF), in which, the products are separated on polyacrylamide gels.
In a second subgroup, the primers used are semi-arbitrary in that they are based upon restriction enzyme sites or sequences that are interspersed in the genome, such as repetitive elements, transposable elements and microsatellites.
The use of primers based on restriction sites is the basis of the techniques of selective restriction fragment amplification (SRFA) or amplified fragment length polymorphism (AFLP), as it is now most commonly known.
In AFLP, DNA is restricted with two restriction enzymes, adaptors are ligated, and then PCR is carried out with generic primers which comprise a common part corresponding to the adaptors and restriction site and a unique part corresponding to selective bases.
Because of the high mutability of simple sequence repeats (SSR), there are many versions, in which, ‘microsatellites’ are used as primers in microsatellite (repeat)-primed PCR (MP-PCR). Unanchored single SSR primer amplification reaction (SPAR) is very similar to RAPD but the primers are SSR-based —however, polymorphisms are apparently not SSR-based.
In inter-SSR amplification (ISA or inter-ISSR PCR) two SSR primers anchored at the 5′ or 3′ end are used —this technique is more reproducible than SPAR or RAPD, but variation is between SSR rather than at the SSR.
Randomly amplified microsatellite polymorphism (RAMP) is based on the random distribution of nucleotide sequences immediately flanking an SSR and is performed between a 5′ anchored mono-, di-, or a tri-repeat and a arbitrary decamer primer —this approach does reflect variation in SSR.
Selective amplification of microsatellite polymorphic loci (SAMPL) is an SSR-based modification of AFLP in which amplification is performed with one labeled SSR primer (anchored through the use of compound repeats) and one unlabeled adopter primer.
Category # 3: Sequence Targeted and Single Locus PCR:
“DNA markers are a short-cut to sequence information” and increased efficiency in screening is exchanged for incomplete sequence information. A limitation of artbitrarily amplified DNA is the lack of allelic information, both in terms of dominance and in terms of the assignment of alleles to loci.
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These problems are overcome with PCR directed to specific single locus targets, for which, a necessary prerequisite is knowledge of the sequence of the target or flanking target regions. In plants and animals there are three sources of potential sequences for a PCR-targeted approach: the chloroplast (cpDNA), mitochondrial (mtDNA) and nuclear (nDNA) genomes.
Nucleotide sequences contain both phylogenetic and, frequency information and are thus, extremely important markers for ecological and evolutionary studies. Sequencing is, however, labour and resource intensive and often insufficient polymorphisms are detected at the cultivar, breed or race level.
DNA sequencing to identify genetic variation for making markers is rather inefficient- only 0.5 to 2 nucleotide differences are expected per kb depending upon species and gene (genome). RAPD, RFLP, SDS – PAGE are described in detail.
Random Amplified Polymorphic DNA (RAPD):
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RAPD (random amplified polymorphic DNA) analysis is a PCR (polymerase chain reaction) based molecular marker technique by amplification of genomic DNA using at least one short oligonucleotide primer in low stringency conditions, results in multiple amplification products from loci distributed throughout the genome.
Depending on the specific conditions of amplification or product separation and detection, the arbitrary primer amplification methods were termed RAPD. AP-PCR (arbitrarily primer PCR; Welsh and McClelland, 1990) or DAF is generated from a region, which is flanked by a part of 10 bp priming sites in the appropriate orientation.
Genomic DNA from two different individuals often produces different amplification patterns. A particular fragment generated for one individual, but not for other represents DNA polymorphism and can be used as a genetic marker. RAPD amplification is performed in conditions resembling those of polymerase chain reaction using genomic DNA from the species of interest and a single short oligonucleotide (a 10 base primer).
Polymerase Chain Reaction:
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The polymerase chain reaction is used to amplify a segment of DNA that lies between the two regions of known sequence, where two oligonucleotides as primers can bind the opposite strands of DNA due to complementary nature of base sequence.
It is based on the features of semi-conservative DNA replication carried out by DNA polymerases in prokaryotic and eukaryotic cells. The only requirement is that the sequences at the borders of the selected DNA region must be known, so that, two short oligonucleotides could anneal to the target DNA molecule for amplification.
It involves three steps:
1. Denaturation 94°C
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2. Annealing 35°-65°C
3. Extension 72°C
The amplification reaction is catalysed by Taq DNA polymerase. The template DNA is first denatured by heating at 94°C. The reaction mixture is then cooled to a temperature that allows the primers to anneal to their target sequences. These annealed primers are then extended. (i.e. synthesis of DNA) with Taq DNA polymerase.
The cycle of denaturation. annealing and DNA synthesis is repeated many times, because the product of one round of amplification serves as template for the next, each successive cycle essentially, doubles the amount of the desired DNA product.
Components of PCR:
1. Oligomer primer:
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Primer should have high GC content. Primers are used at a concentration of 1 µM, which is sufficient for at least 30 cycles of amplifications.
2. Amplification buffer:
This buffer contains KCl, Tris Cl and 1.5 µM magnesium chloride
3. Deoxyribonucleoside triphosphates:
dNTPs are used at a saturating concentration of 200 µM for each dNTP. It consists of 4 dNTPs such as ATP, CTP, GTP and TTP.
4. Target sequence/genomic DNA:
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The concentration of the target sequence is 5-100 ng.
5. Taq DNA polymerase:
This enzyme is purified from Thermus aquaticus.
It consists of 5′ – 3′ polymerase dependent exonucleasc activity. Addition of excess enzyme may lead to amplification of non-target sequences.
Equipment:
a. Pestle mortar
b. Eppendorf tubes
c. Micropipettes
d. Tips
e. Water bath
f. Refrigerated centrifuge (10,000 – 15,000 rpm)
g. Electrophoresis system (horizontal)
h. Bio-photometer for the determination of DNA concentration
i. PCR machine (thermal cycler)
j. Gel documentation system
Reagents:
1 M Tris pH (8.0)
Tris – 12.11 g
Distilled water – 80 ml
After adjusting the pH (8.0), volume is made up to 100 ml.
10% Sodium dodecyl sulphate (SDS)
10 g SDS is dissolved in 100 ml distilled water.
0.5 M EDTA (ethylene diamine tetra acetic acid) -pH 8.0.
EDTA – 18.62 g
Distilled water – 80 ml
After adjusting the pH (8.0), volume is made up to 100 ml.
5 M Potassium acetate (pH 5.8)
Potassium acetate – 49.12 g
Distilled water – 80 ml
After adjusting the pH (5.8), volume is made up to 100 ml.
3 M Sodium acetate (pH 5.8)
Readymade solution available
RNase – 10 mg/ml
Proteinase K – 20 mg/ml
Chloroform: isoamyl-alcohol (24 : 1)
Isopropanol
70% Ethanol
SDS extraction buffer
1 M Tris (pH 8.0) – 100 ml
0.5 M EDTA (pH 8.0) – 167 ml
Polyvinyl pyrolidone(PVP) – 10 g
SDS (10%) – 170 ml
The volume is made up to 1000 ml.
Tris-EDTA buffer (TE)
1 M Tris (pH 8.0) – 1 ml
0.5 M EDTA (pH 8.0) – 0.2 ml
The volume is made up to 100 ml
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Tris-acetate (TAE) 50 X (pH 8.0)
Tris base – 24.2 g
Glacial acetic acid – 5.71 ml
0.5 M EDTA – 10.0 ml
The pH is adjusted to 8.0 with concentrated HCl and the volume is made up to 100 ml. This stock solution is diluted 50 times for 1 X working solution.
Ethidium bromide – 10 mg/ml
100 mg ethidium bromide is dissolved in 10 ml distilled water. The bottle is wrapped in aluminium foil paper.
DNA loading dye
Bromophenol blue (0.25%) – 0.025 g
Glycerol (30%) – 2 ml
The volume is made up to 10 ml with distilled water.
Agarose
Primers (usually 10 base nucleotide sequence to be obtained from standard suppliers based on specified sequence)
PCR amplification kit
λ Hind-III digested marker DNA
