RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD)

RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD) or Arbitrarily Primed PCR (AP – PCR)

• Single short Primer of Random Arbitrary Sequence (10 bp)
• No sequence data is required; hence can be initiated with minimal infrastructural facilities.
• The primer will bind to many sites at low PCR stringency conditions
• Low annealing temp (36 – 40°C)
• High MgCl2 concentration (0.5 – 1.0microlitres)
• Dominant Marker

Advantages:

• Sequence data is not required.
• RAPD studies in a new species can be initiated with minimal infrastructure facilities.

Disadvantages:

• Short primer (of generally 10mer) with random sequence will bind to many sites within the template DNA at low PCR stringency conditions. Due to this property, a slight change in the parameters of PCR would result in a change in RAPD-PCR product patterns. Hence, for obtaining the reproducibility of the results, strict standardization of the PCR is required.

Points to be taken care of

• With every PCR, set up a PCR reaction without genomic DNA (called negative PCR reaction) to monitor the DNA contamination during PCR set up.
• RAPD primer to be used is first tested for the amplification of clear and repeated bands.
• Utmost care has to be taken during set up of RAPD-PCR, to avoid contamination of PCR products
• For repeatability of results, to strictly follow:
• Proper lab techniques.
• Strict standardization of all reagents and protocols

Analysis of Data

• The presence or absence of each fragment recorded as 1 or 0.
• Faint or poorly amplified fragments to be excluded from analysis as well as bands above 2000bp and below 300bp.

Assumptions

• Two allele system: presence ++/+ –; absence – –
• Fragments migrated at the same position had the same molecular weight are homologous bands from the same allele and alleles from different loci do not co-migrate.
• Populations conform to Hardy-Weinberg Equilibrium (in an indefinitely large randomly mating population, the allelic/gene frequencies (i.e. the proportion of different alleles of a gene) remain unchanged generation after generation in the absence of mutation, migration, selection & genetic drift; ( p + q)2 = 1.

Analysis of data (RAPD)

Genetic variability

• Percentage Polymorphic loci (a gene is polymorphic if the frequency of the most common alleles is < 0.95 ie P < 0.95)
• Average Gene Diversity (H = Average heterozygosity), Mean heterozygosities (h) of all the loci

Genetic Uniqueness/differentiation

• GST (Masatoshi Nei) coefficient of genetic differentiation = FST (Sewall Wright) 
• (FST is the index of genetic differentiation that describes how much variation in allele frequencies is present between the local population. It is a measure of population differentiation and ranges from 0 where all the population have the same allele frequencies at all the loci to 1.0, where all the populations are fixed for different alleles at all loci)
• Significance is tested for departure from zero
• Nm (Gene flow = no.of migrants between population). If the values of GST /FST are high, Nm values will be less

• Pairwise genetic distance(GD) = 1 – Similarity Index (SI)
• Calculated from SI = 2 NAB/(NA + NB)

NAB = is the number of bands shared by 2 populations

NA = total bands for population A

NB = total bands for population B

• Paired T-test/ANOVA
• Dendrogram – Unweighted Pair Group Method for Arithmetic Mean (UPGMA); to test the confidence level, bootstrapping 1000 times.
• Software used: Popgene, TFPGA, Gen Alex