Randomly
amplified polymorphic dna (RAPD) as the name suggests are the products of a PCR reaction primed by arbitrarily selected primers.
How is it
different from conventional PCR?
In
conventional PCR, one designs the forward and reverse primers with the aim that
they will anneal to the gene of interest. In order to accomplish this, it is
necessary that one knows the sequence of the gene of interest. The next few
steps follow a cycle wherein the primers first anneal, then a polymerase
extends them and again the extended products are denatured making them ready
for the next cycle of primer annealing.
In case of
RAPD, the knowledge of the gene sequence is not a pre-requisite. One selects
random primers which at low temperature display lower fidelity and hence anneal
to many arbitrary sites on the DNA template with a variety of mismatches. After
a few initial steps of annealing at lower temperature the amplification may
then be carried out according to the conventional PCR or may also be continued
even at the same temperature. When these amplified products are run on a gel, a
pattern of bands is obtained which is unique for a particular species and is
dependent upon the primers used.
Selection of
primers
A single
random primer will seldom be informative. When we use many such primers and
then score them; will the RAPD profile make some sense in terms of
polymorphism. Polymorphism maybe detected when the fingerprint of one sample
shows an amplified band while the other sample does not even when the same primer
is used with both the samples.
RAPD may be
generated due to mutation in template (insertions/deletions) or presence of
alleles in heterozygous individuals. However, these cannot be pinpointed as
such because RAPD markers are dominant i.e they amplify many loci in a single
go with a single primer.
Some of the amplified bands are easily recognizable while some others may be difficult to interpret. The ambiguity arises due to the fact that every primer will possess different power to distinguish between different sites of amplification. Also there will exist competition between primers and different sites. Certain amplified fragments may interfere in the amplification or separation of other segments. Reproducibility of the RAPD pattern becomes problematic below a certain concentration of genomic DNA and may produce smears. On the other hand, there maybe poor resolution if the genomic concentration is very high
Reference:
Molecular Biomethods Handbook, second edition, John M Walker, Ralph Rapley, chpt 10, pg 132-147
