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RAPD Fingerprinting 117
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Randomly Amplified Polymorphic DNA Fingerprinting
The Basics
Ranil S. Dassanayake and Lakshman P. Samaranayake
1. Introduction
The study of genetic polymorphism among diverse populations of organisms is
a complex task. However, this can be accomplished by using newer tools, such as
randomly amplified polymorphic DNA (RAPD). RAPD is a polymerase chain reaction
(PCR) technique that relies on the generation of amplification products for a given
nucleic acid using an amplification-based scanning technique driven by arbitrary
priming oligonucleotides. The result is the generation of amplification products
(amplicons) that represent a multiplicity of anonymous sites that are characteristic of
the studied genome (Fig. 1A,B).
In RAPD, the first few cycles are performed at a low stringency, which ensures the
generation of products by priming with mismatches between the primer and the template.
The subsequent PCR cycles are performed at a higher stringency (see Note 1),
yielding products that have ends complementary to the primer. The amplified region
consists of unstructured, hypervariable, mostly noncoding sequences that vary in length
from one species to another. As the arbitrary priming depends upon the complimentary
regions in the DNA template, differences in these regions lead to uniquely characteristic
fingerprinting patterns (1). These differences permit any organism to be characterized
at the species or the strain level. However, it is noteworthy that the clarity of the species
or strain discrimination, fingerprint complexity, and detection of DNA polymorphism
are dependent on the primer that is selected for the RAPD assay.
A single primer approx 10 bp in size (40–70% guanine–cytosine content) is generally
used in PCR fingerprinting. For the amplification of the target region, the distance
between priming regions has to be not more than 3 to 4 Kb. Specificity in RAPD is
defined as the ability to produce “consensus” fingerprints on multiple occasions as
a result of a multiplicity of targeted arbitrary sites. To achieve such consensus and
“stringent” fingerprints in arbitrary priming protocols, a decrease in primer length (2)
or blocking of the interactions between amplicons by incorporating a mini-hairpin at
the 5′ terminus of the oligonucleotide is required (3).
There are a number of limitations in the routine use of RAPD profiles for detailed
evaluation of the genomes of organisms. The necessarily short primers require stringent
From: Methods in Molecular Biology, Vol. 226: PCR Protocols, Second Edition
Edited by: J. M. S. Bartlett and D. Stirling © Humana Press Inc., Totowa, NJ
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