Random Amplified Polymorphic DNA (RAPD)

Introduction

Random Amplified Polymorphic DNA (RAPD)

markers are DNA fragments from PCR amplification of random segments of genomic DNA with single primer of arbitrary nucleotide sequence.

How It Works

Unlike traditional PCR analysis, RAPD (pronounced "rapid") does not require any specific knowledge of the DNA sequence of the target organism: the identical 10-mer primers will or will not amplify a segment of DNA, depending on positions that are complementary to the primers' sequence. For example, no fragment is produced if primers annealed too far apart or 3' ends of the primers are not facing each other. Therefore, if a mutation has occurred in the template DNA at the site that was previously complementary to the primer, a PCR product will not be produced, resulting in a different pattern of amplified DNA segments on the gel.

Example

RAPD is an inexpensive yet powerful typing method for many bacterial species.

Silver-stained polyacrylamide gel showing three distinct RAPD profiles generated by primer OPE15 for Haemophilus ducreyi isolates from Tanzania, Senegal, Thailand, Europe, and North America.

Selecting the right sequence for the primer is very important because different sequences will produce different band patterns and possibly allow for a more specific recognition of individual strains.

Limitations of RAPD

• Nearly all RAPD markers are dominant, i.e. it is not possible to distinguish whether a DNA segment is amplified from a locus that is heterozygous (1 copy) or homozygous (2 copies). Co-dominant RAPD markers, observed as different-sized DNA segments amplified from the same locus, are detected only rarely.
• PCR is an enzymatic reaction, therefore the quality and concentration of template DNA, concentrations of PCR components, and the PCR cycling conditions may greatly influence the outcome. Thus, the RAPD technique is notoriously laboratory dependent and needs carefully developed laboratory protocols to be reproducible.
• Mismatches between the primer and the template may result in the total absence of PCR product as well as in a merely decreased amount of the product. Thus, the RAPD results can be difficult to interpret.

Developing Locus-specific, Co-Dominant Markers from RAPDs

• The polymorphic RAPD marker band is isolated from the gel.
• It is amplified in the PCR reaction.
• The PCR product is cloned and sequenced.
• New longer and specific primers are designed for the DNA sequence, which is called the Sequenced Characterized Amplified Region Marker (SCAR).

References

» Mbwana J, et al. Molecular characterization of Haemophilus ducreyi isolates from different geographical locations. J Clin Microbiol. 2006 Jan;44(1):132-7. PMID: 16390960

» Williams JG, et al. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res. 1990 Nov 25;18(22):6531-5. PMID: 1979162

Resources

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