RAPD (pronounced "rapid") stands for 'Random Amplification of Polymorphic DNA'. It is a type of PCR reaction, but the segments of DNA that are amplified are random. The scientist performing RAPD creates several arbitrary, short primers (8–12 nucleotides), then proceeds with the PCR using a large template of genomic DNA, hoping that fragments will amplify. By resolving the resulting patterns, a semi-unique profile can be gleaned from an RAPD reaction.
Contents
- Introduction
- How it works
- Example
- Limitations of RAPD
- Developing locus specific co dominant markers from RAPDs
- References
No knowledge of the DNA sequence of the targeted genome is required, as the primers will bind somewhere in the sequence, but it is not certain exactly where. This makes the method popular for comparing the DNA of biological systems that have not had the attention of the scientific community, or in a system in which relatively few DNA sequences are compared (it is not suitable for forming a cDNA databank). Because it relies on a large, intact DNA template sequence, it has some limitations in the use of degraded DNA samples. Its resolving power is much lower than targeted, species-specific DNA comparison methods, such as short tandem repeats. In recent years, RAPD has been used to characterize, and trace, the phylogeny of diverse plant and animal species.
Introduction
RAPD markers are decamer (10 nucleotide length) DNA fragments from PCR amplification of random segments of genomic DNA with single primer of arbitrary nucleotide sequence and which are able to differentiate between genetically distinct individuals, although not necessarily in a reproducible way. It is used to analyse the genetic diversity of an individual by using random primers. Due to problems in experiment reproducibility, many scientific journals do not accept experiments merely based on RAPDs anymore. RAPD requires only one primer for amplification.
How it works
Unlike traditional PCR analysis, RAPD 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. The image visible at the link [1] is a 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.