Random amplification of polymorphic DNA ( RAPD ) , referred to as “rapid”, [1] is a type of polymerase chain reaction (PCR), but the segments of DNA that are amplified are random. [2] The scientist performing RAPD creates several arbitrary, short primers (8–12 nucleotides), then proceeds with PCR using a large template of genomic DNA, with the expectation that the fragments will be amplified. By resolving the resulting pattern, a quasi-unique profile can be obtained from the RAPD response.
No knowledge of the DNA sequence of the target genome is necessary, as the primer will bind somewhere in the sequence, but it is not certain where exactly. This popularizes the method for comparing DNA from biological systems that have not received the attention of the scientific community, or in systems where relatively short DNA sequences are compared (not suitable for creating cDNA databanks). ). Since 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 that of 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 various plant and animal species.
Introduction
RAPD markers are decamers (10 nucleotides long) DNA fragments resulting from PCR amplification of random segments of genomic DNA with a single primer of arbitrary nucleotide sequence and which are able to differentiate between genetically distinct individuals, although not necessarily that in a reproducible manner. It is used to analyze the genetic diversity of an individual using random primers. Due to problems in experiment reproducibility, many scientific journals no longer accept experiments based solely on RAPD. RAPD requires only one primer for amplification.
How it works
Unlike conventional PCR analysis, RAPD does not require any specific knowledge of the target organism’s DNA sequence: identical 10-mer primers will or will not amplify a segment of DNA that is complementary to the sequence of the primers. For example, if the primers are too far apart or the 3′ ends of the primers are not facing each other, no fragment is formed. Therefore, if a mutation occurred in the template DNA at a site that was complementary to the first primer, a PCR product would not be produced, resulting in a different pattern of amplified DNA segments on the gel.
Borders
- Almost all RAPD markers are dominant, i.e. it is not possible to distinguish whether the DNA segment is amplified from a locus that is heterozygous (1 copy) or homozygous (2 copies). The codominant RAPD markers, seen as DNA segments of different sizes amplified from the same locus, are rarely detected.
- PCR is an enzymatic reaction, so the quality and concentration of template DNA, the concentration of PCR components and PCR cycling conditions can greatly affect the result. Thus, RAPD techniques are notoriously laboratory dependent and require carefully developed laboratory protocols to be reproducible.
- Mismatch between primer and template can result in total absence of PCR product as well as only small amount of product. Thus, unlike conventional PCR analysis, RAPD results can be difficult to interpret.
