Random Amplified Polymorphic DNA (RAPD)
RAPDs are DNA fragments amplified by PCR using short synthetic primers (generally 10 bp) of random sequence. These oligonucleotides serve as both forward and reverse primer, and are usually able to amplify fragments from 1-10 genomic sites simultaneously. Amplified fragments, usually within the 0.5-5 kb size range, are separated by agarose gel electrophoresis, and polymorphisms are detected, after ethidium bromide staining, as the presence or absence of bands of particular sizes. These polymorphisms are considered to be primarily due to variation in the primer annealing sites, but they can also be generated by length differences in the amplified sequence between primer annealing sites.
StrengthsThe main advantage of RAPDs is that they are quick and easy to assay. Because PCR is involved, only low quantities of template DNA are required. Since random primers are commercially available, no sequence data for primer construction are needed. Moreover, RAPDs have a very high genomic abundance and are randomly distributed throughout the genome.
WeaknessesThe main drawback of RAPDs is their low reproducibility, and hence highly standardized experimental procedures are needed because of their sensitivity to the reaction conditions. RAPD analyses generally require purified, high molecular weight DNA, and precautions are needed to avoid contamination of DNA samples because short random primers are used that are able to amplify DNA fragments in a variety of organisms. Altogether, the inherent problems of reproducibility make RAPDs unsuitable markers for transference or comparison of results among research teams working in a similar species and subject. As for most other multilocus techniques, RAPD markers are not locus-specific, band profiles cannot be interpreted in terms of loci and alleles (dominance of markers), and similar sized fragments may not be homologous.
ApplicationsRAPDs have been used for many purposes, ranging from studies at the individual level (e.g. genetic identity) to studies involving closely related species. RAPDs have also been applied in gene mapping studies to fill gaps not covered by other markers. Variants of the RAPD technique include Arbitrarily Primed Polymerase Chain Reaction (AP-PCR) which uses longer arbitrary primers than RAPDs, and DNA Amplification Fingerprinting (DAF) that uses shorter, 5-8 bp primers to generate a larger number of fragments. Multiple Arbitrary Amplicon Profiling (MAAP) is the collective term for techniques using single arbitrary primers.
Applications of random amplified polymorphic DNA (RAPD) in molecular ecology Hadrys, H., Balick, M and Schierwater, B. (1992). Molecular Ecology, 1: 55-63. doi: 10.1111/j.1365-294X.1992.tb00155.x.
DNA polymorphisms amplified by arbitrary primers are useful as genetic markers Williams, J.G.K., Kubelik, A.R., Livak, K.J., Rafalski, J.A. and Tingey, S.V. (1990). Nucleic Acid Research, 18: 6531-6535. doi: 10.1093/nar/18.22.6531.