DescriptionRFLPs are bands that correspond to DNA fragments, usually within the range of 2-10 kb, that have resulted from the digestion of genomic DNA with restriction enzymes. DNA fragments are separated by agarose gel electrophoresis and are detected by subsequent Southern blot hybridization to a labelled DNA probe. Labeling of the probe may be performed with a radioactive isotope or with alternative non-radioactive stains, such as digoxigenin or fluorescein. The locus specific RFLP probes consist of a homologous sequence of a specific chromosomal region. Probes are generated through the construction of genomic or complementary DNA (cDNA) libraries and therefore may be composed of a specific sequence of unknown identity (genomic DNA) or part of the sequence of a functional gene (exons only, cDNA). RFLP probes are maintained as clones in suitable bacterial vectors that conveniently allow the isolation of the DNA fragments they hold. Probes from related species may be used (heterologous probes). DNA sequence variation affecting the absence or presence of recognition sites of restriction enzymes, and insertions and deletions within two adjacent restriction sites, form the basis of length polymorphisms.
StrengthsRFLPs are generally found to be moderately polymorphic. In addition to their high genomic abundance and their random distribution, RFLPs have the advantages of showing codominant alleles and having high reproducibility.
WeaknessesThe main drawbacks of RFLPs are the requirement of laborious and technically demanding methodological procedures, and high expense. In general, if research is conducted with poorly studied groups of wild species or poorly studied crops (orphan crops) suitable probes may not yet be available, so considerable investments are needed for development. Moreover, large quantities of purified, high molecular weight DNA are required for each DNA digestion. Larger quantities are needed for species with larger genomes, and for the greater number of times needed to probe each blot. RFLPs are not amenable to automation and collaboration among research teams requires distribution of probes.
ApplicationsRFLPs can be applied in diversity and phylogenetic studies ranging from individuals within populations or species, to closely related species. RFLPs have been widely used in gene mapping studies because of their high genomic abundance due to the ample availability of different restriction enzymes and random distribution throughout the genome. They also have been used to investigate relationships of closely related taxa, as fingerprinting tools, for diversity studies, and for studies of hybridization and introgression, including studies of gene flow between crops and weeds.
Restriction fragment length polymorphism mapping in conifers and applications to forest genetics and tree improvement Neale, D.B. and Williams, C.G. (1991). Canadian Journal of Forest Research, 21: 545-554. doi: 10.1139/x91-076.