RFLP is a simple method of detecting variability in the sequences of DNA within the genomes of a population of individuals (ie, polymorphism) using mutations at the sites of action of restriction enzymes (1, 2). Restriction enzymes are endonucleases that cut DNA at specific, very short base sequences, producing restriction fragments whose average size reflects the frequency with which the specific restriction sequence is found along the DNA. Point mutations may cause restriction sites to disappear, may cause the restriction fragment affected to increase in size by addition of the adjacent one, or may cause new restriction sites to appear, splitting one restriction fragment into two smaller fragments. By separating the resulting restriction fragments by gel electrophoresis in agar, on the basis of their lengths, the patterns of DNA bands can be compared in different individuals. This technique is limited by its ability to detect only mutation events occurring within enzyme restriction sites, which are sequences of only 4 to 6 nucleotides separated by hundreds or thousands of bases. This limitation can be overcome to some extent by using several restriction enzymes; however, in a study of vertebrate mitochondrial DNA using 20 restriction enzymes that are specific for sequences of six bases, only about 3% of the genome will be sampled (3).
Consequently, RFPL is not a very powerful method for analyzing polymorphisms. If applied to a random selection of DNA, it will reveal, approximately, a level of polymorphism comparable to that found with protein isozymes. An improvement is the use in Southern blotting of specific probes revealing fragments belonging to a restricted region whose polymorphism is under study. In the case of a species whose genome is practically unknown, but for which a large number of probes is available, randomly cloned genome fragments may generate RFLP patterns of some interest. It is best, however, to isolate the section of DNA of interest, expand it by the polymerase chain reaction (PCR), subject it to the subsequent action of several restriction enzymes, and analyze the pattern of bands obtained by electrophoresis. Given the very large number of restriction enzymes that are available now, it will often be possible to design simple RFLP tests to reveal the alternatives.
