Restriction Fragment Length Polymorphism (RFLP) is a technique in which organisms may be differentiated by analysis of patterns derived from cleavage of their DNA. If two organisms differ in the distance between sites of cleavage of a particular restriction endonuclease, the length of the fragments produced will differ when the DNA is digested with a restriction enzyme. The similarity of the patterns generated can be used to differentiate species (and even strains) from one another.
Restriction endonucleases are enzymes that cleave DNA molecules at specific nucleotide sequences depending on the particular enzyme used. Enzyme recognition sites are usually 4 to 6 base pairs in length. Generally, the shorter the recognition sequence, the greater the number of fragments generated. If molecules differ in nucleotide sequence, fragments of different sizes may be generated. The fragments can be separated by gel electrophoresis. Restriction enzymes are isolated from a wide variety of bacterial genera and are thought to be part of the cell's defenses against invading bacterial viruses. These enzymes are named by using the first letter of the genus, the first two letters of the species, and the order of discovery.
PCR is a technique for amplifying a specific region of DNA, defined by a set of two "primers" at which DNA synthesis is initiated by a thermostable DNA polymerase. Usually, at least a million-fold increase of a specific section of a DNA molecule can be realized and the PCR product can be detected by gel electrophoresis. The regions amplified are usually between 150-3,000 base pairs in length.
Isolation of sufficient DNA for RFLP analysis is time-consuming and labor intensive. However, PCR can be used to amplify very small amounts of DNA, usually in 2-3 hours, to the levels required for RFLP analysis. Therefore, more samples can be analyzed in a shorter time.
Dr. Walter Hill (formally at SEA/SPRC)