RFLP (Restriction Fragment Length Polymorphism)

RFLP, or restriction fragment length polymorphism, is a molecular biological technique used to compare DNA from two samples. Special enzymes that cleave the DNA in specific locations are used to digest strands of DNA. Mutations within the DNA result in strands of different lengths. Electrophoresis is then used to separate the strands according to their length. RFLP is used as part of DNA fingerprinting, to detect genetic diseases and to determine genetic relationships between species.

The DNA molecule is made up of a sequence of four smaller molecules called nucleotides. The four nucleotides are adenine (A), guanine (G), cytosine (C), and thymine (T). The sequence of these nucleotides is extremely important, as it determines the structure of all of the molecules in an individual. Differences in individuals result from small variations, called mutations, in the sequence of DNA. There are a variety of types of mutations in DNA. Insertions are regions of DNA where nucleotides have been added to a sequence. Deletions are regions where nucleotides have been removed. In vertebrates (animals with a backbone), there are regions of DNA that contain many repetitions of the same sequence. Two families of these repeats are found quite often in DNA: variable number of tandem repeats (VNTRs) and short tandem repeats (STR). Point mutations may also occur in DNA. This is simply the replacement of a single nucleotide by a different one.

A special type of protein called a restriction enzyme, or a restriction endonuclease, can recognize specific sequences of nucleotides on DNA and then cleave the DNA at these locations. For example, the restriction enzyme HaeIII recognizes the sequence GCGC and it cleaves the bond between middle cytosine and guanine. Bacteria naturally produce restriction enzymes and they use them to cleave the DNA from foreign organisms. Over 90 different restriction enzymes have been isolated from different species of bacteria. Each of these enzymes cleaves DNA between different, and specific, sequences of nucleotides.

When performing RFLP, the target DNA is usually subjected to polymerase chain reaction, which produces millions of copies of strands of DNA identical to the original. This amplified DNA is then combined with a set of restriction enzymes, which cleave the DNA in specific locations. For example, consider the strand of DNA from one individual with the sequence GCGCAAGGCGAATTCGCGC. The restriction enzymes HaeIII and EcoRI are both added to the mixture. As discussed, HaeIII cleaves between C and G on the sequence GCGC. EcoRI recognizes the sequence GAATTC and it cleaves the bond between the adenine and the thymine. The resulting strands from this RFLP would be GC, GCAAGGCGAA, TTCGC, and CG. Next, consider a sample of the same region of DNA from a second individual. This individual has a point mutation so that their DNA sequence is GCGCAAGGCGAATTCGCCC. After exposure to the same restriction enzymes, the resulting strands of DNA would be GC, GCAAGGCGAA and TTCGCCC.

After exposure to the restriction enzymes, the two mixtures are transferred to a gel and electrophoresis is performed. In gel electrophoresis an electrical current is transmitted through the gel causing the fragments of DNA to migrate through the gel according to their electrophoretic mobility. This distance is roughly proportional to the inverse of the fragment's length. As a result, shorter fragments migrate farther from the origin as they move through the gel.

After the gel is run, the DNA is labeled using a radioactive probe and the gel is exposed to x-ray film, which changes color in the presence of radioactivity. The locations of the fragments of DNA show up on the film as bands. Different samples can be loaded onto the gel in different lanes so that the banding patterns can be compared side-by-side. In the example above, if the digested DNA is loaded into two lanes on the same gel, three bands will appear in both lanes but the pattern will be different. Both lanes will have a band very far from the origin containing the small sequence GC and a band close to the origin containing the sequence GCAAGGCGAA. Both lanes will also have a third band between these two. However, the band from the first individual will be farther from the origin than the band from the second individual, because it is shorter.

In cases where the DNA under consideration contains VNTRs or STRs, restriction enzymes that do not cut within the VNTR or STR sequence are used. The resulting gel has bands closer to the origin that represent fragments with more repeats and bands farther from the origin for fragments that contain few repeats.

The applications for RFLP are many. DNA fingerprinting uses the presence of STRs at thirteen different locations on the chromosomes. The lengths of these STRs are detected using RFLP analysis. Several genetic diseases are detected using RFLP analysis including cystic fibrosis, Huntington's chorea and sickle-cell anemia. In particular, sickle-cell anemia is caused by a single mutation of a single nucleotide: thymine is replaced by adenine. This mutation occurs at a point in the DNA sequence that is recognized by the restriction enzyme MstII in a person without the disease. The RFLP from a person suffering from sickle-cell anemia will have a long band instead of two shorter ones because the cleavage by MstII will not occur. Finally, mutations in DNA between species are often investigated using RFLP analysis. Species with more different banding patterns are suspected of being less closely related than species with more similar banding patterns.

see also DNA banks for endangered animals; DNA fingerprint; DNA sequences, unique; Mitochondrial DNA analysis; Y chromosome analysis.