RFLP Analysis

Some genetic defects can be detected by the method of restriction fragment length polymorphisms (RFLPs). This analysis is based upon the presence of genetic polymorphisms, or alleles of a particular gene that are found in more than 1% of the population. When a sample of DNA is incubated with certain endonucleases, the DNA is cleaved into fragments, called restriction fragments, of a particular length. Such endonucleases are derived from bacteria which have them to restrict the entry of foreign DNA. For a given allele, the fragment lengths will be the same. If a mutation has changed one or more nucleotides in a gene, then the endonuclease may not recognize it the same as another allele, and the cleavage will occur at a different point, leading to differences in length of the restriction fragments. Differences in the number of tandem repeats will also affect the action of the endonuclease on the DNA.

RFLP analysis starts with extracting DNA from a sample, typically blood containing leukocytes and incubating it with a restriction endonuclease to digest the DNA into a million strands. Then gel electrophoresis is used to separate the strands by length. These fragments are then transferred (blotted) from the gel to a nitrocelulose membrane, called a "Southern transfer" (after the name of the inventor, not the location) to produce a "Southern blot." A large number of copies of a radiolabelled probe--a specific strand of DNA, is then applied and incubated so that the probe strands will find complementary base pairs with a certain restriction fragment. An autoradiogram is made to identify the specific location of the "bands" made by the pairing of the strands on the membrane. A polymorphism associated with a disease may yield restriction fragments longer or shorter than the "normal" allele.

In the above example, both parents are heterozygotes for a genetic disease manifested as an autosomal recessive disorder. One child at the left is also a heterozygote, the next is an affected homozygote, and the last child on the right is unaffected. The allele with the mutation has longer restriction fragments and does not migrate as far in the direction of migration (arrow) as the restriction fragments from the normal allele.