Restriction Enzymes

The ability of restriction enzymes to reproducibly cut DNA at specific sequences has led to the widespread use of these tools in many molecular genetics techniques. Restriction enzymes can be used to map DNA fragments or genomes. Mapping means determining the order of the restriction enzyme sites in the genome. These maps form a foundation for much other genetic analysis. Restriction enzymes are also frequently used to verify the identity of a specific DNA fragment, based on the known restriction enzyme sites that it contains.

Perhaps the most important use of restriction enzymes has been in the generation of recombinant DNA molecules, which are DNAs that consist of genes or DNA fragments from two different organisms. Typically, bacterial DNA in the form of a plasmid (a small, circular DNA molecule) is joined to another piece of DNA (a gene) from another organism of interest. Restriction enzymes are used at several points in this process. They are used to digest the DNA from the experimental organism, in order to prepare the DNA for cloning. Then a bacterial plasmid or bacterial virus is digested with an enzyme that yields compatible ends. These compatible ends could be blunt (no overhang), or have complementary overhanging sequences. DNA from the experimental organism is mixed with DNA from the plasmid or virus, and the DNAs are joined with an enzyme called DNA ligase. As noted above, the identity of the recombinant DNA molecule is often verified by restriction enzyme digestion.

Restriction enzymes also have applications in several methods for identifying individuals or strains of a particular species. Pulsed field gel electrophoresis is a technique for separating large DNA fragments, typically fragments resulting from digesting a bacterial genome with a rare-cutting restriction enzyme. The reproducible pattern of DNA bands that is produced can be used to distinguish different strains of bacteria, and help pinpoint if a particular strain was the cause of a widespread disease outbreak, for example.

Restriction fragment length polymorphism (RFLP) analysis has been widely used for identification of individuals (humans and other species). In this technique, genomic DNA is isolated, digested with a restriction enzyme, separated by size in an agarose gel, then transferred to a membrane. The digested DNA on the membrane is allowed to bind to a radioactively or fluorescently labeled probe that targets specific sequences that are bracketed by restriction enzyme sites. The size of these fragments varies in different individuals, generating a "biological bar code" of restriction enzyme-digested DNA fragments, a pattern that is unique to each individual.

Restriction enzymes are likely to remain an important tool in modern genetics. The reproducibility of restriction enzyme digestion has made these enzymes critical components of many important recombinant DNA techniques.