16 - Restriction Mapping

Summary

One of the ways biologists begin to analyze a long sequence of DNA is to develop a restriction site map. Restriction sites are the locations at which the sequence is cut by enzymes known as restriction enzymes – or, more precisely, restriction endonucleases. Restriction enzymes are found in bacteria, where they provide some protection against viral invasion by destroying viral DNA. Each bears a name such as EcoRI and HindIV, derived from the bacterium in which it was discovered. Each restriction enzyme can cut DNA at any location containing a specific short sequence. Examples of some commonly used restriction enzymes and the sequences they cut are given in Figure 16.1. The sequence is typically a palindrome of even length. In most cases, the enzyme cuts the double strand unevenly so that a small group of unpaired nucleotides – a sticky end – remains on both sides of the cut. In nature, this feature may facilitate further degradation of the invading DNA by other enzymes; in the laboratory, it is often used to “cut and paste” new sequences into DNA at known locations.

A restriction map of a sequence is simply a list of the locations at which one or more restriction enzymes are known to cut the sequence. Such a map can be used to pinpoint the origin of a gene or other subsequence within a larger sequence.

Restriction maps are generally constructed by performing digestion experiments: The sequence to be mapped is replicated by PCR and then exposed to one or more restriction enzymes, together and/or separately.