Restriction Site Mapping

The first preliminary characterization of any newly isolated DNA fragment usually involves restriction site mapping. For this, a family of bacterial enzymes called restriction endonucleases, or restriction enzymes, are utilized. These enzymes recognize specific sequences of DNA, usually four or six base pairs, and cut the DNA at that sequence. For example, the restriction enzyme Eco RI recognizes the sequence: and makes a staggered cut in both strands of the DNA at the positions shown by the arrows. Every time that particular sequence occurs in a fragment of DNA, Eco RI will make a cut and generate a number of smaller fragments. Restriction site mapping entails locating the positions along a stretch of DNA that are recognized and cleaved by a set of restriction enzymes. Almost 300 restriction enzymes covering 100 or so different recognition sequences are now known. However, only a subset of about three to six enzymes are usually used for mapping in the first instance. This is a first-order analysis of the DNA sequence (the next step being the total sequencing of the DNA), and serves as a fingerprint for the particular DNA fragment.