16 - Cloning Vectors and Techniques for Exonuclease–Hybridization Restriction Mapping

Abstract

Constructing restriction maps of cloned DNA fragments is often a tedious, but very necessary task frequently encountered in the course of gene isolation or chromosome walking. There are two methods for constructing maps of this type. The first requires digesting the cloned DNA with two different restriction enzymes, both singly and together. After separating the resulting fragments by agarose gel electrophoresis, a uniquely ordered arrangement that accounts for the observed array of bands in each digest can be determined by trial and error method. The amount of time required for this process is substantial, increasing as the size of the insert DNA increases. A second approach to restriction mapping makes use of a method initially developed by Smith and Birnstiel. The DNA to be mapped is uniquely labeled at one end of the molecule. Then, partial digestion with a restriction enzyme, followed by electrophoresis and autoradiography, reveals a series of fragments that can be ordered from the labeled terminus.