Long-range restriction mapping of genomic DNA

Abstract

Abstract Long-range restriction maps are important tools to determine the arrangement and distance of gene markers, and a prerequisite before committing oneself to costly and time-consuming cloning and sequencing projects in order to identify gene organization and rearrangements. For the construction of a low-resolution physical map of a genome or chromosomal region, the intact DNA is digested with a restriction endonuclease that cleaves infrequently and the fragments are separated by pulsed-field gel electrophoresis (PFGE) (1, 2). Restriction fragment patterns of small genomes, such as those of prokaryotes or lower eukaryotes, can be visualized directly by gel staining. With larger genomes, the complex mixture of fragments is blotted onto membranes after electrophoresis, and hybridized with suitable probes to detect fragments of interest. The order of probes and of fragments is established from the combinatorial analysis of partial and complete digests with one or more restriction enzymes. In the simplest case, two probes will hybridize to the same restriction fragment, thus revealing physical linkage between them. However, independent confirmation must always be sought to exclude hybridization to distinct but co-migrating bands. This may be accomplished by, for example, hybridization of the two probes in question to the fragments produced by partial digestion with the same enzyme as was used in the complete digest: both probes should detect the same bands (and with the same relative intensities) in this more complex mixture of fragments. Combined usage of several different enzymes should then allow construction of a macrorestriction map around both loci, which may be refined by double digestion analysis.