Genomic long-range restriction mapping

Abstract

With the development of pulsed-field gel electrophoresis methods and the availability of rare-cutting restriction endonucleases, it has become possible to construct restriction maps spanning very large genomic regions. In the past few years, methods for the construction of long-range maps have been applied and refined in many different laboratories. Although a number of specific protocols have been developed, all of them rely on a basic set of procedures combining pulsed-field gel electrophoresis, Southern blotting, and detection of specific fragments using various types of molecular probes. Long-range maps have many uses and have proven to be particularly valuable for the positional cloning and identification of genes involved in various genetic disorders. The restriction endonucleases most useful for constructing such maps include CpG dinucleotides in their recognition sequences and provide landmarks helpful for locating transcribed sequences. This article presents a collection of methods that have been successfully applied to the construction of long-range maps. Strategic considerations important for the success of a mapping study are discussed, and various alternative protocols that exist in the literature are evaluated. Finally, some suggestions for facilitating data analysis and for trouble-shooting common problems are made.