Two-dimensional Pulsed-field Gel Electrophoresis

Abstract

Two key techniques were responsible for the rapid progress in bacterial genome mapping during the last decade: the identification of infrequently cleaving restriction endonucleases which produce large DNA of defined size (see Chapt 24), and the development of pulsed field gel electrophoresis (PFGE) to separate high molecular weight DNA (see Chapt 25). As pointed out in Chapter 25, the relatively small bacterial genome size (0.5–10 Megabase pairs) makes these organisms ideal objects for the construction of low-resolution physical maps by both one-dimensional and two-dimensional combinatorial analyses. The creation of distinct “spots” enables physical genome analysis by two-dimensional gel electrophoresis as has already been applied to plasmid analysis by conventional continuous field gel electrophoresis some twenty years ago (Villems et al., 1978). The macrorestriction map is assembled from a few two-dimensional PFGE gels of partial-complete single digestions or complete double digestions (Bautsch, 1988). The fragment order can be established without any genetic data and without using hybridization methods, which is particularly advantageous for the mapping of poorly characterized genomes. This chapter describes the essentials of bacterial genome mapping by two-dimensional PFGE.