6 - Macrorestriction Mapping and Analysis of Bacterial Genomes

Abstract

This chapter focuses on the construction of macrorestriction maps of bacterial genomes. The relatively small genome of 0.5–10 Mbp makes bacteria an appropriate target for the comprehensive study of genome organization by pulsed-field gel electrophoresis (PFGE) techniques. The number, size, and topology of genetic entities are the fundamental characteristics of a genome. PFGE permits visualization of chromosomes and plasmids. Unsheared bacterial DNA is prepared by the inclusion of intact bacteria into agarose blocks prior to cell lysis. It is found that determining the size of circular chromosomes requires the additional step of linearizing the DNA prior to PFGE, since large circular DNA remains trapped in the plug instead of migrating through the gel. It is observed that after PFGE of embedded samples, circular chromosomes will produce an intense fluorescent signal from the agarose plug, which reflects the trapped circles, and faint bands in the megabase range. For the construction of a macrorestriction map, the genomic DNA is digested with restriction endonucleases that cut only rarely and the fragments are subsequently separated by PFGE. It is suggested that the mapping strategy requires an optimal quality of the agarose-embedded DNA and optimal separation conditions.