Document Type
Article
Abstract
The techniques described in this chapter provide a framework with which to begin an analysis of important features of bacterial genome organization and gene function. In this report we describe the preparation of chromosomal DNA, conditions for digestion with restriction endonucleases, and electrophoretic methods that result in the optimal resolution of large DNA restriction fragments. The sizes of the separated fragments can be summed to accurately determine genome size. The fragments can also be probed in hybridization experiments to produce low-resolution genomic restriction maps, and the precise locations of insertion or deletion mutations can be rapidly identified. In addition, PFGE technology can be applied to analyses of in vivo chromosome replication. We describe how replication forks can be synchronized and how to radiolabel the newly replicated DNA. The sequential incorporation of labeled nucleotides into DNA fragments separated on pulsed-field gels allows the identification of the chromosomal origin of replication, the rate and direction of replication fork movement, and the localization of the replication terminus in a single experiment.
Digital Object Identifier (DOI)
Publication Info
Published in Methods, Volume 1, Issue 2, 1990, pages 160-168.
Rights
© 1990 by Academic Press, Inc. Under a Creative Commons license
APA Citation
Dingwall, A., Shapiro, L., & Ely, B. (1990). Analysis of bacterial genome organization and replication using pulsed-field gel electrophoresis. Methods, 1(2), 160–168.https://doi.org/10.1016/S1046-2023(05)80131-8