Double-strand breakage of DNA is a process central to life and death in DNA-coded organisms. Its sensitive and quantitative detection is realized by pulsed-field gel electrophoresis of a huge (Mb) circular chromosome. A single double-strand break at one of its millions of potential sites will make it linear and release it from branches of an agarose jungle. Then the huge fragments will move according to their size. We developed this method to analyze formation of DNA double-strand breaks and their processing in E. coli. Here we detail our protocol taking the example of chromosome breaks caused by action of a restriction enzyme in vivo. It is important to prevent formation of irrelevant double-strand breaks.
Keywords: Bacterial chromosome; DNA double-strand break; DNA recombination; DNA repair; DNA replication; Restriction endonuclease.