A general method for detecting rearrangements in a bacterial genome.

Abstract

An effective method was developed to monitor genome rearrangement in bacteria. The whole procedure consists of five steps. (i) Genomic DNAs of reference cells and test cells are digested with the same restriction enzyme. (ii) The DNA restriction fragments from the test cells are radioactively labeled. (iii) The labeled DNA fragments of test cells are mixed with unlabeled DNA fragments from reference cells that are 100- to 1000-fold in excess and the mixture is electrophoresed in an agarose gel. (iv) After electrophoresis, DNA fragments are alkali-denatured; this is followed by renaturation in situ in the gel. The labeled rearranged DNA fragments from the test cells will renature much slower, as compared with the nonrearranged fragments, since in this location of the gel these rearranged fragments do not have a counterpart in the driver DNA, which is in excess. (v) The DNA gel is electrophoresed in a second dimension perpendicular to the first dimension after renaturation. The denatured rearranged DNAs are revealed after autoradiography, since single-stranded DNA fragments have higher electrophoretic mobility than double-stranded fragments of the same sizes. This process of detection has been demonstrated in this reoprt by using Escherichia coli HB101 as the reference strain and E. coli HB101 carrying .lambda. phage DNA (1:1 genomic ratio) as the test strain.