Mapping by insertion mutagenesis without cloning.

Abstract

This paper describes a new strategy for positioning specific loci on known genomic maps or for generating high-resolution physical maps of organisms that are susceptible to transposable elements. The strategy does not require cloning and thus saves time and effort. It is based on isolating cell lines containing appropriate insertions of a DNA element (transposon) carrying a selectable marker and one or more restriction sites. DNA from independent cell lines is digested to completion with a restriction enzyme that cuts within the transposon and the adjacent genomic DNA. The fragments thus produced are analyzed by partial digestions with a panel of restriction enzymes, separated and probed sequentially with oligonucleotides complementary to the ends of the transposon. Algorithms that compare and order the different restriction fingerprints are used to either place the unknown locus on an existing restriction map or, in the case of a new genome, to form contigs to generate a map. The usefulness of this strategy was demonstrated by mapping an Escherichia coli insertion mutation that was difficult to map by more standard procedures.