Mapping Transposon Insertion Sites by Inverse Polymerase Chain Reaction and Sanger Sequencing.

Abstract

Inverse polymerase chain reaction (PCR) is a method designed to amplify a segment of DNA for which only a portion of the sequence is known. The method consists of circularizing the DNA fragment by self-ligation and performing PCR with primers annealing inside the known sequence but pointing away from each other (hence the technique is also called "inside-out PCR"). Here we describe how inverse PCR can be used to identify the site of transposon insertion in the bacterial chromosome. This protocol, implemented here with a class of transposons generating reporter gene fusions, involves (i) preparing genomic DNA from the strain harboring the unknown insertion, (ii) cleaving the genomic DNA with a restriction enzyme, (iii) performing a ligation reaction under conditions favoring circularization of the DNA fragments, and (iv) performing inverse PCRs with inside-out primers annealing near either or both termini of the transposon. This last step results in the amplification of the chromosomal sequences immediately adjacent to the transposon, which can then be identified by Sanger sequencing. The protocol can be performed in parallel on several strains providing an effective and economic way for rapidly identifying multiple transposon insertion sites.