Single-primer PCR procedure for rapid identification of transposon insertion sites.

Transposons have been used in many laboratories worldwide as a powerful tool for insertional mutagenesis to investigate gene function in bacteria (8). The technique has recently been adapted in which a transposon, such as mini-Tn5, delivers a unique identifying sequence of 40 nucleotides (1). This technique, termed signature-tagged mutagenesis (STM), is widely used in the search for virulence-associated genes via negative selection and allows the large-scale analysis of pooled mutants. Different procedures have been developed for identification of the transposon insertion regions. Single-primer PCR (4) requires restriction endonuclease digestion, ligation, PCR and cloning steps. It also depends on the availability of appropriate restriction sites. The rapid amplification of transposon ends (RATE) technique (6) involves several steps including ligation of DNA fragments to linkers, singlestrand amplification using biotin-labeled primers, isolation of the amplified products and another PCR before the sequencing. A single-primer PCR technique was originally developed by Parks et al. (5) and modified by Spector et al. (7). However, both techniques required intermediate cloning and selection steps before sequencing. Here, we report a technique that does not require cloning and allows rapid identification of transposon insertion sites using a single PCR primer and a uniquely designed PCR procedure. To test this method, we used mini-Tn5 insertion mutants from a recently constructed Yersinia pseudotuberculosis STM library (3). Figure 1 outlines the strategy for sequencing the transposon insertion regions. The procedure involves just one transposon-specific primer and a single PCR consisting of three rounds of amplification. The first round is designed for linear amplification of single-stranded, transposon-specific templates from the transposon that are to be used in the following steps and is carried out at a standard annealing temperature (50°C). The second round involves a low annealing temperature (30°C) for amplification of the region adjacent to the transposon insertion site. Amplification of this region is possible because, at low annealing temperatures, the primer will bind to sites of limited sequence comBenchmarks