PCR Screening in Signature-Tagged Mutagenesis of Essential Genes

Signature tagged-mutagenesis (STM) is a functional genomics technique that identifies microbial genes required for infection within an animal host, or within host cell (1 ,2 ). As first described by Hensel et al., 1995 (3 ), transposon mutants are generated and each one tagged with a unique DNA sequence. Originally, STM used comparative hybridization to isolate mutants unable to survive in specified environmental conditions and to identify genes critical for survival in the host (3 ). The original STM has been modified to use defined oligonucleotides for tag construction into mini-Tn5 and to use polymerase chain reaction (PCR) instead of hybridization for rapid screening of bacterial mutants in vivo (4 ). The modified STM technique has been called PCR-based signature-tagged mutagenesis (PBSTM).