7.3 Signature Tagged Mutagenesis

Abstract

This chapter provides an overview of signature tagged mutagenesis (STM). STM combines the strength of mutational analysis with the ability to follow the fate of a large number of different mutants within a single animal. In its original form, a transposon was used for random insertional mutagenesis of the genome of Salmonella typhimurium , but there is no reason the technique could not be applied to other pathogens, so long as they are haploid, can undergo insertional mutagenesis, and infect their experimental host as a mixed population. In STM, each transposon contains a different DNA sequence tag, which allows mutants to be differentiated from each other. The tags comprise 40 bp variable central regions flanked by invariant “arms” of 20 bp, which allow the amplification and labeling of the central portions by the polymerase chain reaction (PCR). Mutants are assembled into 96-well microtiter dishes, which are used to prepare replica DNA colony blots. The mutants are then mixed to form the inoculum or “input” pool before inoculation into an animal. After infection is established, cells of the pathogen are isolated from the animal and pooled to form the “recovered pool.” Tags in the recovered pool and tags in the input pool are separately amplified, labeled, and used to probe DNA colony blots of the inoculum. The chapter further describes virulence gene cloning and DNA sequencing.