Further strategies for signature-tagged mutagenesis and the application of oligonucleotide microarrays for the quantitation of DNA-tagged strains.

The original signature-tagged mutagenesis (STM) technique has certain technical limitations, including high variation in signal intensities after hybridization, owing to the variability of the tag sequences, necessitating further rounds of screening. This chapter describes the basic STM approach to identify potential virulence determinants in Yersinia pseudotuberculosis and to monitor the survival rate of Helicobacter pylori mutants under different environmental conditions. In principle, this technology could be more widely applied to measure the relative abundance of tagged microbial strains in any complex environment. This chapter reasoned that the detection of DNA tagged strains could be improved by using (1) the DNA sequence tags designed to have similar hybridization properties, (2) two tags for each mutant, and (3) sequences corresponding to both strands of the tags arrayed on an affymetrix “bar coding” microarray, containing immobilized oligonucleotide tag sequences.