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High throughputin vivoscreens: Signature-tagged mutagenesis

Elsevier Ltd
DOI: 10.1016/s0580-9517(02)33010-1
  • Comparative Nucleic Acid Analysis
  • Biology


Publisher Summary This chapter discusses the approaches for using tagging mutagenesis and some of the technical refinements that have been developed. The mutants are tagged with a unique identifying DNA sequence, allowing the analysis of multiple mutants in parallel, opening the way for high-throughput mutational analysis in vivo. Thus, signature-tagged mutagenesis (STM) is as applicable to the problems of functional genomics as the search for pathogenicity determinants. Furthermore, STM has distinct advantages over other methods; especially those based on transcript analysis, as it involves the generation of knockout strains, allowing a direct attribution of genotype to phenotype. The fundamental methodology of STM, following the production of the mutant library, is separated into two phases; an “input” and an “output” phase. The two phases of the experiment are essentially identical except that, prior to the output phase, a round of selection is incorporated. The presence of mutants is detected at each stage by extracting genomic DNA from the pool of mutants and amplifying the tags by polymerase chain reaction (PCR) with primers based on the regions flanking the tag. The flanking regions are removed from the resultant product by endonuclease digestion and the labelled tag is hybridized to a dot blot comprised of individual tags from each mutant.

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