Mutational specificity analysis: Assay for mutations in the yeast SUP4 -o gene

Abstract

Mutational specificity analysis can yield valuable insights into processes that generate genetic change or maintain genetic stability. Powerful diagnostic tools for such analysis have been created by combining genetic assays for mutation with DNA sequencing. Here, steps for isolating spontaneous mutations in the yeast (Saccharomyces cerevisiae) suppressor tRNA gene SUP4 -o as a prelude to sequence characterization are described (modifications of this protocol can be used to study induction of mutations by various physical or chemical agents). Mutations in SUP4 -o are selected on drug-containing medium by virtue of their inactivation of suppressor activity. The small size, detailed knowledge of detectably mutable sites, and other features of the target gene facilitate subsequent analysis of these mutations.