High-throughput creation of a whole-genome collection of yeast knockout strains

Abstract

Gene disruption methods have proved to be a valuable tool for studying gene function in yeast. Gene replacement with a drug-resistant cassette renders the disruption strain selectable and is stable against, reversion. Polymerase chain reaction-generated deletion cassettes are designed with homology sequences that flank the target gene. These deletion cassettes also contain unique "molecular bar code© sequence tags. Methods to generate these mutant strains are scalable and facile, allowing for the production of a collection of systematic disruptions across the Saccharomyces cerevisiae genome. The deletion strains can be studied individually or pooled together and assayed in parallel utilizing the sequence tags with microarray-based methods. © Humana Press Inc.