High-Throughput Creation of a Whole-Genome Collection of Yeast Knockout Strains

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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.