Microbial Gene Essentiality: Protocols and Bioinformatics

Volume 416 of the series Methods in Molecular Biology™ pp 205-220

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

  • Angela M. ChuAffiliated withDepartment of Biochemistry, Stanford University School of Medicine
  • , Ronald W. DavisAffiliated withDepartments of Biochemistry and Genetics, Stanford University School of MedicineStanford Genome Technology Center

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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.

Key Words

gene disruption homologous recombination sequence tags systematic disruption yeast deletion yeast knockout