Efficient PCR-based gene disruption in Saccharomyces strains using intergenic primers

Gene disruptions are a vital tool for understanding Saccharomyces cerevisiae gene function. An arrayed, library of gene disruption strains has been produced by a consortium of yeast laboratories, however their use is limited to a single genetic background. Since the yeast research community works with several different strain backgrounds, disruption libraries in other common laboratory strains are desirable. We have developed simple PCR-based methods that allow transfer of gene disruptions from the S288C-derived strain library into any Saccharomyces strain. One method transfers the unique sequence tags that flank each of the disrupted genes and replaces the kanamycin resistance marker with a recyclable URA3 gene from Kluyveromyces lactis. All gene-specific PCR amplifications for this method are performed using a pre-existing set of primers that are commercially available. We have also,extended this PCR technique to develop a second general gene disruption method suitable for any transformable strain of Saccharomyces. Copyright (C) 2002 John Wiley Sons, Ltd.