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.
Reid
Robert J D
RJ
Department of Genetics and Development, Columbia University College of Physicians and Surgeons, New York, NY 10032-2704, USA.
Sunjevaric
Ivana
I
Keddache
Mehdi
M
Rothstein
Rodney
R
Kedacche
Mehdi
M
D017931:DNA Primers; D017353:Gene Deletion; D007716:Kluyveromyces / Q000235:genetics; D008967:Molecular Biology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014170:Transformation, Genetic
D017931:DNA Primers