Strategy for deletion of complete open reading frames in Saccharomyces cerevisiae.
The classical disruption method for yeast genes is by using in vitro deletion of the gene of interest, or of a part of it, with restriction enzymes. We are now routinely using a strategy that takes advantage of polymerase chain reactions (PCRs) which amplify large pieces of DNA. Since this approach results in a complete, precise deletion of the open reading frame, which is replaced by a unique restriction site, the ligated PCR can be used for the insertion of different markers or for two-step gene disruptions without an inserted marker. As we have now used this strategy for the deletion of more than ten genes we have in this report included some hints based on our experience.
Eberhardt
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Laboratorium voor Moleculaire Celbiologie, Katholieke Universiteit te Leuven, Flanders, Belgium.
Hohmann
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D017353:Gene Deletion; D008957:Models, Genetic; D016366:Open Reading Frames; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014170:Transformation, Genetic