PCR-synthesis of marker cassettes with long flanking homology regions for gene disruptions in S. cerevisiae.
A PCR-method for fast production of disruption cassettes is introduced, that allows the addition of long flanking homology regions of several hundred base pairs (LFH-PCR) to a marker module. Such a disruption cassette was made by linking two PCR fragments produced from genomic DNA to kanMX6, a modification of dominant resistance marker making S. cerevisiae resistant to geneticin (G418). In a first step, two several hundred base pairs long DNA fragments from the 5'- and 3'- region of a S. cerevisiae gene were amplified in such a way that 26 base pairs extensions homologous to the kanMX6 marker were added to one of their end. In a second step, one strand of each of these molecules then served as a long primer in a PCR using kanMX6 as template. When such a LFH-PCR-generated disruption cassette was used instead of a PCR-made disruption cassette flanked by short homology regions, transformation efficiencies were increased by at least a factor of thirty. This modification will therefore also help to apply PCR-mediated gene manipulations to strains with decreased transformability and/or unpredictable sequence deviations.
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Institut fur Angewandte Mikrobiologie, Biozentrum, Universitat Basel, Switzerland.
D001483:Base Sequence; D005819:Genetic Markers; D016681:Genome, Fungal; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D014170:Transformation, Genetic
D005819:Genetic Markers