PCR‐synthesis of marker cassettes with long flanking homology regions for gene disruptions in S. cerevisiae

• Published in: Yeast (Chichester, England) Vol. 12; no. 3; pp. 259 - 265
• Main Author: Wach, Achim
• Format: Journal Article
• Language: English
• Published: Chichester, UK John Wiley & Sons, Ltd 15.03.1996
• Subjects: Base Sequence; DNA; Functional analysis; G418 resistance; gene targeting; genes; Genetic Markers; geneticin; Genome, Fungal; Molecular Sequence Data; PCR‐based gene disruption; Polymerase Chain Reaction; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Sequence Homology, Nucleic Acid; Transformation, Genetic; Yeast genome analysis
• ISSN: 0749-503X; 1097-0061
• DOI: 10.1002/(SICI)1097-0061(19960315)12:3<259::AID-YEA901>3.0.CO;2-C

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.