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PCR‐synthesis of marker cassettes with long flanking homology regions for gene disruptions in S. cerevisiae
Author(s) -
Wach Achim
Publication year - 1996
Publication title -
yeast
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 102
eISSN - 1097-0061
pISSN - 0749-503X
DOI - 10.1002/(sici)1097-0061(19960315)12:3<259::aid-yea901>3.0.co;2-c
Subject(s) - biology , homology (biology) , genetics , gene , primer (cosmetics) , base pair , polymerase chain reaction , dna , genomic dna , homologous recombination , microbiology and biotechnology , computational biology , chemistry , organic chemistry
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.