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A simplified method for the repeated replacement of yeast chromosomal sequences with in vitro mutations
Author(s) -
SzentGyorgyi Christopher
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(19960615)12:7<667::aid-yea955>3.0.co;2-a
Subject(s) - biology , selectable marker , locus (genetics) , genetics , plasmid , gene , dna , mutant , polymerase chain reaction , cycloheximide , microbiology and biotechnology , cell culture
A strategy for gene replacement in Saccharomyces cerevisiae has been modified to facilitate the repeated substitution of a chromosomal locus with in vitro generated variant sequences, so that the resulting locus contains only the desired mutation and is free of extraneous vector DNA. The construction of an internally deleted chromosomal target locus carrying the counterselectable CYH2 S marker and a second positively selectable marker has been simplified; the design of the locus has been altered to increase the frequency of authentic gene replacements obtained upon the subsequent integration of in vitro mutated DNA. The modified chromosomal target locus is amenable to replacement using either of two transformation protocols: (i) integration of a second positively selectable plasmid carrying mutant sequences to form a tandem intermediate structure at the locus; upon counterselection on cycloheximide, all vector sequence is excised to give the desired replacement at high frequency (>70%); (ii) single‐step integration of a linear segment of mutated genomic DNA by selection for cycloheximide resistance. A subsequent screen for the loss of the positively selectable target locus marker detects the desired replacement at modest frequency (>2%). Polymerase chain reaction using multiple primers in a single amplification reaction is useful for monitoring these variously modified chromosomal loci.