A rapid method for localized mutagenesis of yeast genes.
We have developed a simple procedure for the localized mutagenesis of yeast genes. In this technique the region of interest is first amplified under mutagenic polymerase chain reaction (PCR) conditions. Cotransformation of the PCR product with a gapped plasmid containing homology to both ends of the PCR product allows in vivo recombination to repair the gap with the mutagenized DNA. This procedure is efficient, allows targeting of specific regions for mutagenesis, and requires no subcloning steps in Escherichia coli.
Muhlrad
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Department of Molecular and Cellular Biology, University of Arizona, Tucson 85721.
Hunter
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Parker
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D004271:DNA, Fungal / Q000235:genetics; D005800:Genes, Fungal; D000072235:Mating Factor; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014170:Transformation, Genetic
D004271:DNA, Fungal; D010455:Peptides; D000072235:Mating Factor