Gene disruption with PCR products in Saccharomyces cerevisiae

• Published in: Gene Vol. 158; no. 1; pp. 113 - 117
• Main Authors: Lorenz, Michael C., Muir, R.Scott, Lim, Eric, McElver, John, Weber, Shane C., Heitman, Joseph
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 1995
• Subjects: aua1 gene; Base Sequence; binding proteins; Carrier Proteins - genetics; Carrier Proteins - metabolism; cnb1 gene; deletions; DNA; DNA Primers; fk506 binding protein; fpr2 gene; genetic transformation; homologous recombination; Molecular Sequence Data; Mutagenesis; PCR amplification; Polymerase Chain Reaction; polymerase chain reaction targeting; proteins; Recombinant DNA; Recombination, Genetic; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Sequence Deletion; structural genes; Tacrolimus Binding Proteins; yeast genome; aa; ORF; CNB1; nt; EtdBr; Recombinant DNA; Cn; bp; CsA; FKBP; S; homologous recombination; Δ; kb; yeast genome; oligo; : CPRG; wt; PCR amplification; CMP1 and CMP2; PCR
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/0378-1119(95)00144-U

We describe here the generation of gene disruption constructs using PCR amplification of selectable markers with primers that provide homology to the target gene of interest. We find that regions of homology as short as 38 to 50 bp suffice to mediate homologous recombination in yeast. We describe applications of this technology to three specific yeast genes that would have been difficult to disrupt with current methods. By dispensing with the need to either clone the gene of interest or engineer a standard disruption construct, this method should facilitate analysis of sequenced genes of unknown function, which will soon include the entire yeast genome.