PCR-Mediated Direct Gene Disruption in Schizosaccharomyces Pombe

Kaur, R. ; Ingavale, S. S. ; Bachhawat, A. K. (1997) PCR-Mediated Direct Gene Disruption in Schizosaccharomyces Pombe Nucleic Acids Research, 25 (5). pp. 1080-1081. ISSN 0305-1048

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Official URL: http://doi.org/10.1093/nar/25.5.1080

Related URL: http://dx.doi.org/10.1093/nar/25.5.1080


We have examined the feasibility and efficiency of PCR-mediated direct gene disruptions in the fission yeast Schizosaccharomyces pombe . In the present study, the S.pombe ura4+ gene was amplified by PCR with oligonucleotides that had short flanking regions (∼40 bp) to the target gene. Using this purified PCR product we were able to disrupt genes in an S.pombe strain bearing a ura4 deletion, with an efficiency ranging between 1 and 3% among selected transformants. The results indicated that despite S.pombe's preference for non-homologous or illegitimate recombination, even very short stretches of homologous regions could be used to target genes at a defined frequency in this organism. The successful disruption of four independent genes ( sts1+ , gcs1+ , gsh2+ and hmt1+ ) by this method further demonstrates that, despite the relatively low efficiency, the method is very feasible, and it's simplicity, especially when coupled to phenotype-based screening, should greatly facilitate disruption of genes in S.pombe.

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