PCR-mediated direct gene disruption in Schizosaccharomyces pombe
PCR-mediated direct gene disruption in Schizosaccharomyces pombe
复制标题
DOI：
10.1093/nar/25.5.1080
复制
发表时间：
1997-03-01
影响因子：
14.9
通讯作者：
Bachhawat, AK
中科院分区：
文献类型：
--
作者：
Kaur, R;Ingavale, SS;Bachhawat, AK
本研究以裂殖酵母裂殖酵母（Schizosophyomycespombe）为研究对象，采用PCR技术，以裂殖酵母ura4（+）基因为模板，设计了一个短侧翼区的寡核苷酸引物，对裂殖酵母ura4（+）基因进行了PCR扩增使用该纯化的PCR产物，我们能够破坏携带ura4缺失的粟酒裂殖酵母菌株中的基因，结果表明，尽管粟酒裂殖酵母偏好非同源或非法重组，但即使是非常短的同源区片段也可以用于在该生物体中以确定的频率靶向基因。通过该方法成功地破坏了4个独立的基因（sts1（+）、gcs1（+）、gsh2（+）和hmt1（+）），进一步证明了尽管效率相对较低，但该方法是非常可行的，并且它的简单性，特别是当与基于表型的筛选相结合时，将极大地促进裂殖酵母中基因的破坏。
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 (similar to 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.