PRECISE GENE DISRUPTION IN SACCHAROMYCES-CEREVISIAE BY DOUBLE FUSION POLYMERASE CHAIN-REACTION

被引：117
作者：
AMBERG, DC
BOTSTEIN, D
BEASLEY, EM
机构：
[1] Department of Genetics, Stanford University School of Medicine, Stanford, California
来源：
YEAST | 1995年 / 11卷 / 13期
关键词：
GENE DISRUPTION; FUSION PCR; SACCHAROMYCES CEREVISIAE;
D O I：
10.1002/yea.320111307
中图分类号：
Q5 [生物化学]; Q7 [分子生物学];
学科分类号：
071010 ; 081704 ;
摘要：
We adapted a fusion polymerase chain reaction (PCR) strategy to synthesize gene disruption alleles of any sequenced yeast gene of interest. The first step of the construction is to amplify sequences flanking the reading frame we want to disrupt and to amplify the selectable marker sequence. Then we fuse the upstream fragment to the marker sequence by fusion PCR, isolate this product and fuse it to the downstream sequence in a second fusion PCR reaction. The final PCR product can then be transformed directly into yeast. This method is rapid, relatively inexpensive, offers the freedom to choose from among a variety of selectable markers and allows one to construct precise disruptions of any sequenced open reading frame in Saccharomyces cerevisiae.
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页码：1275 / 1280
页数：6
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