PRECISE GENE DISRUPTION IN SACCHAROMYCES-CEREVISIAE BY DOUBLE FUSION POLYMERASE CHAIN-REACTION
PRECISE GENE DISRUPTION IN SACCHAROMYCES-CEREVISIAE BY DOUBLE FUSION POLYMERASE CHAIN-REACTION
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DOI：
10.1002/yea.320111307
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发表时间：
1995-10-01
期刊：
影响因子：
2.6
通讯作者：
BEASLEY, EM
中科院分区：
文献类型：
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作者：
AMBERG, DC;BOTSTEIN, D;BEASLEY, EM
我们采用融合聚合酶链反应（PCR）策略来合成任何感兴趣的测序酵母基因的基因破坏等位基因。构建的第一步是放大我们想要破坏的阅读框两侧的序列并放大可选择标记序列。然后，我们通过融合 PCR 将上游片段与标记序列融合，分离该产物并在第二次融合 PCR 反应中将其与下游序列融合。最终的 PCR 产物可直接转化至酵母中。这种方法快速、相对便宜，提供了从多种选择标记中进行选择的自由，并允许人们构建对酿酒酵母中任何测序开放阅读框的精确破坏。
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.