Amberg D C, Botstein D, Beasley E M
Department of Genetics, Stanford University School of Medicine, CA 94305-5120, USA.
Yeast. 1995 Oct;11(13):1275-80. doi: 10.1002/yea.320111307.
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.
我们采用了一种融合聚合酶链反应（PCR）策略来合成感兴趣的任何已测序酵母基因的基因破坏等位基因。构建的第一步是扩增我们想要破坏的阅读框两侧的序列，并扩增选择标记序列。然后通过融合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产物随后可直接转化到酵母中。该方法快速、相对便宜，提供了从多种选择标记中进行选择的自由，并允许构建酿酒酵母中任何已测序开放阅读框的精确破坏。
