A SIMPLE METHOD FOR SITE-DIRECTED MUTAGENESIS USING THE POLYMERASE CHAIN-REACTION
A SIMPLE METHOD FOR SITE-DIRECTED MUTAGENESIS USING THE POLYMERASE CHAIN-REACTION
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DOI：
10.1093/nar/17.16.6545
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发表时间：
1989-08-25
影响因子：
14.9
通讯作者：
GALAS, DJ
中科院分区：
文献类型：
--
作者：
HEMSLEY, A;ARNHEIM, N;GALAS, DJ
我们开发了一种通用且简单的方法，通过聚合酶链反应 (PCR) 进行引物扩增来指导质粒中的特定序列变化。该方法基于使用包含所需变化的引物扩增整个质粒。该方法快速、执行简单，并且仅需要微量的质粒模板DNA。重要的是，对于要操作的序列中适当放置的限制性位点没有特殊要求。在我们的系统中，转化体的产量很高，并且携带仅具有所需变化的质粒的比例始终约为 80%。该方法的通用性使其可用于直接改变大多数克隆基因。唯一的限制可能是要操作的质粒的总长度。在研究过程中，我们发现用于 PCR 的 Taq DNA 聚合酶在大部分新合成链的末端添加了一个额外碱基（通常是 A）。这些必须被 DNA 聚合酶的 Klenow 片段去除，以确保基因序列的恢复。
We have developed a general and simple method for directing specific sequence changes in a plasmid using primed amplification by the polymerase chain reactin (PCR). The method is based on the amplification of the entire plasmid using primers that include the desired changes. The method is rapid, simple in its execution, and requires only minute amounts of plasmid template DNA. It is significant that there are no special requirements for appropriately placed restriction sites in the sequence to be manipulated. In our system the yield of transformants was high and the fraction of them harboring plasmids with only the desired change was consistently about 80%. The generality of the method should make it useful for the direct alteration of most cloned genes. The only limitation may be the total length of the plasmid to be manipulated. During the study we found that the Taq DNA polymerase used for PCR adds on a single extra base (usually an A) at the end of a large fraction of the newly synthesized chains. These had to be removed by the Klenow fragment of DNA polymerase to insure restoration of the gene sequence.