A RAPID AND EFFICIENT ONE-TUBE PCR-BASED MUTAGENESIS TECHNIQUE USING PFU DNA-POLYMERASE
A RAPID AND EFFICIENT ONE-TUBE PCR-BASED MUTAGENESIS TECHNIQUE USING PFU DNA-POLYMERASE
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DOI：
10.1093/nar/22.13.2587
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发表时间：
1994-07-11
影响因子：
14.9
通讯作者：
BOCK, SC
中科院分区：
文献类型：
--
作者：
PICARD, V;ERSDALBADJU, E;BOCK, SC
描述了一种在干净的序列背景上有效地产生位点定向突变的快速方法。这种对巨引物PCR诱变方法的改进可以在不到4.5小时的时间内在一管中完成，并且不需要纯化中间产物。采用Pfu DNA聚合酶，将总扩增周期限制在30个以内，获得了高保真的DNA序列复制。该程序的诱变效率足够高，可以通过限制性酶切或测序技术快速、直接地鉴定突变体。
A rapid method for efficiently generating site-directed mutations on a clean sequence background is described. This modification of the megaprimer PCR mutagenesis approach can be performed in one tube in less than 4.5 hours, and does not require purification of intermediate products. High fidelity of DNA sequence replication is obtained by employing Pfu DNA polymerase and limiting the total number of amplification cycles to 30. The mutagenesis efficiency of the procedure is high enough to allow rapid, direct identification of mutants by restriction digest or sequencing techniques.