Sayers J R, Krekel C, Eckstein F
University College of North Wales.
Biotechniques. 1992 Oct;13(4):592-6.
Several improvements to the existing phosphorothioate-based site-directed mutagenesis methodology are reported, and here it is demonstrated that the new procedure is able to produce large deletions, insertions and point mutations rapidly and with very high efficiency. The time required for the polymerization step has been reduced by using T7 DNA polymerase to extend the mutant oligonucleotide primer-template. The reaction produces good yields of double-stranded closed-circular DNA and some partially polymerized template. The reaction was treated with T5 D15 exonuclease to selectively destroy partially polymerized single-stranded phage DNA that may otherwise contribute to an increased background of wild-type transformants. The use of these enzymes greatly facilitates the implementation of the phosphorothioate-based site-directed mutagenesis method by requiring less template DNA and by allowing all the in vitro manipulations to be completed in a day. In its present form the method may easily be automated, enabling large systematic site-directed mutagenesis projects to be undertaken.
本文报道了对现有基于硫代磷酸酯的定点诱变方法的多项改进，并且在此证明新方法能够快速且高效地产生大片段缺失、插入和点突变。通过使用T7 DNA聚合酶来延伸突变寡核苷酸引物模板，聚合步骤所需的时间得以减少。该反应能产生高产率的双链闭环DNA以及一些部分聚合的模板。用T5 D15核酸外切酶处理该反应，以选择性地破坏部分聚合的单链噬菌体DNA，否则这些DNA可能会导致野生型转化体背景增加。这些酶的使用极大地促进了基于硫代磷酸酯的定点诱变方法的实施，因为所需的模板DNA较少，并且所有体外操作都能在一天内完成。以其目前的形式，该方法可以很容易地实现自动化，从而能够开展大规模的系统性定点诱变项目。
