Primer extension mutagenesis powered by selective rolling circle amplification.
Primer extension mutagenesis powered by selective rolling circle amplification.
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DOI：
10.1371/journal.pone.0031817
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Lamminmäki U
中科院分区：
文献类型：
--
作者：
Huovinen T;Brockmann EC;Akter S;Perez-Gamarra S;Ylä-Pelto J;Liu Y;Lamminmäki U
引物延伸诱变是一种用于体外进化实验的文库构建的常用工具。在这里，我们描述了由T.A. Kunkel使用含尿嘧啶的单链DNA作为模板，通过额外的尿嘧啶-DNA糖基化酶处理和滚环扩增（RCA）步骤进行引物延伸。结果表明，从模板中去除尿嘧啶碱基导致通过phi 29 DNA聚合酶选择性扩增携带所需突变的nascently合成的环状DNA链。在Kunkel诱变中形成的DNA异源双链体的选择性RCA（sRCA）将诱变效率从50%提高到接近100%，并且转化体的数量增加了300倍，而没有显著的多样性偏差。我们还观察到，突变和野生型DNA都存在于至少三分之一的直接转化Kunkel异源双链体的细胞中。相反，用sRCA产物转化的细胞仅含有突变的DNA。在sRCA中，突变链的复杂的基于细胞的选择被更可控的基于酶的选择所取代，并且文库创建所需的DNA更少。用240纳克DNA作为起始材料，用所述方法证明了100亿个成员的基因文库的构建。
Primer extension mutagenesis is a popular tool to create libraries for in vitro evolution experiments. Here we describe a further improvement of the method described by T.A. Kunkel using uracil-containing single-stranded DNA as the template for the primer extension by additional uracil-DNA glycosylase treatment and rolling circle amplification (RCA) steps. It is shown that removal of uracil bases from the template leads to selective amplification of the nascently synthesized circular DNA strand carrying the desired mutations by phi29 DNA polymerase. Selective RCA (sRCA) of the DNA heteroduplex formed in Kunkel's mutagenesis increases the mutagenesis efficiency from 50% close to 100% and the number of transformants 300-fold without notable diversity bias. We also observed that both the mutated and the wild-type DNA were present in at least one third of the cells transformed directly with Kunkel's heteroduplex. In contrast, the cells transformed with sRCA product contained only mutated DNA. In sRCA, the complex cell-based selection for the mutant strand is replaced with the more controllable enzyme-based selection and less DNA is needed for library creation. Construction of a gene library of ten billion members is demonstrated with the described method with 240 nanograms of DNA as starting material.
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