Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland.
PLoS One. 2012;7(2):e31817. doi: 10.1371/journal.pone.0031817. Epub 2012 Feb 15.
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.
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 T.A. Kunkel 描述的方法的进一步改进，该方法使用含有尿嘧啶的单链 DNA 作为引物延伸的模板，通过额外的尿嘧啶-DNA 糖基化酶处理和滚环扩增（RCA）步骤。结果表明，模板中尿嘧啶碱基的去除导致带有所需突变的新合成的环状 DNA 链被 phi29 DNA 聚合酶选择性扩增。Kunkel 诱变中形成的 DNA 异源双链体的选择性 RCA（sRCA）将突变效率从接近 50%提高到接近 100%，转化子数量提高了 300 倍，而没有明显的多样性偏差。我们还观察到，在用 Kunkel 的异源双链体直接转化的细胞中，至少有三分之一的细胞中存在突变型和野生型 DNA。相比之下，用 sRCA 产物转化的细胞仅含有突变 DNA。在 sRCA 中，对突变链的复杂基于细胞的选择被更可控的基于酶的选择所取代，并且用于文库构建所需的 DNA 更少。使用描述的方法，从 240 纳克 DNA 起始材料构建了十亿个成员的基因文库。
