Hanson-Manful Paulina, Patrick Wayne M
Institute of Natural Sciences, Massey University, Auckland, New Zealand.
Methods Mol Biol. 2013;996:251-67. doi: 10.1007/978-1-62703-354-1_15.
In contrast to site-directed mutagenesis and rational design, directed evolution harnesses Darwinian principles to identify proteins with new or improved properties. The critical first steps in a directed evolution experiment are as follows: (a) to introduce random diversity into the gene of interest and (b) to capture that diversity by cloning the resulting population of molecules into a suitable expression vector, en bloc. Error-prone PCR (epPCR) is a common method for introducing random mutations into a gene. In this chapter, we describe detailed protocols for epPCR and for the construction of large, maximally diverse libraries of cloned variants. We also describe the utility of an online program, PEDEL-AA, for analyzing the compositions of epPCR libraries. The methods described here were used to construct several libraries in our laboratory. A side-by-side comparison of the results is used to show that, ultimately, epPCR is a highly stochastic process.
与定点诱变和理性设计不同，定向进化利用达尔文原理来鉴定具有新特性或改进特性的蛋白质。定向进化实验的关键第一步如下：(a) 向目标基因中引入随机多样性，以及 (b) 通过将所得分子群体整体克隆到合适的表达载体中来捕获这种多样性。易错PCR（epPCR）是一种向基因中引入随机突变的常用方法。在本章中，我们描述了epPCR以及构建大型、最大程度多样化的克隆变体文库的详细方案。我们还描述了一个在线程序PEDEL-AA在分析epPCR文库组成方面的用途。这里描述的方法已在我们实验室用于构建多个文库。通过对结果的并列比较来表明，最终，epPCR是一个高度随机的过程。
