Creating randomized amino acid libraries with the QuikChange® Multi Site-Directed Mutagenesis Kit
Creating randomized amino acid libraries with the QuikChange® Multi Site-Directed Mutagenesis Kit
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DOI：
10.2144/02335pf01
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发表时间：
2002-11-01
期刊：
影响因子：
2.7
通讯作者：
Allen, RM
中科院分区：
文献类型：
--
作者：
Hogrefe, HH;Cline, J;Allen, RM
QuikChange（R）多位点定向诱变试剂盒是一种简单有效的方法，可在质粒DNA模板中同时引入多达5个位点的点突变。在这里，我们使用QuikChange Multi试剂盒和简并（单密码子）引物在lacZ基因的选定位点引入所有可能的氨基酸。在使用两个或三个简并引物的反应中，产生由两个或三个不同位点处的突变的随机组合组成的多样化文库（10（4）-10（5）个突变体/反应）。这种方法提供了一个为期一天的程序进行定点饱和诱变，当加上一个合适的筛选试验，应大大促进过程中的关键残基的替代氨基酸链取代和进化的蛋白质功能的评估。
The QuikChange(R) Multi Site-Directed Mutagenesis Kit is a simple and efficient method for introducing point mutations at up to five sites simultaneously in plasmid DNA templates. Here we used the QuikChange Multi kit with degenerate (one codon) primers to introduce all possible amino acids at selected sites in the lacZ gene. In reactions employing two or three degenerate primers, diverse libraries (10(4)-10(5) mutants/reaction) are created consisting of random combinations of mutations at two or three different sites. This method provides a one-day procedure for performing site-directed saturation mutagenesis and, when coupled with a suitable screening assay, should greatly facilitate the process of evaluating alternative amino acid chain substitutions at key residues and evolving protein function.