An easy-to-use site-directed mutagenesis method with a designed restriction site for convenient and reliable mutant screening
An easy-to-use site-directed mutagenesis method with a designed restriction site for convenient and reliable mutant screening
复制标题
一种易于使用的定点诱变方法，具有设计的限制性位点，可方便可靠地进行突变体筛选
DOI：
10.1631/jzus.b0820367
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发表时间：
2009-06-01
影响因子：
5.1
通讯作者：
Tong, Yi-gang
中科院分区：
文献类型：
--
作者：
Zhang, Bao-zhong;Zhang, Xin;Tong, Yi-gang
定点突变(SDM)是研究蛋白质功能-结构关系的重要方法。在这项研究中，我们介绍了一种简单易用的、基于聚合酶链式反应(PCR)的双链质粒DNA SDM方法，并设计了一个限制性内切酶切点，以确保简单高效地筛选突变。首先将待突变的DNA序列翻译成氨基酸序列，然后将氨基酸序列反向翻译成含有简并密码子的DNA序列，从而产生大量含有各种限制性内切酶(RE)位点的沉默突变序列。选择具有合适RE位点的突变序列作为靶DNA序列，设计一对突变引物，通过反向聚合酶链式反应扩增全长质粒。扩增产物经5‘-磷酸化、环化后转化到大肠杆菌宿主中。用设计的RE对转化子进行消化筛选。该方法只使用一对引物，只进行一次聚合酶链式反应，不需要与危险的同位素杂交进行突变筛选或亚克隆步骤。
Site-directed mutagenesis (SDM) has been a very important method to probe the function-structure relationship of proteins. In this study, we introduced an easy-to-use, polymerase chain reaction (PCR)-based SDM method for double-stranded plasmid DNA, with a designed restriction site to ensure simple and efficient mutant screening. The DNA sequence to be mutated was first translated into amino acid sequence and then the amino acid sequence was reversely translated into DNA sequence with degenerate codons, resulting in a large number of sequences with silent mutations, which contained various restriction endonuclease (RE) sites. Certain mutated sequence with an appropriate RE site was selected as the target DNA sequence for designing a pair of mutation primers to amplify the full-length plasmid via inverse PCR. The amplified product was 5'-phosphorylated, circularized, and transformed into an Escherichia coli host. The transformants were screened by digesting with the designed RE. This protocol uses only one pair of primers and only one PCR is conducted, without the need for hybridization with hazardous isotope for mutant screening or subcloning step.