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首页> 美国卫生研究院文献>Journal of Zhejiang University. Science. B >An easy-to-use site-directed mutagenesis method with a designed restriction site for convenient and reliable mutant screening
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An easy-to-use site-directed mutagenesis method with a designed restriction site for convenient and reliable mutant screening

机译：一种易于使用的定点诱变方法具有设计的限制性酶切位点可方便可靠地进行突变体筛选

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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.
机译：定点诱变（SDM）已经成为探测蛋白质功能结构关系的一种非常重要的方法。在这项研究中，我们为双链质粒DNA引入了一种易于使用的，基于聚合酶链反应（PCR）的SDM方法，并设计了限制位点以确保简单有效的突变体筛选。首先将要突变的DNA序列翻译为氨基酸序列，然后将氨基酸序列反向翻译为具有简并密码子的DNA序列，从而导致大量具有沉默突变的序列，其中包含各种限制性核酸内切酶（RE）位点。选择具有适当RE位点的某些突变序列作为靶DNA序列，以设计一对突变引物，以通过反向PCR扩增全长质粒。将扩增的产物进行5'-磷酸化，环化，并转化到大肠杆菌宿主中。通过用设计的RE消化来筛选转化体。该方案仅使用一对引物，并且仅进行一次PCR，而无需与危险同位素杂交以进行突变筛选或亚克隆步骤。

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