A rapid and efficient PCR-based mutagenesis method applicable to cell physiology study
A rapid and efficient PCR-based mutagenesis method applicable to cell physiology study
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DOI：
10.1152/ajpcell.00517.2004
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发表时间：
2005-06-01
影响因子：
5.5
通讯作者：
Ma, JJ
中科院分区：
文献类型：
--
作者：
Ko, JK;Ma, JJ
基于PCR的诱变是分子生物学和蛋白质工程研究的基石。在此，我们描述了一种使用IIs型限制性内切酶的快速高效诱变方法。使用两对锚和诱变引物将模板基因扩增成两个单独的PCR片段。突变序列位于IIs型限制酶的识别位点附近。在用IIs型酶消化两个片段后，然后将彼此互补的暴露的粘性末端连接在一起以产生突变基因。我们应用这种方法在EGFP和Bcl-2家族基因中引入多个定点突变，并在所需位点观察到完美的诱变效率。这种高效且经济的诱变方法可应用于细胞生理学中各种各样的结构和功能研究。
PCR-based mutagenesis is a cornerstone of molecular biology and protein engineering studies. Herein we describe a rapid and highly efficient mutagenesis method using type IIs restriction enzymes. A template gene is amplified into two separate PCR fragments using two pairs of anchor and mutagenic primers. Mutated sequences are located near the recognition site of a type IIs restriction enzyme. After digestion of two fragments with a type IIs enzyme, exposed cohesive ends that are complementary to each other are then ligated together to generate a mutated gene. We applied this method to introduce multiple site-directed mutations in EGFP and Bcl-2 family genes and observed perfect mutagenesis efficiency at the desired sites. This efficient and cost-effective mutagenesis method can be applied to a wide variety of structural and functional studies in cell physiology.