Carrigan Patricia E, Ballar Petek, Tuzmen Sukru
Biodesign Institute, Arizona State University, Phoenix, AZ, USA.
Methods Mol Biol. 2011;700:107-24. doi: 10.1007/978-1-61737-954-3_8.
The technique of site-directed mutagenesis has been used to characterize gene and protein structure-function relationships, protein-protein interactions, binding domains of proteins, or active sites of enzymes for the last three decades. In this technique, a nucleotide sequence of interest is experimentally altered using synthetic oligonucleotides. The most commonly used approach is to use an oligonucleotide that is complementary to part of a single-stranded DNA template, but containing an internal mismatch to direct the mutation. In addition to single point mutations, this approach may also be used to construct multiple mutations, insertions, or deletions. As a result of its broad applicability in disease gene characterization studies, numerous commercial kits are now available, making this technique quick, straightforward, and reliable. In this chapter, we detail the steps involved in site-directed mutagenesis and highlight the essentials of this versatile technique based upon our experience.
在过去三十年中，定点诱变技术一直被用于表征基因与蛋白质的结构-功能关系、蛋白质-蛋白质相互作用、蛋白质的结合结构域或酶的活性位点。在这项技术中，利用合成寡核苷酸对感兴趣的核苷酸序列进行实验性改变。最常用的方法是使用与单链DNA模板的一部分互补，但含有内部错配以指导突变的寡核苷酸。除了单点突变外，该方法还可用于构建多个突变、插入或缺失。由于其在疾病基因表征研究中的广泛适用性，现在有许多商业试剂盒可供使用，使得这项技术快速、直接且可靠。在本章中，我们详细介绍定点诱变所涉及的步骤，并根据我们的经验强调这项通用技术的要点。
