Protein engineering: single or multiple site-directed mutagenesis.
Protein engineering: single or multiple site-directed mutagenesis.
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DOI：
10.1007/978-1-62703-293-3_13
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发表时间：
2013-01-01
期刊：
影响因子：
--
通讯作者：
Vaisvila, Romualdas
中科院分区：
文献类型：
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作者：
Hsieh, Pei-Chung;Vaisvila, Romualdas
定点诱变技术是分子生物学中研究蛋白质结构和功能特性的宝贵工具。为了加快所需的时间和简化诱变方法，我们在本章中推荐两种方案。第一种方法是单位点定向突变，包括点突变、插入或缺失，可以通过反向PCR策略与诱变引物和高保真Phusion() DNA聚合酶来实现，以极高的效率引入位点定向突变。第二种方法是将多个突变设计成感兴趣的基因。这可以一步完成PCR与诱变引物和组装所有诱变PCR产品使用Gibson组装主混合。该方法允许同时改变多个核苷酸，与传统的诱变方法相比，不仅节省了时间，而且节省了试剂。
Site-directed mutagenesis techniques are invaluable tools in molecular biology to study the structural and functional properties of a protein. To expedite the time required and simplify methods for mutagenesis, we recommend two protocols in this chapter. The first method for single site-directed mutagenesis, which includes point mutations, insertions, or deletions, can be achieved by an inverse PCR strategy with mutagenic primers and the high-fidelity Phusion() DNA Polymerase to introduce a site-directed mutation with exceptional efficiency. The second method is for engineering multiple mutations into a gene of interest. This can be completed in one step by PCR with mutagenic primers and by assembling all mutagenized PCR products using the Gibson Assembly Master Mix. This method allows multiple nucleotides to be changed simultaneously, which not only saves time but also reagents compared to traditional methods of mutagenesis.