Seamless Ligation Cloning Extract (SLiCE) Method Using Cell Lysates from Laboratory Escherichia coli Strains and its Application to SLiP Site-Directed Mutagenesis
Seamless Ligation Cloning Extract (SLiCE) Method Using Cell Lysates from Laboratory Escherichia coli Strains and its Application to SLiP Site-Directed Mutagenesis
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DOI：
10.1007/978-1-4939-6472-7_23
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发表时间：
2017-01-01
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影响因子：
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通讯作者：
Motohashi, Ken
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作者：
Motohashi, Ken
来自实验室大肠杆菌菌株的细胞裂解物内源性地表现出同源重组活性，其可用于体外无缝DNA克隆。这种方法，称为无缝连接克隆提取物（SLiCE）克隆，能够同时整合两个未纯化的DNA片段到载体中的高克隆效率。此外，SLiCE方法具有很高的成本效益，因为几个实验室E。大肠杆菌菌株可用作SLiCE的来源。以前，SLiCE技术已被应用于定点诱变，以开发一种新的技术，称为SLiCE介导的聚合酶链反应（PCR）为基础的定点诱变（SLiP定点诱变）。含有突变位点的两个DNA片段可以同时整合到载体中，同时避免在载体中引入不期望的突变。因此，SLiP定点诱变简化了基于PCR的定点诱变中涉及的多个程序，例如重叠延伸方法PCR或Megaprimer方法。
Cell lysates from laboratory Escherichia coli strains endogenously exhibit homologous recombination activity, which can be utilized for seamless DNA cloning in vitro. This method, termed Seamless Ligation Cloning Extract (SLiCE) cloning, enables high cloning efficiency with simultaneous integration of two unpurified DNA fragments into a vector. In addition, the SLiCE method is highly cost-effective, as several laboratory E. coli strains may be utilized as sources of SLiCE. Previously, the SLiCE technique has been applied to site-directed mutagenesis to develop a novel technique termed SLiCE-mediated polymerase chain reaction (PCR)-based site-directed mutagenesis (SLiP site-directed mutagenesis). Two DNA fragments containing a mutation site can be simultaneously integrated into a vector while avoiding the introduction of undesirable mutations in the vector. Therefore, SLiP site-directed mutagenesis simplifies multiple procedures involved in PCR-based site-directed mutagenesis such as overlap extension method PCR or the Megaprimer method.