Motohashi Ken
Department of Bioresource and Environmental Sciences, Faculty of Life Sciences, Kyoto Sangyo University, Kamigamo Motoyama, Kita-ku, Kyoto, 603-8555, Japan.
Methods Mol Biol. 2017;1498:349-357. doi: 10.1007/978-1-4939-6472-7_23.
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.
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，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，能够实现高克隆效率，同时将两个未纯化的DNA片段整合到载体中。此外，SLiCE方法具有很高的成本效益，因为几种实验室大肠杆菌菌株可用作SLiCE的来源。此前，SLiCE技术已应用于定点诱变，以开发一种称为基于SLiCE介导的聚合酶链反应（PCR）的定点诱变（SLiP定点诱变）的新技术。两个含有突变位点的DNA片段可以同时整合到载体中，同时避免在载体中引入不需要的突变。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，如重叠延伸法PCR或大引物法。
