Seamless Ligation Cloning Extract (SLiCE) Method Using Cell Lysates from Laboratory Escherichia coli Strains and its Application to SLiP Site-Directed Mutagenesis.

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.