Seamless ligation cloning extract (SLiCE) method using cell lysates from laboratory escherichia coli strains and its application to slip site-directed mutagenesis

Abstract

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 S eamless L igation C loning E xtract (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 sitedirected 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.