Journal Article

A simple and efficient seamless DNA cloning method using SLiCE from Escherichia coli laboratory strains and its application to SLiP site-directed mutagenesis

Motohashi, Ken

2015

Overview

Background Seamless ligation cloning extract (SLiCE) is a simple and efficient method for DNA assembly that uses cell extracts from the Escherichia coli PPY strain, which expresses the components of the λ prophage Red/ET recombination system. This method facilitates restriction endonuclease cleavage site-free DNA cloning by performing recombination between short stretches of homologous DNA (≥15 base pairs). Results To extend the versatility of this system, I examined whether, in addition to bacterial extracts from the PPY strain, other E. coli laboratory strains were suitable for the SLiCE protocol. Indeed, carefully prepared cell extracts from several strains exhibited sufficient cloning activity for seamless gene incorporation into vectors with short homology lengths (approximately 15–20 bp). Furthermore, SLiCE was applied to the polymerase chain reaction (PCR)-based site-directed mutagenesis method, in a process termed “SLiCE-mediated PCR-based site-directed mutagenesis (SLiP site-directed mutagenesis)”. SLiP site-directed mutagenesis simplifies the steps of PCR-based site-directed mutagenesis, as it exploits the capability of the SLiCE method to insert multiple fragments. Conclusions SLiCE can be performed in the laboratory with no requirement for a special E. coli strain, and the technique is easily established. This method increases the cloning efficiency, shortens the time for DNA manipulation, and greatly reduces the cost of seamless DNA cloning.

• Publisher: BioMed Central,BioMed Central Ltd
• Subject: Applied Microbiology / Arabidopsis Proteins - genetics / Biochemical Engineering / Biomedical Engineering/Biotechnology / Biotechnology / Cell Extracts - genetics / Chemistry / Chemistry and Materials Science / Cloning / Cloning, Molecular - methods / DNA / DNA assembly / Escherichia coli / Escherichia coli - genetics / Escherichia coli - metabolism / genes / Genetic aspects / Genetic Engineering / Genetic vectors / Health aspects / Homologous Recombination / Methodology / Methodology Article / Methods / molecular cloning / Mutagenesis, Site-Directed - methods / Nucleases / Plant Breeding/Biotechnology / polymerase chain reaction / Protein and enzyme technology / Recombinant Proteins - genetics / Recombinant Proteins - metabolism / restriction endonucleases / site-directed mutagenesis