期刊
BMC BIOTECHNOLOGY
卷 15, 期 -, 页码 -出版社
BMC
DOI: 10.1186/s12896-015-0162-8
关键词
Homologous recombination; Seamless DNA cloning; SLiCE; Site-directed mutagenesis; Plant redox-related gene
资金
- JSPS KAKENHI [25650037]
- Science Research Promotion Fund from the Promotion and Mutual Aid Corporation for Private Schools of Japan
- Grants-in-Aid for Scientific Research [25650037] Funding Source: KAKEN
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 lambda 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 Escherichia 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 Escherichia 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.
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