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モトハシ タケシ
MOTOHASHI TAKESHI
本橋 健 所属 京都産業大学 生命科学部 先端生命科学科 職種 教授 |
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| 言語種別 | 英語 |
| 発行・発表の年月 | 2015 |
| 形態種別 | 研究論文 |
| 査読 | 査読あり |
| 標題 | A simple and efficient seamless DNA cloning method using SLiCE from Escherichia coli laboratory strains and its application to SLiP site-directed mutagenesis |
| 執筆形態 | その他 |
| 掲載誌名 | BMC Biotechnology |
| 出版社・発行元 | BIOMED CENTRAL LTD |
| 巻・号・頁 | 15(1),pp.47 |
| 著者・共著者 | Motohashi, K. |
| 概要 | 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. |
| DOI | 10.1186/s12896-015-0162-8 |
| ISSN | 1472-6750 |
| Put Code(ORCID) | 21490296 |
| PermalinkURL | http://www.scopus.com/inward/record.url?eid=2-s2.0-84930619645&partnerID=MN8TOARS |