A simple and efficient seamless DNA cloning method using SLiCE from Escherichia coli laboratory strains and its application to SLiP site-directed mutagenesis.
A simple and efficient seamless DNA cloning method using SLiCE from Escherichia coli laboratory strains and its application to SLiP site-directed mutagenesis.
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DOI：
10.1186/s12896-015-0162-8
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发表时间：
2015-06-03
影响因子：
3.5
通讯作者：
Motohashi K
中科院分区：
文献类型：
--
作者：
Motohashi K
无缝连接克隆提取物（Seamless ligation cloning extract，SLiCE）是一种简单有效的DNA组装方法，它使用大肠杆菌PPY菌株的细胞提取物，表达λ原噬菌体Red/ET重组系统的组分。该方法通过在同源DNA的短片段（≥15个碱基对）之间进行重组，促进了限制性内切酶切割位点游离DNA克隆。为了扩展该系统的通用性，我检查了除了PPY菌株的细菌提取物外，是否还可以使用其他E。大肠杆菌实验室菌株适用于SLiCE方案。事实上，从几种菌株中仔细制备的细胞提取物显示出足够的克隆活性，用于将基因无缝掺入具有短同源长度（约15-20 bp）的载体中。此外，将SLiCE应用于基于聚合酶链反应（PCR）的定点诱变方法，在称为“SLiCE介导的基于PCR的定点诱变（SLiP定点诱变）"的过程中。SLiP定点诱变简化了基于PCR的定点诱变的步骤，因为它利用了SLiCE方法插入多个片段的能力。SLiCE可以在实验室中进行，不需要特殊的E。大肠杆菌菌株，并且该技术易于建立。该方法提高了克隆效率，缩短了DNA操作的时间，并大大降低了无缝DNA克隆的成本。本文的在线版本（doi：10.1186/s12896-015-0162-8）包含补充材料，可供授权用户使用。
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). 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. 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. The online version of this article (doi:10.1186/s12896-015-0162-8) contains supplementary material, which is available to authorized users.
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影响因子：
4.8
作者：
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