Site-directed, Ligase-Independent Mutagenesis (SLIM): a single-tube methodology approaching 100% efficiency in 4 h
Site-directed, Ligase-Independent Mutagenesis (SLIM): a single-tube methodology approaching 100% efficiency in 4 h
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DOI：
10.1093/nar/gnh172
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发表时间：
2004-01-01
影响因子：
14.9
通讯作者：
Tillett, D
中科院分区：
文献类型：
--
作者：
Chiu, J;March, PE;Tillett, D
定点连接酶非依赖性突变（SLIM）是一种新型的PCR介导的突变方法，可以适应所有三种序列修饰类型（插入，缺失和取代）。该方法利用在单个反应中通过两个加尾长引物和两个短引物对模板进行反向PCR扩增，所有步骤在一个试管中进行。设计加尾引物以在PCR产物末端的互补突出端上含有所需突变。在扩增后变性和再退火时，混合PCR产物之间的异源双链体形成产生所需的可克隆突变质粒。该技术是高度稳健的，适用于高通量基因工程和文库构建的应用。在这项研究中，SLIM被用来创建噬菌体T7基因5内的序列插入、缺失和替换。获得所需产品的总效率> 95%。
Site-directed, Ligase-Independent Mutagenesis (SLIM) is a novel PCR-mediated mutagenesis approach that can accommodate all three sequence modification types (insertion, deletion and substitution). The method utilizes an inverse PCR amplification of the template by two tailed long primers and two short primers in a single reaction with all steps carried out in one tube. The tailed primers are designed to contain the desired mutation on complementary overhangs at the terminus of PCR products. Upon post-amplification denaturation and re-annealing, heteroduplex formation between the mixed PCR products creates the desired clonable mutated plasmid. The technique is highly robust and suitable for applications in high-throughput gene engineering and library constructions. In this study, SLIM was employed to create sequence insertions, deletion and substitution within bacteriophage T7 gene 5. The overall efficiency for obtaining the desired product was >95%.