Restriction site-free insertion of PCR products directionally into vectors
Restriction site-free insertion of PCR products directionally into vectors
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DOI：
10.2144/00283st08
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发表时间：
2000-03-01
期刊：
影响因子：
2.7
通讯作者：
Page, MGP
中科院分区：
文献类型：
--
作者：
Chen, GJ;Qiu, N;Page, MGP
已经开发了一种无限制性位点克隆方法，用于将PCR产物以高效率灵活且精确地插入载体的任何所需位置。该方法使用一对具有两个部分的DNA整合引物。3'部分通过PCR分离插入物，3'部分将PCR产物整合到载体的同源区中。为了诱变，可以将突变产生序列的第三部分置于3'和5'部分之间。该方法已用于克隆E.大肠杆菌编码肽基-tRNA水解酶的基因，将其表达为天然蛋白和谷胱甘肽S-转移酶融合蛋白。它也被用来转换E.将具有N-末端六组氨酸标签的大肠杆菌脂肪酸生物合成蛋白转化为具有C-末端六组氨酸标签的构建体。
A restriction site-free cloning method has been developed for inserting a PCR product into a vector flexibly and precisely at any desired location with high efficiency. The method uses a pair of DNA integration primers with two portions. The 3' portion isolates the inserts by PCR, and the 3' portion integrates the PCR products into the homologous region of the vector For mutagenesis, a third portion of mutation-generating sequences can be placed in between the 3' and 5' portions. This method has been used to clone the E. coli gene that codes for peptidyl-tRNA hydrolase, expressing it as a native protein and as a glutathione S-transferase fusion protein. It was also applied to convert a construct of the E. coli fatty acid biosynthesis protein with an N-terminal hexa-histidine tag into a construct with a C-terminal hexa-histidine tag.