Use of exonuclease for rapid polymerase-chain-reaction-based in vitro mutagenesis.
Use of exonuclease for rapid polymerase-chain-reaction-based in vitro mutagenesis.
复制标题
使用核酸外切酶进行基于快速聚合酶链反应的体外诱变。
DOI：
10.1016/0378-1119(91)90002-s
复制
发表时间：
1991
期刊：
影响因子：
3.5
通讯作者：
Ames,GF
中科院分区：
文献类型：
--
作者：
Shyamala,V;Ames,GF
我们描述了一种改善位点特异性诱变的简单策略。我们将聚合酶链式反应 (PCR) 与噬菌体 λ 核酸外切酶 (Exoλ) 处理相结合，产生大于 2.5 kb 的突变片段。该方法的适用性已通过两个重叠突变引物得到证明。通过使用单一突变引物，该程序也变得更具成本效益，这被称为 SMP-PCR 程序。激酶引物、PCR 扩增、Exoλ 消化和 PCR 第二步的整个过程可以在不到 6 小时内完成。我们已经使用这种方法在鼠伤寒沙门氏菌组氨酸转运操纵子的hisP基因中产生了许多突变。
We describe a simple strategy for improving site-specific mutagenesis. We have combined the polymerase chain reaction (PCR) with a phage λ exonuclease (Exoλ) treatment to produce mutated fragments larger than 2.5 kb. The applicability of this approach has been proven with two overlapping mutated primers. The procedure has also been made more cost-effective by the use of a single mutated primer, which is referred to as SMP-PCR procedure. The entire procedure of kinasing the primer, amplification by PCR, Exoλ digestion and second step of PCR can be performed in less than 6 h. We have used this approach to generate a number of mutations in theSalmonella typhimurium hisPgene of the histidine transport operon.