Wong F, Komaromy M
Research Institute, Palo Alto Medical Foundation, CA 94301, USA.
Biotechniques. 1995 Jun;18(6):1034-8.
The two-primer method for site-directed mutagenesis facilitates the mutation of targets in double-stranded DNA. We have encountered difficulties using the original method for the mutagenesis of DNA cloned into pBluescript vectors, which is possibly due to the presence of regions of secondary structure that cannot be efficiently copied by the enzymes used. We report a modification of the two-primer method using the thermostable VentR DNA polymerase and Thermus aquaticus ligase, allowing an increase in reaction temperatures. The modified method is functional with the pBluescript family of vectors.
用于定点诱变的双引物方法有助于双链DNA中靶标的诱变。我们在使用原始方法对克隆到pBluescript载体中的DNA进行诱变时遇到了困难，这可能是由于存在二级结构区域，所用酶无法有效复制这些区域。我们报告了一种使用耐热的VentR DNA聚合酶和嗜热栖热菌连接酶对双引物方法的改进，从而提高了反应温度。改进后的方法适用于pBluescript载体家族。
