Handa P, Varshney U
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India.
Indian J Biochem Biophys. 1998 Apr;35(2):63-6.
Oligonucleotide based site directed mutagenesis (SDM) is an invaluable technique in molecular biology. Among the various methods developed for SDM, the PCR-based approach, Kunkel's and the Eckstein's procedures are widely used. The Kunkel's method, on account of its cost effectiveness and simplicity, is preferred by many a scientist. However, a general drawback of this method is the high background due to persistence of the parent template resulting in low efficiency of mutagenesis. In this report, we describe a modification of the Kunkel's method to increase the efficiency of selecting against the wild type strand. We have used Sequenase for the extension reaction, and introduced an in vitro UDG step to enhance the biological selection against the parent strand. Consequently, the efficiency of the modified method is enhanced to allow screening of the mutants directly by DNA sequencing. A step by step single tube protocol which is over in less than three hours makes it a method of choice for efficient and cost-effective site directed mutagenesis.
基于寡核苷酸的定点诱变（SDM）是分子生物学中一项非常重要的技术。在为SDM开发的各种方法中，基于PCR的方法、昆克尔法和埃克斯坦法被广泛使用。昆克尔法因其成本效益高和操作简单，受到许多科学家的青睐。然而，该方法的一个普遍缺点是由于亲本模板的持续存在导致背景较高，从而导致诱变效率较低。在本报告中，我们描述了对昆克尔法的一种改进，以提高针对野生型链的选择效率。我们使用测序酶进行延伸反应，并引入了体外尿嘧啶-DNA糖基化酶（UDG）步骤，以增强对亲本链的生物学选择。因此，改进方法的效率得到提高，允许直接通过DNA测序筛选突变体。一个不到三小时就能完成的单管分步方案使其成为高效且经济高效的定点诱变的首选方法。
