Weiner M P, Costa G L, Schoettlin W, Cline J, Mathur E, Bauer J C
Stratagene Cloning Systems, La Jolla, CA 92037.
Gene. 1994 Dec 30;151(1-2):119-23. doi: 10.1016/0378-1119(94)90641-6.
We have developed a facile procedure for rapid PCR-based site-directed mutagenesis of double-stranded DNA. Increasing the initial template concentration and decreasing the PCR cycles to 5-10 allows us to reduce the rate of undesired second-site mutations and dramatically increase the time savings. Following PCR, DpnI treatment is used to select against parental DNA molecules. The DpnI (target sequence 5'-Gm6ATC) is specific for methylated and hemimethylated DNA and is used to digest parental DNA and select for mutation-containing amplified DNA. DNA isolated from almost all common Escherichia coli strains is Dam methylated and therefore susceptible to DpnI digestion. Pfu DNA polymerase is used, prior to intramolecular ligation of the linear template, to remove any bases extended onto the 3' ends of the PCR product by Taq DNA polymerase. The recircularized vector DNA incorporating the desired mutations is transformed into E. coli. This method can be used independently of any host strain and vector.
我们开发了一种简便的方法，用于基于PCR的双链DNA快速定点诱变。提高初始模板浓度并将PCR循环次数减少到5-10次，使我们能够降低不期望的第二位点突变率，并显著节省时间。PCR之后，使用DpnI处理来筛选去除亲本DNA分子。DpnI（靶序列5'-Gm6ATC）对甲基化和半甲基化DNA具有特异性，用于消化亲本DNA并筛选含突变的扩增DNA。从几乎所有常见大肠杆菌菌株中分离的DNA都是Dam甲基化的，因此易受DpnI消化。在对线性模板进行分子内连接之前，使用Pfu DNA聚合酶去除Taq DNA聚合酶延伸到PCR产物3'末端的任何碱基。将包含所需突变的环化载体DNA转化到大肠杆菌中。该方法可独立于任何宿主菌株和载体使用。
