Sanchis Joaquin, Fernández Layla, Carballeira J Daniel, Drone Jullien, Gumulya Yosephine, Höbenreich Horst, Kahakeaw Daniel, Kille Sabrina, Lohmer Renate, Peyralans Jérôme J-P, Podtetenieff John, Prasad Shreenath, Soni Pankaj, Taglieber Andreas, Wu Sheng, Zilly Felipe E, Reetz Manfred T
Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470, Mülheim/Ruhr, Germany.
Appl Microbiol Biotechnol. 2008 Nov;81(2):387-97. doi: 10.1007/s00253-008-1678-9. Epub 2008 Sep 27.
Saturation mutagenesis constitutes a powerful method in the directed evolution of enzymes. Traditional protocols of whole plasmid amplification such as Stratagene's QuikChange sometimes fail when the templates are difficult to amplify. In order to overcome such restrictions, we have devised a simple two-primer, two-stage polymerase chain reaction (PCR) method which constitutes an improvement over existing protocols. In the first stage of the PCR, both the mutagenic primer and the antiprimer that are not complementary anneal to the template. In the second stage, the amplified sequence is used as a megaprimer. Sites composed of one or more residues can be randomized in a single PCR reaction, irrespective of their location in the gene sequence.The method has been applied to several enzymes successfully, including P450-BM3 from Bacillus megaterium, the lipases from Pseudomonas aeruginosa and Candida antarctica and the epoxide hydrolase from Aspergillus niger. Here, we show that megaprimer size as well as the direction and design of the antiprimer are determining factors in the amplification of the plasmid. Comparison of the results with the performances of previous protocols reveals the efficiency of the improved method.
饱和诱变是酶定向进化中的一种强大方法。传统的全质粒扩增方案，如Stratagene公司的QuikChange，在模板难以扩增时有时会失败。为了克服这些限制，我们设计了一种简单的双引物、两阶段聚合酶链反应（PCR）方法，该方法是对现有方案的改进。在PCR的第一阶段，不互补的诱变引物和反引物都与模板退火。在第二阶段，扩增的序列用作大引物。由一个或多个残基组成的位点可以在单个PCR反应中随机化，而不管它们在基因序列中的位置如何。该方法已成功应用于多种酶，包括巨大芽孢杆菌的P450-BM3、铜绿假单胞菌和南极假丝酵母的脂肪酶以及黑曲霉的环氧水解酶。在这里，我们表明大引物大小以及反引物的方向和设计是质粒扩增的决定因素。将结果与先前方案的性能进行比较，揭示了改进方法的效率。
