Sarkar G, Sommer S S
Mayo Clinic/Foundation.
Biotechniques. 1990 Apr;8(4):404-7.
We describe a simple and efficient method of mutagenesis which we term the "megaprimer" method. The method utilizes three oligonucleotide primers to perform two rounds of polymerase chain reaction. In the method, the product of the first polymerase chain reaction is used as one of the polymerase chain reaction primers (a "megaprimer") for the second polymerase chain reaction. When a phage promoter and a translational initiation signal are attached to the appropriate oligonucleotide primer, the mutant protein can be generated without any in vivo manipulations. To illustrate the method, two mutations in the catalytic domain of the human factor IX gene have been generated. The substitution of megaprimers for oligonucleotide primers may have utility in other polymerase chain reaction-based methods.
我们描述了一种简单高效的诱变方法，我们称之为“大引物”法。该方法利用三种寡核苷酸引物进行两轮聚合酶链反应。在该方法中，第一轮聚合酶链反应的产物用作第二轮聚合酶链反应的聚合酶链反应引物之一（“大引物”）。当将噬菌体启动子和翻译起始信号连接到合适的寡核苷酸引物上时，无需任何体内操作即可产生突变蛋白。为了说明该方法，已在人因子IX基因的催化结构域中产生了两个突变。用大引物替代寡核苷酸引物可能在其他基于聚合酶链反应的方法中有用。
