The "megaprimer" method of site-directed mutagenesis.

TLDR

The paper introduces the “megaprimer” method, a simple and efficient approach to site‑directed mutagenesis. The method uses three primers and two rounds of PCR, with the first PCR product serving as a megaprimer in the second round, allowing in‑vitro generation of mutant proteins when a promoter and initiation signal are incorporated. The authors successfully introduced two mutations into the human factor IX catalytic domain and suggest that megaprimer substitution could benefit other PCR‑based techniques.

Abstract

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.