Megaprimer Polymerase Chain Reaction (PCR)-Based Mutagenesis

The megaprimer method is a simple and versatile approach that can be adopted to create a single mutation in a specific target region as well as to create site-specific insertions, deletions, and gene fusions. This method uses three oligonucleotide primers, two rounds of polymerase chain reaction (PCR), and a DNA template containing the gene to be mutated. The first round of PCR generates a fragment with the desired mutation introduced by using one of the flanking primers and the mutant primer. This amplified fragment-the megaprimer-is used in the second PCR along with the remaining external primer to amplify a longer region of the template DNA. The final product is purified and can be cloned into an appropriate vector. By designing flanking primers with universal restriction site sequences, compatible with the vector of choice, it is possible to create different mutant clones by changing only the mutant primer. Recently, this approach has been improved by the use of forward and reverse flanking primers with significantly different melting temperatures. This allows researchers to perform both PCR steps in a single tube. This protocol has been successfully applied on templates with either low or high G + C content to amplify megaprimers 71-800 bp in length and final products ranging from 400 to 2500 bp.