The 'megaprimer' method of site-directed mutagenesis uses three oligonucleotide primers and two rounds of polymerase chain reaction (PCR)1. One oligonucleotides is mutagenic; the others are forward and reverse primers that lie upstream and downstream from the binding site for the mutagenic oligonucleotide. The mutagenic primer and the nearer of the external primers are used in the first PCR to generate and amplify a mutated fragment of DNA. This amplified fragment—the megaprimer—is used in the second PCR in conjunction with the remaining external primer to amplify a longer region of the template DNA. This protocol is based on a method that uses forward and reverse external primers with significantly different melting temperatures (Tm)2.
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References
Kammann, M., Laufs, J., Schell, J. & Gronenborn, B. Rapid insertional mutagenesis of DNA by polymerase chain reaction (PCR). Nucleic Acids Res. 17, 5404 (1989).
Ke, S.H. & Madison, E.L. Rapid and efficient site-directed mutagenesis by single-tube “megaprimer” PCR method. Nucleic Acids Res. 25, 3371–3372 (1997).
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Rapid and efficient site-directed mutagenesis by the single-tube megaprimer PCR method. Nat Methods 1, 181–182 (2004). https://doi.org/10.1038/nmeth1104-181
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DOI: https://doi.org/10.1038/nmeth1104-181
