1.Barik, S. and M.S. Galinski. 1991. “Megaprimer” method of PCR: increased template concentration improves yield. BioTechniques 10:489-490. 2.Colosimo, A., Z. Xu, G. Novelli, B. Dallapiccola, and D.C. Gruenert. 1999. Simple version of “megaprimer” PCR for site-directed mutagenesis. BioTechniques 26:870-873. 3.Datta, A.K. 1995. Efficient amplification using “megaprimer” by asymmetric polymerase ...

The technique of site-directed mutagenesis (SDM) is widely used in the field of molecular biology to introduce mutations into DNA (5). The development of polymerase chain reaction (PCR)-based SDM has eliminated the need to clone DNA, therefore simplifying the technique and enabling the desired results to be achieved more quickly (3). The “megaprimer” method of PCR-based SDM incorporates three p...

BACKGROUND Site-directed mutagenesis is an efficient method to alter the structure and function of genes. Here we report a rapid and efficient megaprimer-based polymerase chain reaction (PCR) mutagenesis strategy that by-passes any intermediate purification of DNA between two rounds of PCR. RESULTS The strategy relies on the use of a limiting concentration of one of the flanking primers (reve...

3.Higuchi, R., B. Krummel and R.K. Saiki. 1988. A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res. 16:7351-7367. 4.Sarkar, G. and S.S. Sommer. 1990. The “megaprimer” method of site-directed mutagenesis. BioTechniques 8:404-407. 5.Smith, M. 1985. In vitro mutagenesis. Annu. Rev. Genet. 19:423-462. 6.Upende...

BACKGROUND Molecular cloning is an essential step in biological engineering. Methods involving megaprimer-based PCR of a whole plasmid are promising alternatives to the traditional restriction-ligation-based molecular cloning. Their widespread use, however, is hampered by some of their inherent characteristics, e.g., linear amplification, use of self-annealing megaprimers and difficulty with pe...

No molecular cloning technique is considered universally reliable, and many suffer from being too laborious, complex, or expensive. Restriction-free cloning is among the simplest, most rapid, and cost-effective methods, but does not always provide successful results. We modified this method to enhance its success rate through the use of exponential amplification coupled with homologous end-join...