Site-Directed Mutagenesis with LA-PCR™ Technology
Part of the
Springer Protocols Handbooks
book series (SPH)
Protocols for site-directed mutagenesis are widely used in molecular biology and include many polymerase chain reaction (PCR)-based methods that have been developed in order to achieve efficient mutagenesis of a target DNA sequence (1, 2, 3, 4, 5, 6, 7, 8). This chapter describes an efficient and economic PCR-based site-directed mutagenesis method, which is designed to introduce a series of mutations into long DNA cloned in pUC vectors (pUC 18, 19, 118, 119), pBluescript® vectors (Stratagene, Cambridge, UK) (pBluescript® II SK(±), KS (±)) and pGEM® vectors (Promega, Madison, WI) (pGEM®-3Zf,4Z) by adding the advantage of LA-PCR technology™ (TaKaRa Shuzo Co., Kyoto, Japan). The protocol uses a combination of a primer designed for introducing a mutation at the target sequence, with primers that may be reused for each mutagenesis reaction (Fig. 1). By using this method, a series of site-directed mutations may be undertaken that only require a single primer for each desired change, and furthermore, no reiterative transformation steps are necessary (9). As based on the Long and Accurate (LA)-PCR-technology with the improved enzyme, TaKaRa LA Taq, this method provides the high fidelity and introduction of site-directed mutation into longer DNA can be achieved.
KeywordsPolymerase Chain Reaction Polymerase Chain Reaction Product Polymerase Chain Reaction Reaction Polymerase Chain Reaction Buffer Filter Cartridge
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
Ito, W., Ishiguro, H., and Kurosawa, Y. (1991) A general method for introducing a series of mutations into cloned DNA using the polymerase chain reaction. Gene
, 67–70.PubMedCrossRefGoogle Scholar
Hemsley, A., Arnheim, N., Toney, M. D., Cortopassi, G., and Galas, D. J. (1989) A simple method for site-directed mutagenesis using the polymerase chain reaction. Nucleic Acids Res.
, 6545–6551.PubMedCrossRefGoogle Scholar
Higuchi, R., Krummel, B., and Saiki, R. (1988) A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res.
, 7351–7367.PubMedCrossRefGoogle Scholar
Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., and Pease, L. R. (1989) Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene
, 51–59.PubMedCrossRefGoogle Scholar
Weiner, M. P., Costa, G. L., Schoettlin, W., Cline, J., Mathur, E., and Bauer, J. C. (1994) Site-directed mutagenesis of double-stranded DNA by the polymerase chain reaction. Gene
, 119–123.PubMedCrossRefGoogle Scholar
Ling, M. and Robinson, B. H. (1995) A one-step polymerase chain reaction site-directed mutagenesis method for large-cassettes with high efficiency, yield, and fidelity. Anal. Biochem.
, 167–172.PubMedCrossRefGoogle Scholar
Datta, A. K. (1995) Efficient amplification using “megaprimer” by asymmetric polymerase chain reaction. Nucleic Acids Res.
, 4530–4531.PubMedCrossRefGoogle Scholar
Seraphin, B. and Kandels-Lewis, S. (1996) An efficient PCR mutagenesis strategy without gel purification step that is amenable to automation. Nucleic Acids Res.
, 3276–3277.PubMedCrossRefGoogle Scholar
Shimada, A. (1996) PCR-based site directed mutagenesis. Methods Mol. Biol.
, 157–165.PubMedGoogle Scholar
Sambrook, J., Fritsch, E. F., and Maniatis, T. (eds.) (1989) Molecular Cloning. A Laboratory Manual
, 2nd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY.Google Scholar
© Humana Press Inc., Totowa, NJ 2000