Abstract
Site-directed mutagenesis is a commonly used tool for identifying the role of specific amino acids in the structure and function of proteins. Various methods of in vitro mutagenesis have been described and are widely used for introducing modified coding sequences (1-7). In comparison, polymerase chain reaction (PCR)-based methods (1-6) are generally faster and more efficient than non-PCR-based methods (7). On the other hand, some PCR-based methods require two or more primers for each round of mutagenesis, whereas others need a single very long oligonucleotide or two round of PCR for introducing one mutation (5-7). These all can increase the cost of mutagenesis.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Chen, B. and Przybyla, E. A. (1994) An efficient site-directed mutagenesis method based on PCR. BioTechniques 17, 657–659.
Sang, N., Condorelli, G., De Luca, A., MacLachlan, T. K., and Giordano, A. (1996) Generation of site-directed mutagenesis by extra long, high-fidelity polymerase chain reaction. Analyt. Biochem. 233, 142–144.
Tomic, M., Sunjevaric, I., Savtchenko, E. S., and Blumenberg, M. (1990) A rapid and simple method for introducing specific mutations into any position of DNA leaving all other position unalerted. Nucl. Acids Res. 18, 1656.
Barik, S. (1993) Site-directed mutagenesis by double polymerase chain reaction: megaprimer method. Meth. Mol. Biol. 15, 277–286.
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 77, 51–59.
Zhong, D. and Bajaj, S. P. (1993) A PCR-based method for site specific domain replacement that does not require restriction recognition sequences. BioTechniques 15, 874–878.
Kunkel, T. A. (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc. Nat. Acad. Sci. USA 82, 488–192.
Striebel, H.-M., Rysavy, P., Adamec, J., Spizek, J., and Kalousek, F. (1996) Mutational analysis of both subunits from rat mitochondrial processing peptidase. Arch. Biochem. Biophys. 335, 211–218.
Adamec, J. and Kalousek, F. (1999) PCR method for generating multiple mutations at adjacent sites. Folia Microbiol. 44, 11–14.
Moreira, R. F. and Noren, C. J. (1995) Minimum duplex requirements for restriction enzyme cleavage near the termini of linear DNA fragments. BioTechniques 19, 58–59.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2002 Humana Press Inc.
About this protocol
Cite this protocol
Adamec, J. (2002). PCR Method for Generating Multiple Mutations at Adjacent Sites. In: Chen, BY., Janes, H.W. (eds) PCR Cloning Protocols. Methods in Molecular Biology™, vol 192. Humana Press. https://doi.org/10.1385/1-59259-177-9:207
Download citation
DOI: https://doi.org/10.1385/1-59259-177-9:207
Publisher Name: Humana Press
Print ISBN: 978-0-89603-969-8
Online ISBN: 978-1-59259-177-0
eBook Packages: Springer Protocols