Abstract
Site-directed mutagenesis and the polymerase chain reaction (PCR) represent two powerful techniques that have led to rapid advances in our understanding of gene expression and function. Early protocols for site-directed mutagenesis depended on the production of single-stranded DNA containing the gene of interest (11), using M13 phage, or phagemids such as pBluescript. One limitation with this method is the presence of inverted repeat sequences and other complementary regions, which form extensive secondary structures within single-stranded DNA, which often severely decrease the ability to extend annealed primers containing the mutation of interest.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Vieira, J. and Messing, J. (1983) Production of single-stranded plasmid DNA. Methods Enzymol. 153, 3ā11.
Higuchi, R., Kurimmel, B., and Sarkt, R. (1988) A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res. 16, 7351ā7367.
Ho, S. N., Hunt, H. D., Morton, R. M., Pullen, J. K., and Pease, L. R. (1989) Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77, 51ā59.
Sarkar, G. and Sommer, S. S. (1990) The āMegaprimerā method of site-directed mutagenesis. BioTechniques 8, 404ā407.
Barik, S. and Galinski, M. S. (1991) āMegaprimerā method of PCR increased template concentration improves yield. BioTechniques 10, 489,490.
Aiyar, A., Ge, Z., and Leis, J. (1994) A specific orientation of RNA secondary structures is required for initiation of reverse transcription. J. Virol. 68, 611ā618.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning A Laboratory Manual, 2nd ed Cold Spring Harbor Laboratories, Cold Spring Harbor, NY.
Aiyar, A. and Leis, J. (1993) Modification of the megaprimer method of PCR mutagenesis improved implification of the final product. BioTechniques 14, 366,367.
Baldino, F., Chesselet, M.-F., and Lewis, M. E. (1989) High resolution in situ hybridization histochemistry. Methods Enzymol. 168, 761ā777.
Rychlik, W., Spencer, W. J., and Rhoads, R. E. (1990) Optimization of the annealing temperature for DNA amplification in vitro. Nucleic Acids Res. 18, 6409ā6412.
Barnes, W. M. (1994) PCR amplification of up to 35 kb DNA with high fidelity and high yield from bacteriophage templates. Proc. Natl. Acad. Sci. USA 91, 2216ā2220.
Kaufman, D. L. and Evans, G. A., (1990) Restriction endonuclease cleavage at the termini of PCR products. BioTechniques 9, 304ā306.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
Ā© 1996 Humana Press Inc.
About this protocol
Cite this protocol
Aiyar, A., Xiang, Y., Leis, J. (1996). Site-Directed Mutagenesis Using Overlap Extension PCR. In: Trower, M.K. (eds) In Vitro Mutagenesis Protocols. Methods In Molecular Medicineā¢, vol 57. Humana Press. https://doi.org/10.1385/0-89603-332-5:177
Download citation
DOI: https://doi.org/10.1385/0-89603-332-5:177
Publisher Name: Humana Press
Print ISBN: 978-0-89603-332-0
Online ISBN: 978-1-59259-544-0
eBook Packages: Springer Protocols