Abstract
Oligonucleotide-directed site-specific mutagenesis is a powerful tool to explore protein structure—function relationships. The single-stranded (M13) method (1,2), the polymerase chain reaction (PCR) (3–6), and the double-stranded plasmids method (7–9) are three basic procedures for these purposes. The single-stranded method developed by Zoller and Smith (10) has been modified to achieve a higher yield of mutants. Recently, the PCR method has become an easy and popular technique for site-directed mutagenesis. Both advantages and disadvantages of these methods have been discussed thoroughly (11,12). Limitations of these methods include the availability of restriction sites for subcloning and the instability of large inserts in M13 vectors (13), the low fidelity of Taq polymerase, the cost of multiple primers in the PCR protocols, and the low mutant yields with the double-stranded plasmid method.
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
Derbyshire, K. M., Salvo, J. J., and Grindley, N. D. F. (1986) A simple and efficient procedure for saturation mutagenesis using mixed oligodeoxynucleotides. Gene 46, 145–152.
Li, B., Langer, J. A., Schwartz, B., and Pestka, S. (1989) Creation of phosphorylation sites in proteins: construction of a phosphorylatable human interferon α. Proc. Natl. Acad. Sci. USA 86, 558–562.
Hignichi, R., Krummel, B., and Saiki, R. K. (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.
Vallette, F., Mege, E., Reiss, A., and Adesnik, M. (1989) Construction of mutant and chimeric genes using the polymerase chain reaction. Nucleic Acids Res. 17, 723–733.
Kadowaki, H., Kadowaki, T, Wondisford, F. E., and Taylor, S. I. (1989) Use of polymerase chain reaction catalyzed by Taq DNA polymerase for site-specific mutagenesis. Gene 76, 161–166.
Ho, S., 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.
Vlasuk, G. P. and Inouye, S. (1983) Site-specific mutagenesis vising synthetic oligodeoxyribonucleotides as mutagens, in Experimental Manipulation of Gene Expression, Appendix 2 (Inouye, M., ed.), Academic, New York, pp. 291–303.
Morinaga, Y., Franceschini, T., Inouye, S., and Inouye, M. (1984) Improvement of oligonucleotide-directed site-specific mutagenesis using double-stranded plasmid DNA. Bio/Technology 2, 636–639.
DeChiara, T. M., Erlitz, F., and Tamowski, S. J. (1986) Procedures for in vitro DNA mutagenesis of human leukocyte interferon sequences. Methods Enzymol. 119, 403–415.
Zoller, M. J. and Smith, M. (1982) Oligonucleotide-directed mutagenesis using M13-derived vectors: an efficient and general procedure for the production of point mutations in any fragment. Nucleic Acids Res. 10, 6487–6500.
Jones, D. H. and Howard, B. H. (1990) A rapid method for site-specific mutagenesis and directional subcloning by using the polymerase chain reaction to generate recombinant circles. BioTechniques 8, 178–183.
Inouye, S. and Inouye M. (1987) Oligonucleotide-directed site-specific mutagenesis using double-stranded plasmid DNA, in Synthesis and Applications of DNA and RNA (Narang, S. A., ed.), Academic, New York, pp. 181–206.
Cordell, B., Bell, G., Tisher, E., DeNoto, F. M., Ullrich, A., Pictet, R., Rutter, W. J., and Goodman, N. M. (1979) Isolation and characterization of a cloned rat insulin gene. Cell 18, 533–543.
Lai, D., Zhu, X., and Pestka, S. K. (1993) A simple and efficient method for site-directed mutagenesis using double-stranded plasmid DNA. Nucleic Acids Res. 21, 3977–3980.
Nishimura, A., Morita, M., Nishimura, Y., and Sugino, Y. (1990) A rapid and highly efficient method for preparation of competent Escherichia coli cells. Nucleic Acids Res. 18, 6169.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Newton, C. R., Graham, A., Heptinstall, L. E., Powell, S. J., Summers, C., Kalsheker, N., Smith, J. C., and Markham, A. F. (1989) Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nucleic Acids Res. 17, 2503–2515.
Sommer, S. S., Cassady, J. D., Sobell, J. L., and Bottema, C. D. K. (1989) A novel method for detecting point mutations or polymorphisms and its application to population screening for carriers of phenylketonuria. Mayo Clin. Proc. 64, 1361–1372.
Kramer, B., Kramer, W., and Fritz, H. J. (1984) Different base/base mismatches are corrected with different efficiencies by the methyl-directed DNA mismatch-repair system of E. coli. Cell 38, 879–887.
Zell, R. and Fritz, H. J. (1987) DNA mismatch repair in Escherichia coli counteracting the hydrolytic deamination of 5-methyl-cytosine residues. EMBO J. 6, 1809–1815.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1996 Humana Press Inc.
About this protocol
Cite this protocol
Lai, D., Pestka, S. (1996). A Simple Method for Site-Directed Mutagenesis with Double-Stranded Plasmid DNA. In: Trower, M.K. (eds) In Vitro Mutagenesis Protocols. Methods In Molecular Medicine™, vol 57. Humana Press. https://doi.org/10.1385/0-89603-332-5:75
Download citation
DOI: https://doi.org/10.1385/0-89603-332-5:75
Publisher Name: Humana Press
Print ISBN: 978-0-89603-332-0
Online ISBN: 978-1-59259-544-0
eBook Packages: Springer Protocols