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A method for introducing site-specific mutations using oligonucleotide primers and its application to site-saturation mutagenesis

Molecular Biotechnology volume 6, pages 179–189 (1996)Cite this article

Abstract

Oligonucleotide primer-directed mutagenesis is a useful molecular biological tool, which is invalubable for the study of the structure/function relationships in proteins and for the creation of mutant proteins possessing modified or novel biological activities. Mutagenesis studies in which a site-saturation approach is employed require a high-efficiency mutagenesis procedure, which will generate a population of mutated molecules containing an even distribution of all possible amino acid changes, or a subset thereof. This article describes such a mutagenesis technique and discusses the adaptations that are necessary to perform sitesaturation mutagenesis.

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References

  1. Hutchison, C. A., Phillips, S., Edgell, M., Gillam, S., Jahnke, P., and Smith, M. (1978) Mutagenesis at a specific position in a DNA sequence.J. Biol. Chem. 253(18), 6551–6560.

    PubMed  CAS  Google Scholar 

  2. Kunkel, T. A. (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection.Proc. Natl. Acad. Sci. USA 82, 488–492.

    PubMed  Article  CAS  Google Scholar 

  3. Deng, W. P. and Nickoloff, J. A. (1992) Site-directed mutagenesis of virtually any plasmid by eliminating a unique site.Anal. Biochem. 200, 81–88.

    PubMed  Article  CAS  Google Scholar 

  4. Taylor, J. W., Ott, J., and Eckstein, F. (1985) The rapid generation of oligonucleotide-directed mutations at high frequency using phosporothioate-modified DNA.Nucl. Acids Res. 13, 8765–8785.

    PubMed  Article  CAS  Google Scholar 

  5. Sayers, J. R. and Eckstein, F. (1989) Site-directed mutagenesis, based on the phosphorothioate approach. InProtein Function: A Practical Approach (Creighton, T. E., ed.), IRL, Oxford, UK, 279–295.

    Google Scholar 

  6. Worrall, A. F. (1994) Site-directed mutagenesis by the cassette method. InMethods in Molecular Biology, vol. 30 (Kneale G. G., ed.), Humana, Totowa, NJ, 199–210.

    Google Scholar 

  7. Kegler-Ebo, D. M., Docktor, C. M., and DiMaio, D. (1994) Codon cassette mutagenesis: a general method to insert or replace individual codons by using universal mutagenic cassettes.Nucl. Acids Res. 22(9), 1593–1599.

    PubMed  Article  CAS  Google Scholar 

  8. Barettino, D., Feigenbutz, M., Valcarcel, R., and Stunnenberg, H. G. (1994) Improved method for PCR-mediated site-directed mutagenesis.Nucl. Acids Res. 22(3), 541, 542.

    PubMed  Article  CAS  Google Scholar 

  9. Mikaelian, I. and Sergeant, A. (1992) A general and fast method to generate multiple site-directed mutations.Nucl. Acids Res. 20(2), 376.

    PubMed  Article  CAS  Google Scholar 

  10. Picard, V., Ersdal-Badju, E., Lu, A., and Clark Bock, S. (1994) A rapid and efficient one-tube PCR-based mutagenesis technique usingPfu DNA polymerase.Nucl. Acids Res. 22(13), 2587–2591.

    PubMed  Article  CAS  Google Scholar 

  11. Goff, S. A., Short-Russell, S. R., and Dice, J. F. (1987) Efficient saturation mutagenesis of a pentapeptide coding sequence using mixed oligonucleotides.DNA 6(4), 381–388.

    PubMed  CAS  Article  Google Scholar 

  12. Alber, T., Bell, J. A., Dao-Pin, S., Nicholson, H., Wozniak, J. A., Cook, S., and Matthews, B. W. (1988) Replacements of Pro86 in phage T4 Iysosyme extend an α-helix but do not alter protein stability.Science 239, 631–635.

    PubMed  Article  CAS  Google Scholar 

  13. Zabin, H. B. and Terwilliger, T. C. (1991) Isolation and characterization of temperature-sensitive mutants of the bacteriophage fl gene V protein.J. Mol. Biol 279, 257–275.

    Article  Google Scholar 

  14. O'Donohue, M. J., Scarlett, G. P., and Kneale, G. G. (1993) Tyr 26 and Phe 73 are essential for full biological activity of the Fd gene V protein.FEMS Microbiol. Letts. 109, 219–224.

    Article  Google Scholar 

  15. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989)Molecular Cloning—A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

    Google Scholar 

  16. Hanahan, D., Jessee, J., and Bloom, F. R. (1991) Plasmid transformation ofEscherichia coli and other bacteria.Methods in Enzymology 104, 63–113.

    Article  Google Scholar 

  17. Taylor, J. W., Schmidt, W., Costick, R., Okrusek, A., and Eckstein, F. (1985) The use of phosphorothioate-modified DNA in restriction enzyme reactions to prepare nicked DNA.Nucl. Acids Res. 13, 8749–8764.

    PubMed  Article  CAS  Google Scholar 

  18. Nakayame, K. L. and Eckstein, F. (1986) Inhibition of restriction endonucleaseNci I cleavage by phosphorothioate groups and its application to oligonucleotide-directed mutagenesis.Nucl. Acids Res. 14, 9679–9698.

    Article  Google Scholar 

  19. Sayers, J. R., Schmidt, W., and Eckstein, F. (1988) 5′-3′ Exonucleases in phosphorothioate-based oligonucleotide-directed mutagenesis.Nucl. Acids Res. 16, 9027–9039.

    Article  Google Scholar 

  20. Jung, R., Scott, M. P., Oliviera, L. O., and Nielsen, N. C. (1992) A simple and efficient method for the oligodeoxyribonucleotide-directed mutagenesis of double-stranded plasmid DNA.Gene 121, 17–24.

    PubMed  Article  CAS  Google Scholar 

  21. Piechocki, M. P. and Hines, R. N. (1994) Oligonucleotide design and optimized protocol for site-directed mutagenesis.Biotechniques 16(4), 702–707.

    PubMed  CAS  Google Scholar 

  22. Hermes, J. D., Parekh, S. M., Blacklow, S. C., Koster, H., and Knowles, J. R. (1989) A reliable method for random mutagenesis: the generation of mutant libraries using spiked oligodeoxyribonucleotide primers.Gene 84, 143–151.

    PubMed  Article  CAS  Google Scholar 

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Affiliations

  1. Laboratoire de Médiateurs Protéiques, INRA Centre de Jouy-en-Josas, Domaine de Vilvert, 78352, Jouy-en-Josas Cedex, France

    Michael J. O’Donohue

  2. Biophysics Laboratories, University of Portsmouth, England

    G. Geoff Kneale

Authors
  1. Michael J. O’Donohue
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  2. G. Geoff Kneale
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O’Donohue, M.J., Geoff Kneale, G. A method for introducing site-specific mutations using oligonucleotide primers and its application to site-saturation mutagenesis. Mol Biotechnol 6, 179–189 (1996). https://doi.org/10.1007/BF02740772

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  • DOI: https://doi.org/10.1007/BF02740772

Index Entries

  • Site-directed mutagenesis
  • site-saturation mutagenesis
  • phosphorothioates