Abstract
Site-directed mutagenesis provides a powerful means for probing protein structure and function. Using this approach, one can introduce specific amino acid changes at any given position in the protein sequence and test the functional consequences of these mutations in vitro or in vivo. If an in vivo test is available, strategies that introduce random base changes at a given codon (or indeed in a larger region of the protein) are particularly valuable.
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References
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.
Kunkel, T. A. (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc. Natl. Acad. Sci. USA 82, 488–492.
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.
Taylor, J. W., Ott, J., and Eckstein, F. (1985) The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA. Nucleic Acids Res. 13, 8765–8785.
Sayers, J. R. and Eckstein, F. (1989) Site-directed mutagenesis, based on the phosphorothioate approach, in Protein Function: A Practical Approach (Creighton, T. E., ed.), IRL, Oxford, UK, pp. 279–295.
Worrall, A. F. (1994) Site-directed mutagenesis by the cassette method, in Methods in Molecular Biology 30 (Kneale G. G., ed.), Humana Press Inc., Totowa, NJ, pp. 199–210.
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. Nucleic Acids Res. 22(9), 1593–1599.
Barettino, D., Feigenbutz, M., Valcarcel, R., and Stunnenberg, H. G. (1994) Improved method for PCR-mediated site-directed mutagenesis. Nucleic Acids Res. 22(3), 541–542.
Mikaelian, I. and Sergeant, A. (1992) A general and fast method to generate multiple site-directed mutations. Nucleic Acids Res. 20(2), 376.
Picard, V., Ersdal-Badju, E., Lu, A., and Clark Bock, S. (1994) A rapid and efficient one-tube PCR-based muatgenesis technique using Pfu DNA polymerase. Nucleic Acids Res. 22(13), 2587–2591.
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.
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 lysosyme extend an α-helix but do not alter protein stability. Science 239, 631–635.
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.
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. Lett. 109, 219–224.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (eds.) (1989) Molecular Cloning—A laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Hanahan, D., Jessee, J., and Bloom, F. R. (1991) Plasmid transformation of Escherichia coli and other bacteria. Methods Enzymol. 104, 63–113.
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. Nucleic Acids Res. 13, 8749–8764.
Nakayame, K. L. and Eckstein, F. (1986) Inhibition of restriction endonuclease Nci I cleavage by phosphorothioate groups and its application to oligonucleotide-directed mutagenesis. Nucleic Acids Res. 14, 9679–9698.
Sayers, J. R., Schmidt, W., and Eckstein, F. (1988) 5′-3′ Exonucleases in phosphoro-thioate-based oligonucleotide-directed mutagenesis. Nucleic Acids Res. 16, 9027–9039.
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 111, 17–24.
Piechocki, M. P. and Hines, R. N. (1994) Oligonucleotide design and optimized protocol for site-directed mutagenesis. Biotechniques 16(4), 702–707.
Hermes, J. D., Parekh, S. M., Blacklow, S. C., Koster, H., and Knowles, J. R. (1989) A reliable method for random muatgenesis: the generation of mutant libraries using spiked oligo-deoxyribonucleotide primers. Gene 84, 143–151.
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© 2000 Humana Press Inc., Totowa, NJ
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O’Donohue, M.J., Kneale, G.G. (2000). Primer-Directed Site-Specific Mutagenesis. In: Rapley, R. (eds) The Nucleic Acid Protocols Handbook. Springer Protocols Handbooks. Humana Press, Totowa, NJ. https://doi.org/10.1385/1-59259-038-1:815
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DOI: https://doi.org/10.1385/1-59259-038-1:815
Publisher Name: Humana Press, Totowa, NJ
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