Abstract
It is now technically possible to create almost any desired mutation in a given DNA sequence. So called site-directed mutagenesis allows the introduction of designed mutations into specific locations. This approach is invaluable for studying gene regulation as well as for functional assessment of proteins and their interactions. Several protocols have been successfully employed to generate such mutants. Here I describe a “linker-scanning” method that I have used to systematically mutate the murine Major Histocompatibility Complex (MHC) class II Eα gene promotor (Fig. 1, ref. 1).
Linker-scanning mutation of the Eα promoter The sequence of each of the 10-bp linker-scanning mutations (numbered 1–20) is shown under the wild-type Eα promoter sequence Class II regulatory motifs are underlined or boxed (reproduced from ref 1).
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References
Viville, S., Jongeneel, V.,Koch,W., Mantovani, R., Benoist, C., and Mathis, D., (1991) The Eα promoter: a linker-scanning analysis. J. Immunol 146, 3211–3217
Wallace, R.B., Schold, M., Johnson, M J., Dembek, P., and Itakura, K. (1981) Oligonucleotide directed mutagenesis of the human β-globin gene: a general method for producing specific point mutations in cloned DNA Nucleic Acids Res.9, 3647–3656
Nelson, R.M. and Long, G.L., (1989) A general method of site-specific mutagenesis using a modification of the Thermus aquaticus polymerase chain reaction. Anal Biochem 180, 147–151
Hemsley, A., Arnheim, N., Toney, M. D., Cortopassi, G., and Galas, D. J., (1989) A simple method for site directed mutagenesis using the polymerase chain reaction. Nucleic Acids Res. 16, 6545–6551
Inouye, S. and Inouye, M. (1987) Oligonucleotide-directed site-specific mutagenesis using double-stranded DNA, in Synthesis and Application of DNA and RNA (Narang, S. A., ed), Academic, Orlando, FL, 181–206.
Hanahan, D. and Meselson, M., (1980) Plasmid screening at high colony density. Gene 10, 63–67.
Stephen, D., Jones, C., and Schofield, J. P., (1990) A rapid method for isolating high quality plasmid DNA suitable for DNA sequencing. Nucleic Acids Res. 18, 7463–7464
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© 1994 Humana Press Inc, Totowa, NJ
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Viville, S. (1994). Site-Directed Mutagenesis Using a Double-Stranded DNA Template. In: Harwood, A.J. (eds) Protocols for Gene Analysis. Methods in Molecular Biology, vol 31. Humana Press. https://doi.org/10.1385/0-89603-258-2:57
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DOI: https://doi.org/10.1385/0-89603-258-2:57
Publisher Name: Humana Press
Print ISBN: 978-0-89603-258-3
Online ISBN: 978-1-59259-518-1
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