Abstract
Generation of fusion proteins is a routine procedure in an increasing number of laboratories worldwide. Generally, the cDNA sequence of the protein under study is subcloned in-frame into a vector containing the sequence of a wellestablished epitope. This procedure, although simple and widespread, presents some important limitations: The vector generally contains a single, unidirectional, multiple-cloning site that allows the epitope to be incorporated into only the N- or C-terminus of the protein; it requires the use of unique restriction endonucleases that have no sites within the inserted cDNA sequence; when working with different expression systems, it is often necessary to acquire different, and potentially expensive, plasmid vectors; and it is a multistep procedure involving polymerase chain reaction (PCR), digestion with restriction enzymes, subcloning, and bacterial transformation and selection.
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References
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. 17, 6545–6551.
Chen, B. and Przybyla, A. E. (1994) An efficient site-directed mutagenesis method based on PCR. BioTechniques 17, 657–659.
Dorrell, N., Gyselman, V. G., Foynes, S., Li, S.-R., and Wren, B. W. (1996) Improved efficiency of inverse PCR mutagenesis. BioTechniques 21, 604–608.
Fisher, C. L. and Pei, G. K. (1997) Modification of a PCR-based site-directed mutagenesis method. BioTechniques 23, 570–574.
Gama, L. and Breitwieser, G. E. (1999) Generation of epitope-tagged proteins by inverse PCR mutagenesis. BioTechniques 26, 814–816.
Turchin, A. and Lawler Jr., J. F. (1999) The Primer Generator: a program that facilitates the selection of oligonucleotides for site-directed mutagenesis. BioTechniques 26, 672–676.
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© 2002 Humana Press Inc.
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Gama, L., Breitwieser, G.E. (2002). Generation of Epitope-Tagged Proteins by Inverse Polymerase Chain Reaction Mutagenesis. In: Braman, J. (eds) In Vitro Mutagenesis Protocols. Methods in Molecular Biology™, vol 182. Humana Press, Totowa, NJ. https://doi.org/10.1385/1-59259-194-9:077
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DOI: https://doi.org/10.1385/1-59259-194-9:077
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-0-89603-910-0
Online ISBN: 978-1-59259-194-7
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