Abstract
A single-stage polymerase-based procedure is described that allows extensive modifications of DNA. The version described here uses the QuikChange Site-Directed Mutagenesis System kit supplied by Stratagene. The original protocol is replaced by a single-stage method in which linear production of complementary strands is accomplished in separate single primer reactions. This has proved effective in introducting insertions and deletions into large gene/vector combinations without subcloning.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Mullis, K., Faloona, F., Scharf, S., Saiki, R., Horn, G., and Ehrlich, H. (1986) Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harbor Symp. Quant. Biol. 51, 262–273.
Cormack, B. (1994) Mutagenesis of cloned DNA. In: Current Protocols in Molecular Biology (Ausubel, F. M., Brent, R. E., Kingston, D. D., et al., eds.), John Wiley and Sons, New York, pp. 8.5.1–8.5.12.
Sarkar, G. and Sommer, S. S. (1990) The “megaprimer” method of site-directed mutagenesis. Biotechniques 8, 404–407.
Aiyar, A. and Leis, J. (1993) Modification of the megaprimer method of PCR mutagenesis: improved amplification of the final product. Biotechniques 15, 366–369.
Barik, S. (1996) Site-directed mutagenesis in virto by megaprimer PCR. Methods Mol. Biol. 57, 203–215.
Wang, W. Y. and Malcolm, B. A. (1999) Two-stage PCR protocol allowing introduction of multiple mutations, deletions and insertions using QuikChange Site-Directed Mutagenesis. Biotechniques 26, 680–682.
Wang, W. Y. and Malcolm, B. A. (2002) Two-stage polymerase chain reaction protocol allowing introduction of multiple mutations, deletions, and insertions, using QuikChange site-directed mutagenesis. Methods Mol. Biol. 182, 37–43.
Makarova, O., Kamberov, E., and Margolis, B. (2000) Generation of deletion and point mutations with one primer in a single cloning. Biotechniques 29, 970–972.
Geiser, M., Gee, R., Drewello, D., and Schmitz, R. (2001) Integration of PCR fragments at any specific site within cloning vectors without the use of restriction enzymes and DNA ligase Biotechniques 31, 88–92.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Salerno, J.C., Jones, R.J., Erdogan, E. et al. A single-stage polymerase-based protocol for the introduction of deletions and insertions without subcloning. Mol Biotechnol 29, 225–232 (2005). https://doi.org/10.1385/MB:29:3:225
Issue Date:
DOI: https://doi.org/10.1385/MB:29:3:225