Abstract
A rapid method is described to efficiently perform site-directed mutagenesis based on overlap extension polymerase chain reaction (OE-PCR). Two template DNA molecules in different orientations relative to only one universal primer were amplified in parallel. By choosing a high dilution of mutagenic primers it was possible to run an overlap extension PCR in only one reaction without purification of intermediate products. This method which we have named one-step overlap extension PCR (OOE-PCR) can in principle be applied to every DNA fragment which can be cloned into a multiple cloning site of any common cloning vector.
Full Text
The Full Text of this article is available as a PDF (40.7 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Barik S., Galinski M. S. "Megaprimer" method of PCR: increased template concentration improves yield. Biotechniques. 1991 Apr;10(4):489–490. [PubMed] [Google Scholar]
- Barik S. Site-directed mutagenesis by double polymerase chain reaction. Mol Biotechnol. 1995 Feb;3(1):1–7. doi: 10.1007/BF02821329. [DOI] [PubMed] [Google Scholar]
- Boles E., Miosga T. A rapid and highly efficient method for PCR-based site-directed mutagenesis using only one new primer. Curr Genet. 1995 Jul;28(2):197–198. doi: 10.1007/BF00315788. [DOI] [PubMed] [Google Scholar]
- Higuchi R., Krummel B., Saiki R. K. A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res. 1988 Aug 11;16(15):7351–7367. doi: 10.1093/nar/16.15.7351. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ho S. N., Hunt H. D., Horton R. M., Pullen J. K., Pease L. R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 1989 Apr 15;77(1):51–59. doi: 10.1016/0378-1119(89)90358-2. [DOI] [PubMed] [Google Scholar]
- Horton R. M., Hunt H. D., Ho S. N., Pullen J. K., Pease L. R. Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene. 1989 Apr 15;77(1):61–68. doi: 10.1016/0378-1119(89)90359-4. [DOI] [PubMed] [Google Scholar]
- Horton R. M. In vitro recombination and mutagenesis of DNA. SOEing together tailor-made genes. Methods Mol Biol. 1997;67:141–149. doi: 10.1385/0-89603-483-6:141. [DOI] [PubMed] [Google Scholar]
- Ito W., Ishiguro H., Kurosawa Y. A general method for introducing a series of mutations into cloned DNA using the polymerase chain reaction. Gene. 1991 Jun 15;102(1):67–70. doi: 10.1016/0378-1119(91)90539-n. [DOI] [PubMed] [Google Scholar]
- Senanayake S. D., Brian D. A. Precise large deletions by the PCR-based overlap extension method. Mol Biotechnol. 1995 Aug;4(1):13–15. doi: 10.1007/BF02907467. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Warrens A. N., Jones M. D., Lechler R. I. Splicing by overlap extension by PCR using asymmetric amplification: an improved technique for the generation of hybrid proteins of immunological interest. Gene. 1997 Feb 20;186(1):29–35. doi: 10.1016/s0378-1119(96)00674-9. [DOI] [PubMed] [Google Scholar]