Abstract
We describe here an improved megaprimer PCR mutagenesis strategy. The cumbersome gel purification step that is usually used can be omitted by appropriately cleaving the first and second DNA templates with restriction enzymes and enzymatically removing remaining primers from the first PCR reaction. We show that this improved procedure is reproducible and highly efficient. Furthermore this method is suitable for automation because all the steps are now carried out in reaction tubes.
Full Text
The Full Text of this article is available as a PDF (33.5 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Armengaud J., Jouanneau Y. Addition of a class IIS enzyme site in the mutagenic primer to improve two-step PCR-based targeted mutagenesis. Nucleic Acids Res. 1993 Sep 11;21(18):4424–4425. doi: 10.1093/nar/21.18.4424. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Good L., Nazar R. N. An improved thermal cycle for two-step PCR-based targeted mutagenesis. Nucleic Acids Res. 1992 Sep 25;20(18):4934–4934. doi: 10.1093/nar/20.18.4934. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kuipers O. P., Boot H. J., de Vos W. M. Improved site-directed mutagenesis method using PCR. Nucleic Acids Res. 1991 Aug 25;19(16):4558–4558. doi: 10.1093/nar/19.16.4558. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sharrocks A. D., Shaw P. E. Improved primer design for PCR-based, site-directed mutagenesis. Nucleic Acids Res. 1992 Mar 11;20(5):1147–1147. doi: 10.1093/nar/20.5.1147. [DOI] [PMC free article] [PubMed] [Google Scholar]