General Information
Site-directed mutagenesis can be used to change particular base pairs in a piece of DNA. There are several methods for achieving this. The approach described here is adapted from the Stratagene site-directed mutagenesis kit, the manual can be found here. Even when using a kit it will be necessary to design primers that are suitable for the specific changes you want to make to your DNA. Most of the contents of the kit can be found in your favorite labs stocks so you may not need to buy the kit itself. If you have problems with this procedure, you can try ‘Round-the-horn site-directed mutagenesis which uses PCR to amplify the desired mutant product.
General Procedure
- Purify template plasmid DNA from a dam+ Escherichia coli strain (to ensure that all GATC sites are methylated for later digestion with DpnI).
- Design forward and reverse primers that will bind to the region of DNA you want to mutate but that contain the modifications you wish to make. See the CAD tool PrimerX.
- Run a primer-extension reaction with a proof-reading, non-displacing polymerase such as Pfu DNA polymerase. This results in nicked circular strands of the plasmid.
- Cut up the template DNA with DpnI.
- Transform the circular nicked DNA into a highly competent strain such as XL1-Blue. These cells will repair the nicks and not restrict the unmodified product DNA.
- Select colonies with the correct DNA.
References
Manuals
- Stratagene QuikChange Site-directed Mutagenesis Kit
Publications
- Zheng L, Baumann U, and Reymond JL. An efficient one-step site-directed and site-saturation mutagenesis protocol. Nucleic Acids Res. 2004 Aug 10;32(14):e115. DOI:10.1093/nar/gnh110 | PubMed ID:15304544 | HubMed [Zheng-NAR-2004]
- Ko JK and Ma J. A rapid and efficient PCR-based mutagenesis method applicable to cell physiology study. Am J Physiol Cell Physiol. 2005 Jun;288(6):C1273-8. DOI:10.1152/ajpcell.00517.2004 | PubMed ID:15659713 | HubMed [ko-ma]
- ISBN:0-87969-577-3 [MolecularCloning]
All Medline abstracts: PubMed | HubMed