Site-directed mutagenesis can be used to change particular base pairs in a piece of DNA. There are a number of methods for achieving this. The approach described here is adapted from the Stratagene site-directed mutagenesis kit, the manual can be found [www.stratagene.com/manuals/200518.pdf 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.
- 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.
- Run a PCR 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 Dpn1.
- Transform the circular nicked DNA into a strain such as XL1-Blue. These cells will repair the nicks.
- Select colonies with the correct DNA.
- This protocol is at a very early stage of development. Any contributions welcome!