Site-directed mutagenesis is useful to study molecular interactions, such as RNA-RNA, and RNA protein interactions. This protocol describes how to perform site-directed mutagenesis with a two or 3-step PCR approach. The main advantages of the method are the diversity of different mutations that can be incorporated, as well as a relative ease and cost for doing so.
To begin, use the wild-type DNA template and primer pairs two, one, and two, three, specific for 2-step, or primer pairs three, one, and three, four, specific for 3-step PCR to obtain PCR product one. To validate PCR products by agarose gel electrophoresis, make a 2%agarose gel by dissolving two grams of agarose in 100 milliliters of 1X TAE buffer, using a microwave oven. Add ethidium bromide at a final concentration of 0.5 microgram per milliliter.
Then cast the agarose gel. When solidified, place it in an electrophoresis unit. Load a DNA ladder of known size, and the PCR samples mixed with DNA loading dye into wells.
Run samples at 75 volts for 45 minutes. And visualize bands on a UV transilluminator or with a gel imaging system. Next, purify the PCR product with a gel extraction kit according to the manufacturers protocol and measure the concentration of the purified DNA with a spectrophotometer.
Then store the purified DNA at minus 20 celsius in TE buffer. To begin site-directed mutagenesis with a 2-step PCR, first use the wild-type DNA template, then the primer pairs two, one and two, two, for five primer mutations, or primer pairs two, two, and two, three for three priment mutations to obtain PCR product two. Validate the PCR product by gel electrophoresis, purify the PCR product, and measure its concentration as described before.
Then use the wild-type DNA template and the PCR product two as the primer, together with primer two, three for five priment mutation, or primer two, one for three priment mutation, to obtain PCR product three. Validate the PCR product by gel electrophoresis, purify the PCR product, and measure its concentration as described before. Then store the purified DNA at minus 20 degrees celsius in TE buffer.
To begin site-direct mutagenesis with a 3-step PCR, first use the wild-type DNA template and primer pairs three, one, and three, two to obtain PCR product two. Use the wild-type DNA template and primer pairs three, three, and three, four, to obtain PCR product three. Use the wild-type DNA template and primer pairs three, three and three, four, to obtain PCR product three.
Validate the PCR product by gel electrophoresis, purify the PCR product, and measure its concentration as described before. Finally use two to five nanograms of PCR products two and three as the template along with primer pairs three, one and three, four, to obtain PCR product four. Validate the PCR product by gel electrophoresis, purify the PCR product, and measure its concentration as described before.
Store the purified DNA at minus 20 degrees celsius in TE buffer. In this study, 2-step and 3-step site-directed mutagenesis were used to investigate RNA interactions regarding post transcriptional regulation of CsgD. The wild-type CsgD was PCR amplified to yield PCR product IA, and the wild-type mcaS was PCR amplified to yield PCR product IB.Using a 3-step PCR strategy, PCR products IIA and IIIA were synthesized in the first two steps, and IVA in the third step to introduce mutations in CsgD.
Similarly, complimentary site-directed mutations were introduced in mcaS to yield PCR products IIB, IIIB, in the first two steps, and IVB in the third step. Western blot analysis showed that while expression of wild-type mcaS prevents translation of wild-type CsgD, mutations in either mcaS or CsgD alleviates the observed repression. However, mutations in both CsgD and mcaS prevent translation of CsgD.
Using a 2-step PCR strategy, PCR products IIC, IID, IIE, IIF, and IIG, were synthesized in the first step, and PCR products IIIC, IIID, IIIE, IIIF, and IIIG, in the second step to introduce mutations to the entire CsgD DNA. EMSA results of the HFQ binding to in vitro transcribed CsgD wild-type and mutant RNAs, synthesized using PCR products IIIC, IIID, IIIE, IIIF, and IIIG, showed the increased KD values of the mutant alleles. This proved that HFQ could bind less efficiently to the mutants.
Furthermore HFQ has several binding sites on CsgD, which is shown by the three shifts observed for the wild-type RNA. However, only two binding sites are observed for different mutant RNAs. Thus the site-directed mutational approach identifies primary and or secondary structures of the CsgD mRNA that are important for complete binding of HFQ.
The method for site-directed mutagenesis described here relies on PCR. Good PCR practices are therefor crucial for the method and might require optimization to be successful. Site-directed mutagenesis is only powerful with all three methods such EMSA and Western Blotting.
Other methods, for instance, include various structural assays and some activity assays, and post translation modification studies.
