Multiple reactions to run, each with their own preferred annealing temperature? Grabbed two primers of different Tm’s to amplify a region and trying to work out the best program?
Touchdown PCR to the rescue.
One of the best things to come out of Australia, it uses decreasing annealing temperatures during the cycling until it ‘touches down’ on the optimal annealing temperature for the PCR primers. Many PCR (and qPCR) machines will have this ability programmed where over the course of the cycling, the annealing temperature drops by 0.5-1 °C per cycle for the first 10 cycles.
The optimal annealing temperature in early cycles means that as the annealing temperature drops, the desired amplicon has already been preferentially amplified, thus increasing specificity.
For instance, you might have several reactions you want to run in one run (and don’t have the independent triple blocks of the labCycler). Say the annealing temperatures are 58°C, 55°C and 54°C – the touchdown program would have the annealing temperature dropping from 58°C to 54°C with a 0.5°C drop per cycle for 8 cycles (4 degrees). The program contains the usual 35 cycles.
In a similar way, you can use primers with different Tm’s and for a quick reaction, avoid doing gradients that use multiple reactions to determine optimal annealing temperatures.
So if you want to avoid non-specific bands on your gel, increase the specificity quickly with Touchdown PCR.
- Don RH, Cox PT, Wainwright BJ, Baker K, Mattick JS. ‘Touchdown’ PCR to circumvent spurious priming during gene amplification. Nucleic Acids Res. 1991 Jul 25;19(14):4008.
- Roux KH. Using mismatched primer-template pairs in touchdown PCR. Biotechniques. 1994 May;16(5):812-4. PMID: 8068332.
- Korbie, D., Mattick, J. Touchdown PCR for increased specificity and sensitivity in PCR amplification. Nat Protoc 3, 1452–1456 (2008). https://doi.org/10.1038/nprot.2008.133