The annealing temperature (Ta
) chosen for PCR relies directly on length and composition of the primers. Generally, you should use an annealing temperature about 5°C below the Tm
of your primers. The optimal annealing temperature (Ta
Opt) for a given primer pair on a particular target can be calculated as follows: Ta
Opt = 0.3 x (Tm
of primer) + 0.7 x (Tm
of product) – 14.9; where Tm
of primer is the melting temperature of the less stable primer-template pair, and Tm
of product is the melting temperature of the PCR product .
One consequence of having Ta
too low is that one or both primers will anneal to sequences other than the intended target, because internal single-base mismatches or partial annealing may be tolerated. This can lead to nonspecific PCR amplification and will consequently reduce the yield of the desired product. Conversely, when Ta
is too high reaction efficiency may be reduced, because the likelihood of primer annealing is reduced significantly. Optimal annealing temperatures give the highest product yield of the correct amplicon.
- Rychlik W, Spencer WJ, Rhoads RE (1990) Optimization of the annealing temperature for DNA amplification in vitro. Nucleic Acids Res 18(21):6409–6412.