The annealing temperature (T
a) chosen for PCR relies directly on length and composition of the primers. Generally, you should use an annealing temperature about 5°C below the T
m of your primers. The optimal annealing temperature (T
a Opt) for a given primer pair on a particular target can be calculated as follows: T
a Opt = 0.3 x (T
m of primer) + 0.7 x (T
m of product) – 14.9; where T
m of primer is the melting temperature of the less stable primer-template pair, and T
m of product is the melting temperature of the PCR product [1].
One consequence of having T
a 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 T
a 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.
Reference
- Rychlik W, Spencer WJ, Rhoads RE (1990) Optimization of the annealing temperature for DNA amplification in vitro. Nucleic Acids Res 18(21):6409–6412.