The annealing temperature (T_a) chosen for a PCR depends directly on length and composition of the primer(s). Generally, an annealing temperature about 5°C below the lowest T_m of the pair of primers is used.¹¹ The optimal annealing temperature for any given primer pair on a particular target can be calculated as follows:¹²

Polymerases require the binding of nucleotides at the 3' end of the primer to begin elongation, and because of this, any nonspecific binding at the 3' end will adversely affect amplification. Non-specific binding that occurs at the 5' end of the primer does not necessarily adversely impact amplifications since polymerases cannot begin elongation until the 3' end binds.

One consequence of having too low a T_a is that one or both primers will anneal to sequences other than the true target, as internal single-base mismatches or partial annealing may be tolerated. This can lead to nonspecific amplification and will consequently reduce the yield of the desired product if the 3' -most base is paired with a target. Conversely, too high a T_a may yield little product, as the likelihood of primer annealing is reduced.

Use of this calculated optimal T_a in the annealing step of the PCR cycle usually results in optimal PCR product yield with minimum false product production.

Back Forward