The melting temperature (T_m) is the temperature at which one-half of a particular DNA duplex will dissociate and become single-strand DNA. The stability of a primer-template DNA duplex can be measured by its T_m. Primers with melting temperatures in the range of 52-58°C generally produce better results than primers with lower melting temperatures. While the annealing temperature can go as high as 72°C, primers with melting temperatures above 65°C have a higher potential for secondary annealing.

Primer length and sequence are of critical importance in designing the parameters of a successful amplification. The melting temperature of a nucleic acid duplex increases both with its length and with increasing GC content. A simple formula for calculation of the (T_m) is:

T_m = 4(G + C) + 2(A + T) °C

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