The melting temperature (Tm) is the temperature at which 50% of the double-stranded DNA is changed to single-stranded DNA. It relies directly on the length and composition of the DNA molecule. A longer strand and a higher guanine-cytosine (GC) content are favorable for a higher melting temperature.

The annealing temperature is the temperature used in the annealing step of a PCR reaction, which is highly dependent on the Tm of primers. The annealing temperature should be low enough to allow both forward and reverse primers to bind to the single-stranded DNA, but not so low as to enable the formation of undesired, non-specific duplexes or intramolecular hairpins. Thereby, the annealing temperature is usually set as a few degrees (3-6) lower than the lowest Tm of the primers.