AAT Bioquest

What is the difference between melting temperature and annealing temperature?

Posted June 22, 2020


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.

Additional resources

6-ROX glycine *25 uM fluorescence reference solution for PCR reactions*

Chen, S. C., Halliday, C. L., & Meyer, W. (2002). A review of nucleic acid-based diagnostic tests for systemic mycoses with an emphasis on polymerase chain reaction-based assays. Medical mycology, 40(4), 333-357.

Chuang, L. Y., Cheng, Y. H., & Yang, C. H. (2013). Specific primer design for the polymerase chain reaction. Biotechnology letters, 35(10), 1541-1549.

Melting Point Predictor