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Primer Melting Temperature

Home > DNA Amplification > Overview > Primer Design > Primer Melting Temperature

The melting temperature (Tm) 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 Tm. 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 (Tm) is:

Tm = 4(G + C) + 2(A + T) °C


The actual (Tm) is influenced by the concentration of Mg2+, K+, and cosolvents. There are numerous computer programs to assist in primer design. The formula given above for (Tm) is simplistic; there are many primer design programs which use more complex nearest-neighbor thermodynamics values.01, 10 Also see for more complex nearest-neighbor thermodynamics values.

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© 2007 NFSTC Science Serving Justice®
NOTE TO USERS: The President’s DNA Initiative DNA Analyst Training program and assessment were completed and published in 2005, in cooperation with the National Institute of Justice. The science and techniques in the program are sound and proven, however, program content has not been updated to include tools and technologies developed and in use after 2005, including many kits and robots. Assessment questions address only content delivered in this program and may not contain the full range of tools in use in your laboratory.