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How to calculate melting temperature of DNA "2+4 rule of thumb"

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Published on Oct 30, 2013

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

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