DNA melting temperature - How to calculate it?

I have developed on-line T_m calculations at Integrated DNA Technologies.  Use our software tool to estimate the T_m of your duplex.

Melting temperatures (T_m) of short DNA duplex oligomers (< 70 base pairs) are calculated using the following equation,

where R is the ideal gas constant (1.9865 cal.mol^-1.K^-1). The concentrations (mol/L) of each strand are denoted C₁ and C₂. The formula is valid if C₁ is greater or equal to C₂. For any self-complementary strand, there is just one strand, so set C₂ to zero. You can also ignore C₂ if C₁ is much higher than C₂. This is often the case in biological assays.

The enthalpy (ΔH^o) and entropy (ΔS^o) of duplex annealing are predicted from the nearest-neighbor model and thermodynamic parameters^1-5 (see table below). These parameters are added for each nearest neighbor doublet (stack) in the duplex. End (initiation) interactions must be included.

where N_ij is the number of times the particular nearest-neighbor stack (ij = A, T, G, C) appears in the duplex sequence.

The following table contains thermodynamic parameters.

The symbol "E" is present at initiation (end) parameters; it represents solvent molecules at the end of the duplex that interact with terminal bases. These parameters predict melting temperatures in 1 M Na⁺ buffer of neutral pH (6.5-8.5). DNA duplexes are typically used in buffers of lower sodium, potassium or magnesium concentrations. T_m values are scaled to these buffers using appropriate salt correction models^6,7.

Further details, limitations, considerations of nucleic acid folding, T_m calculations and thermodynamics can be found in IDT Tech Bulletin and my publications.

1. Owczarzy R., Vallone P.M., Gallo F.J., Paner T.M, Lane M.J., and Benight A.S. (1997) Predicting sequence-dependent melting stability of short duplex DNA oligomers, Biopolymers 44, 217-239.
2. SantaLucia J. Jr. (1998) A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics, Proc. Natl. Acad. Sci. USA 95, 1460-1465.
3. Xia T., SantaLucia J. Jr., Burkard M.E., Kierzek R., Schroeder S.J., Jiao X., Cox C., and Turner D.H. (1998) Thermodynamic parameters for an expanded nearest-neighbor model for formation of RNA duplexes with Watson-Crick base pairs, Biochemistry 37, 14719-14735.
4. Sugimoto N., Nakano S., Katoh M., Matsumura A., Nakamuta H., Ohmichi T., Yoneyama M., and Sasak M. (1995) Thermodynamic parameters to predict stability of RNA/DNA hybrid duplexes, Biochemistry 34, 11211-11216.
5. McTigue P.M., Peterson R.J., and Kahn J.D. (2004) Sequence-dependent thermodynamic parameters for locked nucleic acid (LNA)-DNA duplex formation, Biochemistry 43, 5388-5405.
6. Owczarzy R., You Y., Moreira B.G., Manthey J.A., Huang L., Behlke M.A., and Walder J.A. (2004) Effects of sodium ions on DNA duplex oligomers: Improved predictions of melting temperatures, Biochemistry 43, 3537-3554.
7. Owczarzy R., Moreira B.G., You Y., Behlke M.A., and Walder J.A. (2008) Predicting stability of DNA duplexes in solutions containing magnesium and monovalent cations, Biochemistry 47, 5336-5353.