Richard Owczarzy

DNA melting temperature - How to calculate it?

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

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

Tm equation1

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 (ΔHo) and entropy (ΔSo) of duplex annealing are predicted from the nearest-neighbor model and thermodynamic parameters1-5 (see table below). These parameters are added for each nearest neighbor doublet (stack) in the duplex. End (initiation) interactions must be included.

Tm equation2

where Nij 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.

DNA/DNA duplexRNA/RNA duplex
StackΔHo
(kcal.mol-1)
ΔSo
(cal.mol-1.K-1)
StackΔHo
(kcal.mol-1)
ΔSo
(cal.mol-1.K-1)
5'AA3'
3'TT5'
-7.9 -22.2 5'AA3'
3'UU5'
-6.82 -19.0
5'AT3'
3'TA5'
-7.2 -20.4 5'AU3'
3'UA5'
-9.38 -26.7
5'TA3'
3'AT5'
-7.2 -21.3 5'UA3'
3'AU5'
-7.69 -20.5
5'CA3'
3'GT5'
-8.5 -22.7 5'CA3'
3'GU5'
-10.44 -26.9
5'GT3'
3'CA5'
-8.4 -22.4 5'GU3'
3'CA5'
-11.40 -29.5
5'CT3'
3'GA5'
-7.8 -21.0 5'CU3'
3'GA5'
-10.48 -27.1
5'GA3'
3'CT5'
-8.2 -22.2 5'GA3'
3'CU5'
-12.44 -32.5
5'CG3'
3'GC5'
-10.6 -27.2 5'CG3'
3'GC5'
-10.64 -26.7
5'GC3'
3'CG5'
-9.8 -24.4 5'GC3'
3'CG5'
-14.88 -36.9
5'GG3'
3'CC5'
-8.0 -19.9 5'GG3'
3'CC5'
-13.39 -32.7
5'EG3'
3'EC5'
and
5'EC3'
3'EG5'
0.1 -2.8 5'EG3'
3'EC5'
and
5'EC3'
3'EG5'
1.805 -0.75
5'EA3'
3'ET5'
and
5'ET3'
3'EA5'
2.3 4.1 5'EA3'
3'EU5'
and
5'EU3'
3'EA5'
5.525 9.75
Symmetrya 0.0 -1.4 Symmetrya 0.0 -1.4
aThis is the entropic correction for a duplex made from a self-complementary strand.

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. Tm values are scaled to these buffers using appropriate salt correction models6,7.

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

REFERENCES
  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.
Copyright 2013, Updated July 1, 2013, version 5.11.