The Tm, or melting temperature, characterizes the stability of the DNA hybrid formed between an oligonucleotide and its complementary strand. The Tm is critical for determining the optimal temperature at which to use an oligonucleotide as a primer in PCR applications (annealing temperature), as a probe for in situ hybridization, and in Southern, Northern or Dot blot analyses.
Tm = Temperature at which 50% of a given oligonucleotide is hybridized to its complementary strand.
In the absence of destabilizing agents, like formamide or urea, Tm will depend on 3 major parameters:
- The sequence: a GC-rich sequence has a higher melting temperature.
- The strand concentration: high oligonucleotide concentrations favor hybrid formation, which results in a higher melting temperature.
- The salt concentration: high ionic strength results in a higher Tm as cations stabilize the DNA duplexes.
The most accurate method of estimating the Tm of oligonucleotides, and that used at Retrogen, is based upon a thermodynamic analysis of the melting process from which it can be shown that
| Tm =( |
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S)+Rln(C))-273.15-12.0log[Na+] |
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| The changes in enthalpy( |
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H) and entropy ( |
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S) of duplex formation are calculated from |
nearest-neighbor thermodynamic parameters. R is the molar gas constant (1.987 cal.K-1mole-1), and C is the molar concentration of oligonucleotide.
Reference
- Breslauer K.J., Frank R., Blocker H., Markey L.A. (1986) Predicting DNA duplex stability from the base sequence - Proc. Natl. Acad. Sci. USA 83, 3746-3750
- Freier S.M., Kierzek R., Jaeger J.A., Sugimoto N., Caruthers M.H., Nielson T., Turner D.H. (1986) Improved free-energy parameters for predictions of RNA duplex stabilit. - Proc. Natl. Acad. Sci. USA 83, 9373-9377
- Schildkraut C., Lifson S. (1965) Dependence of the melting temperature of DNA on salt concentration - Biopolymers 3, 195-208
- Rychlik W. - Oligo version 4.0, Reference Manual - National Biosciences, Inc., Plymouth, MN
- Rychlik W., Spencer W.J., Rhoads R.E. - Nucleic Acids Res. 18, 6409-6412
- Freier S.M. (1993) Hybridization: Considerations affecting Antisense Drugs in Antisense Research and Applications, eds. Grooke S.T. and Lebleu B. CRC Press, Inc., 67-82
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