Melting Temperature

There are two algorithms for melting temperature calculation:

  • Rough
  • Primer 3

Note that Rough works only for the DNA alphabet; it does not work for extended DNA and RNA alphabets. Primer3 works for all extended alphabets.

Rough

The melting temperature is calculated as follows. For sequences of length 15 or longer:

Tm = 64.9 + 41 * (nG + nC - 16.4) / (nA + nT + nG + nC)

For shorter sequences:

Tm = (nA + nT) * 2 + (nG + nC) * 4

Here “nA”, “nT”, “nC”, and “nG” denote the number of the corresponding nucleotide.

Primer 3

This calculation algorithm is borrowed from the Primer3 package. The algorithm uses the nearest-neighbor (NN) model or the formula from Bolton and McCarthy, PNAS 84:1390 (1962) (as presented in Sambrook, Fritsch, and Maniatis, Molecular Cloning, p 11.46, 1989, CSHL Press). The algorithm has the following parameters:

  • DNA concentration (nanomolar): A value to use as the nanomolar concentration of each annealing oligo during the PCR. This parameter corresponds to ‘c’ in equation (ii) of the paper [SantaLucia (1998) A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proc Natl Acad Sci 95:1460-1465 http://www.pnas.org/content/95/4/1460.full.pdf+html], where a suitable value (for a lower initial concentration of template) is “empirically determined.”

  • Monovalent concentration (millimolar): The millimolar concentration of monovalent salt cations (usually KCl) in the PCR.

  • Divalent concentration (millimolar): The millimolar concentration of divalent salt cations (usually MgCl₂) in the PCR.

  • DNTP concentration (millimolar): The millimolar concentration of the sum of all deoxyribonucleotide triphosphates. A reaction mix containing 0.2 mM ATP, 0.2 mM CTP, 0.2 mM GTP, and 0.2 mM TTP would have this value equal to 0.8.

  • DMSO concentration (%): The concentration of DMSO in percent.

  • DMSO factor: The melting temperature of primers can be approximately corrected for DMSO:

    Tm = Tm(without DMSO) + DMSO factor * DMSO concentration

  • Formamide concentration (mol/l): The concentration of formamide in mol/l. The melting temperature of primers can be approximately corrected for formamide:

    Tm = Tm(without formamide) + (0.453 * GC% / 100 - 2.88) * Formamide concentration

  • NN Max Length: The maximum sequence length for using the nearest-neighbor model. For sequences longer than this, the algorithm uses the “GC%” formula from Bolton and McCarthy, PNAS 84:1390 (1962):

    Tm = 81.5 - DMSO concentration * DMSO factor + 0.453 * (GC% - 2.88) * Formamide concentration + 16.6 * log10(Monovalent concentration / 1000) + 0.41 * GC% - 600 / Length

  • Thermodynamic table: Specifies the thermodynamic table for the melting temperature calculation:

    • Breslauer: Method for Tm calculations from the paper [Rychlik W, Spencer WJ, and Rhoads RE (1990) “Optimization of the annealing temperature for DNA amplification in vitro”, Nucleic Acids Res 18:6409-12 https://academic.oup.com/nar/article/18/21/6409/2388653?login=false] and the thermodynamic parameters from the paper [Breslauer KJ, Frank R, Blocker H, and Marky LA(1986) “Predicting DNA duplex stability from the base sequence” Proc Natl Acad Sci 83:4746-50 http://dx.doi.org/10.1073/pnas.83.11.3746]
    • SantaLucia: Method for Tm calculations and the thermodynamic parameters from [SantaLucia JR (1998) “A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics”, Proc Natl Acad Sci 95:1460-65 http://dx.doi.org/10.1073/pnas.95.4.1460]

  • Salt Correction Formula: Specifies the salt correction formula for the melting temperature calculation:

    • Schildkraut: [Schildkraut, C, and Lifson, S(1965) “Dependence of the melting temperature of DNA on salt concentration”, Biopolymers 3:195-208 (not available online)]
    • SantaLucia: [SantaLucia JR(1998) “A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics”, Proc Natl Acad Sci 95:1460-65 http://dx.doi.org/10.1073/pnas.95.4.1460]
    • Owczarzy: [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: 5336-53 http://dx.doi.org/10.1021/bi702363u]
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