Unipro UGENE User Manual
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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", "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 nanomolar concentration of each annealing oligo over the course 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 MgCl^(2+)) 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 equals 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, algorithm uses the "GC%" formula from Bolton and McCarthy, PNAS 84:1390 (1962):

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