There are two algorithms for melting temperature calculation:
- Rough
- Primer 3
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", "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