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# Melting tempeature

There are two algorithms for melting temperature calculation:

• Rough
• Primer 3

Note, that Rough works only for DNA alphabet, it does not work for DNA extended 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", "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 * (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:
• 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 on-line)]
• 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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