The melting temperature (*Tm*) is defined as the temperature at which half the DNA strands are in the double-helical state and half are in the "random-coil" state.

The*Tm* for short oligonucleotides with normal or degenerate (mixed) nucleotide combinations are calculated in the default setting using nearest neighbour thermodynamic parameters.

The*Tm* is calculated using a formula based on nearest neighbour thermodynamic theory with unified *dS*, *dH* and *dG* parameters (Allawi and SantaLucia, 1997):

where*dH* is enthalpy for helix formation, *dS* is entropy for helix formation, *R* is molar gas constant (1.987cal/K mol), *c* is the nucleic acid molar concentration (0.25x10^{-9} M), [K^{+}] is salt molar concentration (default value is 0.05 M).

The*Tm* for mixed bases is calculated by averaging nearest neighbour thermodynamic parameters - enthalpy and entropy values - at each mixed site; extinction coefficient is similarly predicted by averaging nearest neighbour values at mixed sites.

The mismatched pairs can be taken into account, the parameters being provided for DNA/DNA duplexes and the dangling ends that are unmatched terminal nucleotides being provided for DNA/DNA duplexes. The melting temperature for primer (probe) self or cross dimers and for*in silico* PCR experiments with oligonucleotides having mismatches to the target is calculated using values for the thermodynamic parameters for a nucleic acid duplex.

For non-thermodynamic*Tm* calculation using simple formulae; the Wallace-Ikatura rule is often used as a rule of thumb when primer *Tm* is to be estimated at the bench.

However, the formula was originally applied to the hybridisation of probes in 1M NaCl and is an estimate of the melting temperature for short oligonucleotides:

for longer 14 bases:

where*G+C* - the number of G's and C's and *L* - primer length.

Marmur and Doty originally established a formula to correlate G+C content to the*Tm* of long duplexes at a given ionic strength.

Chester and Marshak added a term to account for DNA strand length (*L*) to estimate primer *Tm*:

The Marmur–Schildkraut–Doty equation also accounts for ionic strength with a term for the K^{+} concentration. Another modification is that of Wetmur:

von Ahsen et al. (2001) formulae:

Two equations above assume that the stabilizing effects of cations are the same on all base pairs. The melting temperature of the PCR product calculated using the formula:

The

The

where

The

The mismatched pairs can be taken into account, the parameters being provided for DNA/DNA duplexes and the dangling ends that are unmatched terminal nucleotides being provided for DNA/DNA duplexes. The melting temperature for primer (probe) self or cross dimers and for

For non-thermodynamic

However, the formula was originally applied to the hybridisation of probes in 1M NaCl and is an estimate of the melting temperature for short oligonucleotides:

for longer 14 bases:

where

Marmur and Doty originally established a formula to correlate G+C content to the

Chester and Marshak added a term to account for DNA strand length (

The Marmur–Schildkraut–Doty equation also accounts for ionic strength with a term for the K

von Ahsen et al. (2001) formulae:

Two equations above assume that the stabilizing effects of cations are the same on all base pairs. The melting temperature of the PCR product calculated using the formula:

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