PrimerDigital

There are several formulas for calculating melting temperatures (Tm). In all cases these calculations will give to user a good starting point for determining appropriate annealing temperatures for PCR, RT-PCR, hybridization and primer extension procedures. However, a precise optimum annealing temperature must be determined empirically.
The melting temperature (Tm) is defined as the temperature at which half of the stands are in the double-helical state and half are in the “random-coil” state. Tm for oligonucleotide for normal and degenerated (mixed or also know as wobble) nucleotide combination calculated by default following nearest neighbour thermodynamic parameters.
The melting temperature for mixed bases is calculated by averaging nearest neighbour thermodynamic parameters – enthalpy and entropy values as each mixed site and extinction coefficient is similarly predicted by averaging nearest neighbour values at mixed sites.

FastPCR allows choosing other nearest neighbour thermodynamic parameters or simple non thermodynamic Tm calculation formulas. The melting temperature is calculated using the formula based on the nearest neighbour thermodynamic theory Tm is defined as the temperature at which half of strands are in the double-helical state and half are in the "random-coil" state. The melting temperature 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 (determined empirically), [K⁺] is salt molar concentration (default value is 50 mM). 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 dimer and for in silico (virtual) PCR experience with oligonucleotide with a mismatches to target is calculated using values of thermodynamics parameters for a nucleic acid duplex. The non thermodynamic Tm calculation for oligonucleotides we suggest to use simple formulas, 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 hybridization of probes in 1 mol/L NaCl and is an estimate of the denaturation temperature (Td):

Tm=2(A + T + U) + 4(G + C) (for short < 15 bases)

or

Tm=64.9 + 41(G + C - 16.4)/L (for longer 14 bases).

Marmur and Doty originally established a formula to correlate GC content (%GC) to the Tm of long duplexes at a given ionic strength. Chester and Marshak added a term to account for DNA strand length (n in base pairs) to estimate primer Tm:

The Marmur–Schildkraut–Doty equation also accounts for ionic strength with a term for the Na+ concentration:


Values between 500 and 750 have been used for b, a value that may increase with the ionic strength. Another modification is that of Wetmur:


von Ahsen et al. (2001) formula:


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: