Bio-Rad C1000 Touch™ Thermal Cycler - Manual (Page 40)

C1000 Touch TM Thermal Cycler Manual
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In the nearest neighbor method, the melting temperature calculations are based on
the thermodynamic relationship between entropy (order or a measure of the
randomness of the oligonucleotide), enthalpy (heat released or absorbed by the
oligonucleotide), free energy, and temperature, where:
/\H = /\G + T* /\S where
/\H = Enthalpy value, Cal/Mole*K
T = temperature, Kelvin.
/\S = Entropy value, Cal/Mole*K
/\G = Gibbs free energy in Cal/Mole*K
The change in entropy and enthalpy is directly calculated by summing the values for
nucleotide pairs shown in Figure 26 (Breslauer et al. 1986).
The relationship between the free energy and the concentration of reactants and products at
equilibrium is given by:
/\G = R*T*ln ((DNA * Primer)/(DNA + Primer))
where R is the gas constant (1.986 Cal/Mole*K)
Substituting G in the two equations and solving for T gives
T = /\H/(/\S + R*Ln((DNA * Primer)/(DNA + Primer)))
Assumes the concentration of DNA and the concentration of the DNA-primer complex
are equal.
It has been determined empirically that there is a 5 kcal free energy (3.4 kcal
(Sugimoto et al. 1996) change during the transition from single-stranded to B-form
DNA.
This is presumably a helix initiation energy. Finally, adding an adjustment for salt gives
the equation that the T
a
calculator uses:
T = (/\H – 5(KCal/K*Mole))/(/\S + (R * ln(1/(primer)))) + 16.6 log
10
(SaltMolarity)
No adjustment constant for salt concentration is needed, since the various
parameters were determined at 1 M NaCl, and the log
10
of 1 is zero.
The thermodynamic calculations assume that the annealing occurs at pH 7.0. The T
m
calculations assume that the sequences are not symmetric and contain at least one G or C.