THERMOCALCULATOR1 is a web-based program that computes the helical stability of duplex DNA structures. It requires the input values of temperature (°C), salt concentration or monovalent cation concentration (mM) and the base sequence of the DNA of interest. The output of the program includes a nearest-neighbor frequency survey table, the input sequence information, the input condition parameters and the thermodynamic parameters (standard entropy:S°; standard enthalpy:H°; free energy:G) of the DNA duplex.

THERMOCALCULATOR2 is a web-based program that computes and compares the helical stability of duplex DNA structures. It requires the input values of temperature (°C), salt concentration or monovalent cation concentration (mM) and the base sequences of the DNAs of interest. The output of the program includes a nearest-neighbor frequency survey table for each query, the input sequences information, the input condition parameters and a table comparing the thermodynamic parameters (standard entropy:S°;standard enthalpy:H°; free energy:G) of the DNA duplexes. The free energy difference (G) between the two queries is also output. The latter parameter is useful for comparison of a mutation sequence (MUT) with the corresponding wild type (WT) sequence (i.e. G=G_MUT - G_WT) as described for the MUTENERGY program (Lin & Kowalski, 1997). For this purpose, enter the sequence mutated and two flanking nucleotides at a minimum.

The principles upon which these two programs are based are:

Under a given set of solution conditions the relative stability of a DNA duplex structure depends on the primary sequence. More specifically, the stability of a DNA duplex depends primarily on the identity of the nearest-neighbor (NN) bases.
 

Ten different nearest-neighbor (NN) interactions are possible in a Watson-Crick DNA duplex structure: AA/TT; AT/TA; TA/AT; CA/GT; GT/CA; CT/GA; GA/CT; CG/GC; GC/CG; GG/CC. The standard (under conditions of 25°C and 1M monovalent cation) entropy (S°) and standard enthalpy (H°) values of all of these 10 interactions are available from the thermodynamic library reported by Breslauer & Marky (1986).
 

The standard entropy value (S°) of a duplex DNA structure is determined by the sum of the standard entropy values of all of its nearest neighbors. Similary, the standard enthalpy (H°) of a duplex DNA structure is determined by the sum of the standard enthalpy values of all of its nearest neighbors.

H° = SUM {(frequency of NN)*(H° of NN)}

S° = SUM {(frequency of NN)*(S° of NN)}

The melting temperature of the duplex can then be calculated by the following formula once we have determined the S° and H°.

Tm = (H°/S°) + 18log[monovalent cation]

Note that the Tm of interest to us is for local strand separation within a larger duplex DNA and is therefore independent of DNA concentration.

Subsequently, the free energy value (G) of the duplex can be calculated by the following formula:

G =H°[1-(T/Tm)]