Free energy, ∆G, can be used to measure the thermodynamic stability of the primer-binding site. The maximum ∆G value for the five bases from the 3' end is used for this calculation.

For primers shorter than 30 base pairs, ∆G is computed using the following equation:

∆G = ∆H – T · ∆S

∆H = enthalpy

∆S = entropy

T = the user-defined melting temperature or T_m

When ∆G is negative, the reaction is spontaneous. When choosing between various primer pairs, a lower ∆G value is favorable.^{10, 13, 14}

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