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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 Tm
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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