Primer 3 end stability

The 3' end residues contribute strongly to non-specific primer extension by Taq DNA polymerase, especially if the binding of these residues is relatively tight to the non-target template. Therefore, primers with very high 3' terminal stability should be rejected. The binding stability can be calculated from the free energy profile (AG). Typically the more computationally demanding aspects of calculating free energy, such as loop entropy, can be ignored because of the unfavorable energetics of opening small loops (internal denaturation) compared to end melting.

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