Two standard approximation calculations are used:
For sequences less than 14 nucleotides:
Tm = (wA + xT) * 2 + (yG + zC) * 4
where w, x, y, z are the number of the bases A, T, G, C in the sequence, respectively.
For sequences longer than 13 nucleotides:
Tm = 64.9 + 41 * (yG + zC - 16.4) / (wA + xT + yG + zC)
When degenerated nucleotides are included in the primer sequence (Y, R, W, S, K, M, D, V, H, B, or N), those nucleotides will be internally substituted prior to minimum and maximum Tm calculation.
ASSUMPTIONS:
Both equations assume that the annealing occurs under the standard conditions of 50 nM primer, 50 mM Na+, and pH 7.0.
