Unipro UGENE User Manual
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There are two algorithms for melting temperature calculation:

  • Rough
  • Primer 3

Rough

The melting temperature is calculated as follows. For sequences of length 15 or longer:

Tm = 64.9 + 41 * (nG + nC - 16.4) / (nA + nT + nG + nC)

For shorter sequences:

Tm = (nA + nT) * 2 + (nG + nC) * 4

Here "nA", "nT", "nC", "nG" denote the number of the corresponding nucleotide.