Determination of Melting Temperatures

2.5.3 Determination of Melting Temperatures

The melting temperature of a specific oligonucleotide primer (T_m) can be calculated by several different ways. The used primer pairs should be constructed to have a similar T_m. The simplest equation, often used, is

Tm = 2(A + T) + 4(G + C)

where A, C, G, and T symbolise the count of the particular nucleotide in the primer. This formula was developed for hybridisation assays with oligonucleotides at a salt concentration of 1 M [128]. The equation is inaccurate for calculation of T_m of primers longer than 20 nt. Usually, the chosen annealing temperature is about 5℃C below the calculated T_m and is determined by trial and error.

Other equations used are

T = 81.5 + 16.6(log[J+]) + 0.41(%G + C) - (600∕l) m(gc)

where J⁺ is the concentration of monovalenced cations and l is the length of the oligonucleotide [129]. This formula is correct for oligonucleotides with a length between 14 and 70 nucleotides.

Tp = 22 + 1.46(ln)

This formula calculates T_p, the optimal annealing temperature ± 2–5℃C. l_n is the effective length of the primer: 2G + C) + (A + T) [130]. This equation should result in correct values for oligonucleotides with 20 to 35 nucleotides.

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