I wrote about how MacVector calculates the melting temperature of a primer in an earlier blog post. One other common question we get is, “once I have designed a pair of primers, what is the optimum annealing temperature to use in the PCR?” MacVector calculates this and displays the results in the Analyze | Primer Design (Primer3) spreadsheet results tab;
Here, the Tm of both the left and right primers is calculated using the Santa Lucia thermodynamic nearest neighbor algorithm. The Tm of the product is also displayed – because the product is nearly always longer than 35 bp, we use the Baldino algorithm. Finally, the recommended annealing temperature (Ta) for use in the reaction is listed in the right-most column.
This calculation takes into account the Tm of both the primers AND the product, based on the algorithms described in W. Rychlik, W.J. Spencer, and R. E. Rhoads, Nucl.Acids.Res. 18:6409-6412(1990). They determined empirically that the greatest yield was observed in a PCR when the Ta was calculated according to this simple formula;
0.3 x Tm(primer) + 0.7 x Tm(product) – 14.9
where the lowest Tm of the two primers is used in the calculation.
Many scientists use a simple rule-of-thumb where they calculate a Ta by subtracting 10 from average Tm of the two primers – you can see this will often give a similar result to MacVector’s calculation. However, the importance of the product Tm in the Rychlik paper explains why sometimes MacVector will recommended a Ta that might be much higher or lower than you would expect based on the primer Tm. In particular, if the chosen primers are of much lower G+C% content than the product, it can be important to maintain a higher annealing temperature than might be expected to prevent the product from annealing with itself and blocking the primers from binding.
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