Melting temperature differences for PCR primers and sequencing primers
Why does the melting temperature for a given primer differ depending on whether it is analyzed as a PCR primer or sequencing primer?
This difference is because in a PCR reaction there is depletion to consider. Use the Primer calculation for designing primers and the Sequencing calculation for sequencing primers.
This difference is because in a PCR reaction there is depletion of the primers. In the Rychlik and Rhoads papers on calculating Tm in PCR reactions, the initial primer concentration is divided by 8000 before calculating the Tm for PCR primers but not for sequencing primers. This is discussed in Rychlik, W., Spencer, W. J., and Rhoads, R. E. 1990. Optimization of the annealing temperature for DNA amplification in vitro. Nucleic Acids Research 18(21):6409-6412). This correction helps to account for the fact that the primer concentration decreases over the course of a PCR reaction and effectively estimates the concentration at the mid-point of the reaction. The experimental results investigated primers at a concentration of 1.0uM - there is no guarantee that the 8,000-fold adjustment would be valid at very different initial primer concentrations However, ignoring the adjustment would also produce misleading results, based on the work presented in the paper.
For these reasons you should use the Primer calculation for designing primers and the Sequencing calculation for sequencing primers.
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