The melting temperature (Tm) is the most important factor in determining the optimal PCR annealing temperature. An ideal PCR reaction should have forward and reverse primers with similar Tm values. Tm is not only determined by primer sequence, but also by other parameters, such as salt concentration and primer concentration. In recent years, extensive thermodynamic studies have been carried out to accurately determine oligo Tm values.
Currently, the following methods for Tm calculation are adopted by most primer design programs.
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