Monovalent and divalent salt correction algorithms for Tm prediction—recommendations for Primer3 usage

Primer3 is a widely used program for selection of oligonucleotide primers for PCR. The websites used for implementation of Primer3 have recently been updated. PCR requires Mg2+, which has a significant dsDNA stabilizing effect that must be taken into account when designing PCR primers. The data sets and formulas used to correct for salt concentrations have been updated in Primer3 to give better prediction of melting temperature (Tm). The liberal combination of different formulas for monovalent and divalent salt correction can lead to different results, depending on the formula chosen by the user. Using published Tm for 475 different oligonucleotides, it is shown that the combination of the implemented conversion of divalent to monovalent cation concentration works well with one salt correction formula but not with an alternative one. Use of a more recently described alternative formula would lead to equally good Tm predictions if divalent cations are present. The proper selection of compatible primer pairs depends on the choice of a good combination of salt correction formulas. Currently the SantaLucia salt correction formula should be used if Mg2+ is present. The alternative formula should be updated to its recent form for future releases.