Versatility of different melting temperature (Tm) calculator software for robust PCR and real-time PCR oligonucleotide design: A practical guide ☆

Success in molecular biology techniques involving hybridization such as PCR and non-PCR-based techniques depends on annealing temperature prediction which derived itself from the melting temperature (Tm) of primers with a minimal error probability. Such calculation should be derived from the comparison of existing methods in a large benchmark set of sequences, as it was the case in this study. In the present study 22 primer design tools were compared based on the deviation of the predicted Tm values of 158 primers using software versus the experimentally determined Tm values. A paired t-test (False discovery rate (FDR) corrected P-values) and mean square deviation (MSD) were applied to assess all software packages. A significant variation was observed for the Tm values of primers calculated by different tools in comparison with optimal experimental condition, which could end up causing wide error in amplification reactions. Based on FDR and MSD criteria, Primer3 Plus and Primer-BLAST performed the best prediction of Tm. We practically aimed to provide an easy, safe and robust application of the current available Tm predictor tools for all researchers especially for premiers to help them increase their success in different PCR and real-time PCR applications.