I am in the process of screening a set of primers for hetero/homodimer formation.
I have filtered 1200 primers using Primer3 (via it's python API - https://libnano.github.io/primer3-py/quickstart.html#thermodynamic-analysis), and now should have a set of primers that do not form homo/heterodimers with any other primers in the set (I only included primers whose delta G for hetero and homodimerization was calculated by primer3 as above -6kcal).
However, when I screen some of these using the individual entry format, the OligoAnalyser tool from IDT (https://eu.idtdna.com/pages/tools/oligoanalyzer) highlights some primers as having a delta G below -9 kcal (sometimes for heterodimerisation, sometimes for homodimers). I have set the primer3 screening reaction conditions to be the same as those used in Oligoanalyser where possible, but cannot find what temperature Oligoanalyser uses as it's default for simulating dimerization reactions. When I run primer3 with different temperatures, this has large impacts on the calculated delta G for dimerization, so I think the temperature may be the root of the differences I am seeing between my screening thermodynamic results and my Oligoanalyser thermodynamic results.
With the above in mind, does anyone know what temperature Oligoanalyser uses to calculate dimerization? I assume it has a default setting, as I can't see an option to change it anywhere in the Olioanalyser tool settings. I have tried to reverse engineer it to find a Primer3 temperature that consistently gives deltaG values that match Oligoanalser, but the values for a temperature that matches for one sequence does not match for another sequence.
Or can anyone suggest another reason for the large differences (sometimes 2x) in calculated delta g's from primer3 and the IDT oligoanalyser?
Thank you! Tim