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
It is likely just very subtle differences in algorithm implementation. IDTs tool is closed source as far as I know, so you may need to post this question to them.
That said, if your primers are sufficiently specific and long, dimerisation should be a minimal problem. I rarely even check my primers for dimerisation or secondary structure these days (for routine cloning), I just order long primers.
Thanks for the advice, I have emailed them so can see what they say. Yes you're right, the problem is I'm doing an 8-plex PCR (4 primer pairs amplifying different gDNA regions) so I'm worried the interactions might be a bit more likely. Will see how it goes, wish me luck!
Did you solve your problem? OligoAnalyser only checks heterodimerisation of two primers, like your problem I want to check multiple primer dimers. Is it possible to do it with primer3?