Question: IDT vs primer 3 - PCR dimerisation
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gravatar for timothy.kirkwood
10 weeks ago by
timothy.kirkwood10 wrote:

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

pcr primer primer3 idt • 218 views
ADD COMMENTlink modified 9 weeks ago • written 10 weeks ago by timothy.kirkwood10
1

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.

ADD REPLYlink written 9 weeks ago by Joe18k

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!

ADD REPLYlink written 9 weeks ago by timothy.kirkwood10
2
gravatar for timothy.kirkwood
9 weeks ago by
timothy.kirkwood10 wrote:

I got a reply from IDT, in case anyone else has a similar issue - essentially they find all secondary structures and the structure TM to calculate the dG:

Dear Dr Tirkwood,

The tool does not use as input temperature but the parameter settings for the PCR or qPCR reaction are: Target type Oligo Concentration Na+ Concentration Mg++ Concentration dNTPs Concentration

By default there is two settings for automatic filling in of these parameters: SpecSheet(Default) qPCR but the parameters can also be manually entered (see instructions here: https://eu.idtdna.com/calc/Analyzer/Home/Instructions).

According to these parameters the tool calculates the corresponding melting temperature for the oligos and the corresponding melting temperature for potential secondary structures. It calculates the deltaG according to the melting temperature values of the secondary structures of dimers detected.

For more information on this see. https://eu.idtdna.com/pages/education/decoded/article/using-the-oligoanalyzer-program You can also see more on the definitions of the biophysical properties calculated by the tool here: https://eu.idtdna.com/calc/Analyzer/Home/definitions

ADD COMMENTlink written 9 weeks ago by timothy.kirkwood10

Please accept your answer so this question is marked as resolved.

Upvote|Bookmark|Accept

ADD REPLYlink written 6 days ago by RamRS30k

Cheers and sorry, I'll remember for the future!

ADD REPLYlink written 6 days ago by timothy.kirkwood10
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