Hello everybody,
I worked with qPCR using SYBR Green for detection, so primer-dimers were a real bane for me. I tried to design my primers carefully, checked them for homo- and heterodimerization using IDT OigoAnalyzer and tried to avoid any dimers with delta G less than -6 kcal/mole. But this wasn't a perfect method to avoid dimer formation in some cases.
Analyzing melting curves of my amplified samples, I noticed that most primers do not form dimers if the template is present, but can dimerize in no-template control. This can be easily explained by competitive inhibition, because delta G for primer binding with the template is less than for its binding with another primer if there is not enough matches.
But sometimes there were really strange primers that rarely if ever dimerized in negative control, but some of positive samples had small but discernible dimer peak on their melt curve alongside the target peak. Not all samples had this second peak, but in some cases I've seen replicas of the same sample with and without this dimer peak. Interestingly, usually such replicas had the same Ct.
Is there any known mechanism of cDNA template somehow catalyzing primer dimerization?
Doesn't need to be primer dimers: could be mispriming at some other, lower affinity location in your DNA/cDNA.
Important thing to remember about primer dimers and mispriming: these usually appear late, almost always substantially later than the quantitative cycles.
If you're like me, you probably run your qPCRs with 40+ cycles, even though Cq values typically fall between ~16-28 (depending on gene). The extra cycles help show that negatives truly are negative, and can also help identify primer dimers: after 40 cycles of exponential amplification, ANYTHING that can serve as an amplicon, no matter how poorly, will get amplified.
It also means that you may well find primer dimer signal in your test samples, but in most cases you can safely ignore this, because the primer dimers didn't start forming or being amplified until your target amplicon had already been quantified. You only measure the Cq ONCE per well, after all.
If triplicates of a given sample (one with obvious primer dimer signal) are all very similar in measured Cq, then trust the Cq.
If triplicates of a given sample (one with obvious primer dimer signal) are very similar EXCEPT for the one with primer dimers, then don't trust that specific Cq.
If you want to really test it, run a regular PCR with replicates of the same sample and harvest wells at different cycles: if your amplicon has a Cq of 22, then collect one well at cycle 24, one at 28, one at 32 and one at 36.
You might see a band in the first sample, a stronger band in the second, a strong band + hint of primer dimers in the third and a strong band + prominent primer dimers in the fourth.
This tells you that your amplicon appears (and is quantified) long before primer dimer fluorescence can contribute to your signal.
Thank you John.
Honestly, I didn't think about possible false priming because I always blast primers to confirm they are specific. But since I work with Helix lucorum snail, the organism whose genes are poorly studied and very few sequences of its genes are available, of course there may be possibilities for primers binding to some non-target snail cDNA.
I always run PCR for exactly 40 cycles, and usually my target genes have Cq 24-29. If dimers are amplified in water, Cq is 34 and more; RT- controls are sometimes amplified with Cq 32 and more.
Dear Ekaterina
- Apologies for my late answer
- To go back to your original thesis yes you could potentially encourage primer dimer formation in the presence of template by altering ion and dNTP concentration which will have an effect on duplex stability
- To be pragmatic though if you primer dimer formation is a constant problem regardless of making new primers, then that could be target and therefore sequence specific
- In this situation, try adding 5% DMSO to your reaction and also pick a completely different region to design primers to
- Even if you continue to be plagued with primer dimers providing they are > 5 cycles from your real product and esecially if thet are > 10 cycles from your specific amplicon - as is usually the case - I would not worry as this will not interfere with relative gene expressio calculations (this is not tru efor absolute quantitation)
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