I'm trying to design qPCR probes for a detection essay. Our lab has 2 thermocyclers : a StepOnePlus (Applied Biosystems) and a BioRad CFX96.
My question is specifically on the quencher part of the probe. The StepOnePlus software only offers options for TAMRA/NFQMGB/none as quenchers. Is it still possible to use other quenchers like Black Hole Quencher (BHQ) or Deep Dark Quencher ? If yes, what option should I select when designing my essay ?
On the other hand, if I order my probes via AB, will the NFQMGB probes be compatible with the BioRad CFX96 machine?
Hi Sébastien,
I am unfamiliar with the AB system, but our lab routinely uses TaqMan probes on a Bio-rad CFX96 thermocycler.
As far as I know, Life Tech offers their own proprietary quenching chemistries (including MGBs), and I don't think you can add BHQs to the dual-labeled probes.
We typically synthesize all of our oligos through IDT (http://www.idtdna.com/site/Order/Menu/Sub?type=QPCRPROBES). They offer a range of fluorescent probes and quenchers that are compatible with the CFX96 instrument. In our experience, BHQs or IowaBlack quenchers seem to do very well for both FAM and HEX fluorophores. TAMRA works as well, albeit with some reduced quenching capabilities; usually not our first choice although they may work well for AB instruments.
As for MGB probes, we rarely use these and we find that dual-labeled probes work just as well for our needs. However, I do recall having tried using a VIC-TAMRA pair (synthesized by AB/Life Tech) that worked relatively well when read using the HEX channel on CFX96.
I would direct your attention to this publication for a careful analysis of quenching efficiencies: http://www.ncbi.nlm.nih.gov/pubmed/16785617
Hopefully it may shed some light on the situation
Bottom line, different companies will offer different products, at different prices. So it all comes down to the type of application you require.
Hope this helps
Hi, I am facing the same problem now. We have ordered probes labeled with FAM, and BHQ as a quencher. And we also work on STep One Plus. Since these probes come from the kit for the viral detection, I have written to the company (PrimerDesign) what quencher should I mark in the StepOne program. Today I got an email that assay will work perfectly if I mark NFQMGB. Well, I will check it tomorrow.
Thank you Eric for a very complete answer.
I finally ordered 2 probes : a FAM-BHQ1 and a YakimaYellow-BHQ1. They both work perfectly on both the StepOne+ and the CFX96 using a Fast mix. The channel used for the YakimaYellow detection is VIC. On the StepOne+ software, I use NFQMGB as the parameter for the quencher and it works without any problem.
Hey Eric,
I have also FAM and TAMRA dyes with BHQ1 as quencher and running Step one Plus Instrument. I really appreciated for your question.
I am facing with some problems while setting up the program I think. Yesterday I tried to run a multiplex experiment with these dyes and select the NFQ-MGB as quencher but could not receive any melting curve or amplification. I am planing to run the product on conventional PCR and double check If there is no amplification.
Have a great day.
It's the same issue with the ABI 7500 thermocycler. Got two probes FAM-MGB and TAMRA-BHQ2 and will go for NFQ-MGB for both
Does the selection of the quencher option in the software affect the reading at all? I'm wondering because I'm using one of these ZEN double quenched probes from IDT (https://www.idtdna.com/pages/education/decoded/article/modification-highlight-zen-internal-quencher). There is no option in ABI's Quantstudio instruments for this quencher.
They refuse to give information about how the actual raw fluorescence values (in the various filters) are converted into fluorescence values (arbitrary units) based on the calibration. Quenchers are definitely not being calibrated for anyways..
Hello Prashant Kalvapalle,
Wondering if you had any success with the ZEN double quencher probes on your Quantstudio? I am trying to optimize double quencher probes on our Quantstudio 5. Appreciate your feedback.
Thanks,
Abishek
Hi @Abhishek. I have tried FAM labelled ZEN double quenched probe from IDT and it worked fine on Quant studio 3. I just used the same 0.25 uM conc. For both the primers and probe and didn't have to optimize anything.
Although I didn't tried multiplexing yet for lack of cheap probes with other dyes in small quantities. Will try Sigma's dual labelled probes soon - https://www.sigmaaldrich.com/technical-documents/articles/biology/dual-labeled-probes.html
Yes you can select none in part wuencher and use Bhq1 and other and you can use nfq mgb with biorad
Thank you all for these useful exchanges! I am facing a problem too with my probes for the diagnosis of the covid19. I ordered a Fam-Tamra probes but the company synthetized a Fam-BHQ...!! The first tests had no worked, and now i am doing another testing...if you have any idea which can help it will be greatful! I have different devices: ABI7500, CFX96 and Lightcycler 480. We also have smartcyclers! Thank you!
Hi Ndongo, you might try Elitech--they have photostable dyes that cover UV-near IR, and quenchers to go with them. Also MGB, duplex stabilizing bases, duplex stabilizing quencher, universal bases, etc. Hth, Noah
Its always a good idea to go to the company website to check for compatibility and also talk to the company rep or engineer. That way the effort of designing will be rewarded. Follow the guidelines on CDC site. Primer probe details are mentioned for easy reference.
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