We are having a perplexing issue with some IDT double-quenched probes. We have a particular amplicon with a SNP and have control DNA that is wild-type, heterozygous, and homozygous for the SNP. Using a double-quenched FAM/ZEN probe for the wild-type sequence, we see adequate signal amplification and delineation of genotypes. However, using the same exact probe sequence but with Cy5 dye/TAO, we see very poor amplification. Nothing is different between the two probes other than the dye type. The fluorescence plotted there is raw, and both probes were used in the same run with the same reagents at the same time.
I realize that different dyes can have different performances, but we did not think it should make this severe of an impact. We have designed this dual-probe experiment with several other SNPs successfully. It is with only this one sequence that we are having this problem. We have tried several different annealing temperatures, probe concentrations, etc. to no avail. The primers we are using have been validated extensively. We have even tried validating an alternate primer set with a melting temperature more conducive to the probe melting temperature. Still the same problem. Any ideas?
It's possible that the optics of the instrument are not picking up signal as well as they are with the other dye pair. Maybe this could be a calibration issue or just an inherent limitation of the signal strength of the dye?
If you take the end products out of the qPCR reaction and run each reaction out on a gel, do you get roughly the same amount? If so, that might just suggest a detection issue. If not, then that would confirm that it is an amplification issue.
Well I am assuming you double checked your order and that the sequences are in deed correctly made. Your probe sequences listed in this pdf are identical but the reporter and double quencher are also the same. They are both listed as FAM/ZEN. Looking at the graphs indicates to me that no amplification was detected in CY5 channel. So based on this you are not using a correctly labelled probe. Also this was ordered twice which indicates that it was not checked by a second or third pair of eyes before ordering. Also, are you multiplexing both of these probes in the same reaction or separate reactions?
Thank you all for responding! This assay is being used for SNP detection. This SNP happens to be located upstream and adjacent to the internal quencher. Does anyone know if that might cause problems? IDT did not seem to think it would, but perhaps someone has experience otherwise.
@ Govinda Sharma and Mathew Beale - The instrument we use is a RotorGene Q, which does not require calibration. I will try running the amplification on a gel to see if amplification is the issue. I doubt the dye has been over exposed to light because this run was done a day or two after the probe was received from IDT.
@ Allen Teal - Sorry! The dye and internal quencher listed on the second probe are incorrect. I have double checked the orders/sequences and the probes should be listed as:
FAM/ZEN Probe: /56-FAM/AGGAACTTC/ZEN/TTAGTGCTCTCCACA/3IABkFQ/
Cy5/TAO Probe: /5Cy5/AGGAACTTC/TAO/TTAGTGCTCTCCACA/3IAbRQSp/
The graphs in the attached PDF are from a signal run without multiplexing. The same master mix (buffer, dNTPs, Taq, H2O) and control DNA was used for both probes. The only differences between the reactions are are the dyes attached to the probes in the separate reactions.
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