We created a drop off ddPCR for the detection of PIK3CA mutations. FAM probe is perfectly matched with WT. We noticed that when we used wt samples one cloud was formed.When we used mutated samples (mixed wt and mutated) two clouds were formed but with different amplitude. The probe is MGB. Does anyone knows why this occurs? Could MGB effect the fluorescence when a mismatch is present??
We observed also similar results with ddPCR.
One possible explanation is that your FAM WT probe also detects the mutated sequence but with a reduced efficiency. You can have a look to Fig 3 in our paper Article Reliable and robust droplet digital PCR (ddPCR) and RT-ddPCR...
showing similar results and possible explanations
Greetings Ms. Athina Markou ..! Usually, when a variant sequence of a gene is provided, there are chances that the mutated variant one can also amplify. But since the mutated sequence has less match with primers/probes compared to WT samples, a second cluster is formed with less amplitude.
Hence, the topmost cluster is that of WT samples and the 2nd one is mutated one. The role of MGB is to fluoresce when the quenching does not occur, hence the occurred fluorescence is due to amplification of the mutated sequence and it is not the fault of MGB.
If you want to eliminate the occurrence of the mutated sample amplification, it is suggested to increase annealing temperature to prevent off-target amplification. Assay redesigning can also be a good option, but if nothing works, change/redesign the probe to minimize complementarity to the mutated sequence.
For more in-depth understanding than my explanation, visit Page 111 - 112 in the Bio-Rad ddPCR applications Guide (6407a) by using this link: https://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6407.pdf
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