I’m performing qPCR assays to amplify a Leuconostoc mesenteroides gene and for this, I’ve used a probe from the literature that is labeled with FAM and MGB (FAM-TTC ACT CTT TTC AAT ACT TAC TG-MGB). With the possibility of realizing a multiplex in the future, and for reasons of cost, I changed the fluorophores: the fluorophore reporter for JOE and the quencher for BHQ-1. After the qPCR assay we didn’t get any amplification and so I seek answers to that. Our laboratory has the thermocycler StepOne (Applied Biosystems).
The MGB dye increases the Melting Temperature (Tm) of the probe and, as I switched to BHQ, I calculated Tm through OligoAnalyzer 3.1 software (https://www.idtdna.com/calc/analyzer) and is extremely low (
While on the one hand, I would be a little worried that your probe and primer have similar Tms. They really ought be seperated by ~10 degrees or so.
Also, FAM with MGB should multiplex just fine. MGB stands for Minor Groove Binding and does not work as does a second dye. For example, if FAM is coupled with TAMRA, the emission of FAM is the excitation of TAMRA so until the probe is used and cleaved you don't see FAM signal. MGB works under a different principle. I believe that JOE can be quenched by MGB just fine.
If you have your heart set on the BHQ, I'd recommend redesigning your probe with another string of bases to bring the Tm up to 70ish.
Dear Matthew,
Thank you for your quick response.
I'll use the probe FAM-MGB to avoid further problems.
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