FAQ: What is the difference between probe- and dye-based versions of the Luna® qPCR Mixes?

qPCR is typically measured in one of two ways: via an intercalating dye that fluoresces more strongly upon binding to double-stranded DNA, or via a fluorescently-labeled “probe” oligonucleotide that anneals to a specific sequence in the PCR amplicon. Background fluorescence of the probe is prevented by the presence of a quenching group. In the case of hydrolysis probes, the quencher is separated from the fluorophore during amplification by the 5´→3´ nuclease activity of Taq DNA Polymerase. To enable dye-based detection, the Luna Universal qPCR Mix contains an intercalating dye that is detected in the SYBR® Green or FAM channel of most real-time instruments. The Luna Universal Probe qPCR products do not contain this double-stranded DNA binding dye (since it would interfere with the use of FAM-labeled probes). Users must supplement the mix with a labeled probe oligonucleotide in addition to forward and reverse PCR primers. Please note that the passive reference dye present in both mixes does not interfere with the use of ROX labeled probes.