Fig 1. Mechanism of Eprobe mediated PCR.
During the denaturation step of a PCR reaction, an Eprobe is single-stranded and its signal is suppressed by an excitonic interaction between the two dye moieties (indicated in grey). During the annealing step, the Eprobe binds to its target sequence, and therefore emits a strong fluorescent signal (indicated in green). In qPCR experiments, an Eprobe-derived fluorescent signal is detected during the annealing step. Eprobes are designed in such a way that they commonly have a TM below the elongation temperature. Therefore an Eprobe is normally single-stranded during the elongation step and hence does not interfere with the polymerase extension reaction. As indicated in yellow, the 3’ end of an Eprobe is blocked to avoid any primer extension reactions from the probe.