Molecular beacons with JOE dye: Influence of linker and 3' couple quencher.

Molecular beacons carrying JOE dye (4',5'-dichloro-2',7'-dimethoxy-6-carboxyfluorescein) on a rigid or flexible linker and one or two BHQ1 quenchers have been prepared and tested in real-time PCR using Fusarium avenaceum elongation factor 1α DNA template. The probes were different in their structures (loop size and stem length), linkers for dye attachment (6-aminohexanol or trans-4-aminocyclohexanol), quencher composition (single and double BHQ1) to elucidate the influence of all these features. Fluorogenic properties of the probes were studied and compared to those of FAM (fluorescein)-based probes. All the factors - stem length, JOE vs FAM, rigid vs flexible linker, single vs double quencher - appeared to play a considerable role in the probe's fluorescent properties and determine the usability of the probe at two different temperatures of fluorescence detection (55°С and 64°С).