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

- A series of Molecular beacons with different structure carried JOE was tested in qPCR.
- JOE proved to be brighter than FAM with probes with the same sequence.
- The length of MB's stem was varied from 4 to 8 bp to optimize MBs design.
- A rigid linker for JOE attaching is preferable for increasing light-up of fluorescence.
- The second BHQ1 in probe significantly decrease background of fluorescence.

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).