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

Mol Cell Probes. 2016 Oct;30(5):285-290. doi: 10.1016/j.mcp.2016.10.003. Epub 2016 Oct 6.

Abstract

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).

Keywords: Couple quencher; JOE dye; Linkers; Molecular beacons; Real-time PCR.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • DNA Probes / genetics
  • Fluoresceins / chemistry*
  • Fluorescent Dyes / chemistry*
  • Molecular Probes / chemistry*
  • Nucleic Acid Denaturation
  • Polymerase Chain Reaction
  • Reference Standards
  • Temperature
  • Xanthenes / chemistry

Substances

  • 4',5'-dichloro-2',7'-dimethoxy-5(6)-carboxyfluorescein
  • DNA Probes
  • Fluoresceins
  • Fluorescent Dyes
  • Molecular Probes
  • Xanthenes