Fluorescence Quenching & Signal Suppression

Oligonucleotide quenchers for ultra‑low background assays: double‑quenched hydrolysis probes, molecular beacons, FRET pairs, and electrochemical formats. RUO → GMP‑like manufacturing with ISO‑aligned QC.

qPCR hydrolysis (TaqMan‑style)
Molecular beacons
FRET sensors
Electrochemical

Overview

Fluorescence quenching is the intentional suppression of a donor dye’s emission by a nearby quencher. In oligonucleotide probes, this keeps the assay dark until a target‑dependent event (e.g., polymerase cleavage or hairpin opening) separates the pair or disrupts their interaction—yielding high signal‑to‑noise (S/N) precisely when and where it matters.

Quenching can be dominated by contact (static π–π interactions), dynamic (collisional) processes, and/or FRET when a spectral acceptor is used. Modern dark quenchers dissipate energy non‑radiatively and minimize bleed‑through in multiplexed panels.

Bio‑Synthesis, Inc. (Lewisville, Texas) is a U.S. manufacturer of custom fluorescence‑quenched oligonucleotides for research, diagnostics, and therapeutic development. We design and produce double‑quenched qPCR probes, molecular beacons, FRET pairs, and electrochemical probes, with options for internal/3' dark quenchers, spacers, linkers, and alternate backbones.

  • Supply & scale: RUO → GMP‑like; 10 nmol to multi‑µmol; project & campaign support.
  • QC & documentation: HPLC (RP/IE), ESI‑MS, UPLC purity, ds%; extended GLP/cGMP‑like docs on request.
  • Quenchers covered: BHQ‑1/2/3, BBQ‑650, QSY® 7/9/21/35, Iowa Black® FQ/RQ, Eclipse™, ATTO 540Q/580Q/612Q, DYQ series.
  • Design help: dye/quencher mapping for multiplex, probe length & spacing, leakage control.
Experience
45+ years
Supply
RUO → GMP‑like
QC
HPLC • ESI‑MS
Turnaround
Priority lanes
Why it matters
  • Sensitivity: Lower baseline → earlier Ct/Cq and improved LOD.
  • Specificity: Quiet off-state reduces false calls from probe breathing/partials.
  • Multiplexing: Dark quenchers cut bleed-through vs fluorescent acceptors.
Use cases
  • qPCR hydrolysis probes (TaqMan-style) and digital PCR
  • Molecular beacons for SNP genotyping & RNA imaging
  • FRET biosensors and smFRET studies
  • Electrochemical probes (MB/Fc) & point-of-care cartridges
Mechanisms
  • Contact (static): π–π stacking at short spacings suppresses baseline.
  • Dynamic (collisional): Increases with flexible linkers & temperature.
  • FRET: Requires spectral overlap; enables ratiometric options.
Design impacts
  • Positioning: Internal quencher ~8–12 nt from dye; 3' quencher curbs leak.
  • Chemistry: Match quencher to dye band (e.g., BHQ-1/QSY 7 for FAM; BHQ-3/BBQ-650/QSY 35 for Cy5).
  • Architecture: Double-quenched (internal + 3') for >25-mers/high-GC.

Dark Quencher Catalog

Representative offering. Additional brands and placements (5', internal, 3') available on request.

Quencher Mod.Code Recommended Emission Range Notes / Typical Use
BHQ®‑1 [BHQ‑1] ~480–580 nm (FAM/HEX/TET) General purpose for green/yellow dyes; low native fluorescence.
BHQ®‑2 [BHQ‑2] ~560–610 nm (Cy3/TAMRA/ROX) Broad orange‑red coverage; robust for qPCR.
BHQ®‑3 [BHQ‑3] ~620–670 nm (Cy5) Optimized for far‑red emissions.
BlackBerry® Quencher (BBQ‑650®) [BBQ‑650] ~650–750 nm Near‑IR/far‑red; excellent for Cy5/647 channels.
QSY® 7 [QSY7] ~500–560 nm Green channels; alternative to BHQ‑1.
QSY® 9 [QSY9] ~560 nm Cy3/ATTO 550 class dyes.
QSY® 21 [QSY21] ~580–610 nm TAMRA/ROX region.
QSY® 35 [QSY35] ~650–700 nm Cy5/near‑IR; low bleed‑through.
Iowa Black® FQ / RQ [IAB] FQ: ~480–580 nm; RQ: ~580–670 nm Common in qPCR probe kits; rugged and economical.
Epoch Eclipse™ Quencher [Eclipse] ~560–610 nm Good with Cy3/TAMRA; legacy compatibility.
ATTO 540Q [ATTO540Q] ~520–560 nm Pairs with FAM/HEX when ATTO dyes are used elsewhere in panel.
ATTO 580Q [ATTO580Q] ~560–600 nm Cy3/ATTO 550 class.
ATTO 612Q [ATTO612Q] ~600–630 nm ROX/TAMRA vicinity.
Dyomic DYQ‑1 [DYQ‑1] ~560 nm Orange; alternative to BHQ‑2/QSY 9.
Dyomic DYQ‑2 [DYQ‑2] ~560–600 nm Orange‑red coverage.
Dyomic DYQ‑4 [DYQ‑4] ~600–620 nm Red edge; ROX region.
Dyomic DYQ‑425 [DYQ‑425] ~500–540 nm Green; FAM/HEX.
Dyomic DYQ‑505 [DYQ‑505] ~520–560 nm Green‑yellow; FAM/HEX/TET.
Dyomic DYQ‑660 [DYQ‑660] ~650–670 nm Cy5 channel.
Dyomic DYQ‑661 [DYQ‑661] ~660–680 nm Far‑red.
Dyomic DYQ‑700 [DYQ‑700] ~700–720 nm Near‑IR.
Probe Formats
Hydrolysis (qPCR) Probes
5' dye + internal/3' quencher; signal increases upon polymerase‑mediated cleavage.
Molecular Beacons
Hairpin keeps dye near quencher until target hybridization opens the loop.
Double‑Quenched Designs
Internal quencher near dye plus terminal quencher; improves baseline on longer probes.
FRET Pairs
Donor–acceptor combinations for ratiometric readouts; useful in sensors and smFRET.
Electrochemical Probes
Redox labels (MB/Fc) with structural quenching on electrodes for ultra‑low background.

Design Notes

Quick Recipes
  • Hydrolysis probes (qPCR): 5' dye, internal quencher at ~+9 ±2 nt, 3' dark quencher; length 18–30 nt; Tm target ≈ assay temp +10–15 °C; avoid runs of G adjacent to dye.
  • Molecular beacons: 5' dye, 3' quencher; 4–6 bp stem; loop 18–30 nt; stem Tm > assay temp by 7–10 °C; place dye away from guanines.
  • FRET sensors: donor 5' / acceptor internal or 3'; adjust overlap integral; use ratiometric readout to cancel drift.
  • Electrochemical: redox tag (MB/Fc) + quencher/hairpin geometries to suppress off‑state; thiol to gold for electrodes.
Spacer & Positioning

Internal placement 8–12 nt from the dye often minimizes baseline while preserving cleavage/readout. C3/C6 spacers or abasic (rSpacer) can tune flexibility and distance.

Choosing a Quencher
  • FAM/HEX/TET (~500–560 nm): BHQ‑1, Iowa Black FQ, QSY 7, ATTO 540Q, DYQ‑505.
  • Cy3/ATTO 550 (~560 nm): BHQ‑2, Eclipse, QSY 9, ATTO 580Q, DYQ‑1/2.
  • ROX/TAMRA (~580–610 nm): BHQ‑2, QSY 21, ATTO 612Q, DYQ‑4.
  • Cy5/ATTO 647N (~650–670 nm): BHQ‑3, BBQ‑650, QSY 35, DYQ‑660/661.
  • Near‑IR (700 nm+): BBQ‑650, QSY 35, DYQ‑700r.
Double‑Quenched Probes

Internal + 3' quenchers reduce baseline on long probes (>25 nt) and tough amplicons. Internal spacing near the dye (+7 to +10) improves contact quenching; 3' quencher controls exonuclease‑independent leakage.

Controls & QC

Include NTCs, passive references as required, and verify purity (HPLC) and mass (ESI‑MS). For regulated studies, request extended QC (bioburden/endotoxin, residual solvents, ds% by UPLC, stability studies).

Ready to design darker, cleaner probes?

Ordering checklist for quenched oligos
  • Assay & platform — qPCR (hydrolysis) / Molecular beacon / FRET / Electrochemical; instrument model & channels.
  • Dye & Quencher — Donor (e.g., FAM/HEX/ROX/Cy5), dark quencher (BHQ‑1/2/3, QSY 7/9/21/35, BBQ‑650, Iowa Black, Eclipse, ATTO 540Q/580Q/612Q, DYQ).
  • Layout — 5′ dye, internal quencher (+7 to +10), 3′ quencher; single vs double‑quenched; hairpin vs linear.
  • Probe stats — Length (18–30 nt typical), target Tm, GC%, target region, avoid G next to dye.
  • Format & docs — Tubes/plates, buffer/concentration, RUO → GMP‑like documentation.

FAQ

What is fluorescence quenching in oligonucleotide probes?

Reduction of donor fluorescence by a quencher (contact/FRET/collisional), keeping baseline low until target activation.

When do double‑quenched probes help?

On longer probes (>25 nt) or high‑GC targets; they reduce baseline and improve S/N.

Do you support regulated projects?

Yes—request extended QC and documentation for GLP/cGMP‑like needs.

Which quencher should I pick for FAM?

BHQ‑1, QSY 7, Iowa Black FQ, ATTO 540Q, or DYQ‑505 typically perform well with FAM/HEX‑like emissions.

Can I multiplex with multiple quenchers?

Yes. Match each dye’s emission to a compatible quencher to minimize crosstalk and baseline fluorescence.

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References

  1. Molecular beacons (1996). Article PubMed
  2. Real‑time quantitative PCR (1996). PubMed
  3. Real‑time qRT‑PCR 5′‑nuclease assay (1996). PubMed
  4. Fluorescence quenching & FRET efficiency (2002). Article
  5. Eclipse™ dark quencher examples (2007). Article
  6. BHQ overview (tech note). Article
  7. MGB Eclipse probes (2002). PubMed
  8. Review — Molecular beacons in diagnostics (2012). Article
  9. Review — Rationally designed molecular beacons (2015). Article
  10. QSY® quencher applications (examples). Article
  11. Practical BHQ vs TAMRA evaluation (2009). PubMed
Further Reading (Bio‑Synthesis)
  1. MERFISH Read‑Out Probes (service). Page
  2. Designing readout probes for MERFISH/smFISH. Page

Why Choose Bio-Synthesis

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