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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 Design Notes Quencher Catalog Probe Formats Ordering FAQ References Speak to a Scientist

Overview

What it is At‑a‑Glance Why it matters Use cases Mechanisms Design impacts

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.

Anchor Quencher 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.
#BBQ‑650 BlackBerry® Quencher (BBQ‑650®) ~650–750 nm Near‑IR/far‑red; excellent for Cy5/647 channels.
#QSY7 QSY® 7 ~500–560 nm Green channels; alternative to BHQ‑1.
#QSY9 QSY® 9 ~560 nm Cy3/ATTO 550 class dyes.
#QSY21 QSY® 21 ~580–610 nm TAMRA/ROX region.
#QSY35 QSY® 35 ~650–700 nm Cy5/near‑IR; low bleed‑through.
#IAB Iowa Black® FQ / RQ FQ: ~480–580 nm; RQ: ~580–670 nm Common in qPCR probe kits; rugged and economical.
#Eclipse Epoch Eclipse™ Quencher ~560–610 nm Good with Cy3/TAMRA; legacy compatibility.
#ATTO540Q ATTO 540Q ~520–560 nm Pairs with FAM/HEX when ATTO dyes are used elsewhere in panel.
#ATTO580Q ATTO 580Q ~560–600 nm Cy3/ATTO 550 class.
#ATTO612Q ATTO 612Q ~600–630 nm ROX/TAMRA vicinity.
#DYQ‑1 Dyomic DYQ‑1 ~560 nm Orange; alternative to BHQ‑2/QSY 9.
#DYQ‑2 Dyomic DYQ‑2 ~560–600 nm Orange‑red coverage.
#DYQ‑4 Dyomic DYQ‑4 ~600–620 nm Red edge; ROX region.
#DYQ‑425 Dyomic DYQ‑425 ~500–540 nm Green; FAM/HEX.
#DYQ‑505 Dyomic DYQ‑505 ~520–560 nm Green‑yellow; FAM/HEX/TET.
#DYQ‑660 Dyomic DYQ‑660 ~650–670 nm Cy5 channel.
#DYQ‑661 Dyomic DYQ‑661 ~660–680 nm Far‑red.
#DYQ‑700r Dyomic DYQ‑700r ~700–720 nm Near‑IR.
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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.
Speak to a Scientist

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.

Speak to a Scientist

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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

Trusted by biotech leaders worldwide for over 40+ years of delivering high quality, fast and scalable synthetic biology solutions.

Greetings Researchers,

Please be advised that any information, guidelines, writeups, and oral/video/graphic content on our website are intended solely as general guidelines.
It is the researcher's sole responsibility to conduct thorough research on the designs of the products intended for their experiments before submitting
their designs to Biosynthesis for review of production feasibility.
Thank you for your understanding.

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