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Molecular beacons — ultra-low background and sharp single-nucleotide specificity

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Custom Molecular Beacons

Hairpin probes • qPCR & isothermal detection • SNP genotyping • Imaging

Overview Design Guide Dyes & Quenchers Use Cases Synthesis Options QA FAQ Contact
ISO 9001:2015 / ISO 13485:2016 45+ Years of Expertise U.S.A. Facilities-Texas GLP/GMP-Aligned

Overview

Bio-Synthesis designs and manufactures **molecular beacon probes** for real-time PCR, isothermal assays, SNP genotyping, and imaging. Beacons are **hairpin oligos** carrying a 5′ fluorophore and 3′ quencher; when unbound, the stem keeps the dye and quencher in close proximity for **ultra-low background**. Upon perfect target hybridization, the hairpin opens and **fluorescence is restored**.

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At-a-Glance

  • Formats: standard hairpin, short LNA/BNA beacons, multiplex dye panels
  • Backbones: DNA; 2′-OMe/2′-F; LNA/BNA mixmers for higher Tm and specificity
  • Applications: qPCR/dPCR, **isothermal** (LAMP/RPA), SNP/miRNA detection, imaging
  • Purification/QC: HPLC (standard), PAGE optional; **MS confirmation**
  • Scales: 1 mg to grams; tubes or 96-well plate format

Technology & Benefits

Technology

  • Hairpin architecture: Stem holds fluorophore and quencher
    in proximity; loop is complementary to the target.
  • Signal-on binding: Target binding opens the stem–loop,
    separating dye and quencher, restoring fluorescence.
  • Reversible detection: Beacon remains intact (non-cleaving), enabling melt analysis and reuse across cycles.
  • Thermal tuning: LNA/BNA or 2′-modifications raise Tm for
    shorter, more specific beacons.
  • Multiplex-ready: Distinct dyes with minimal spectral overlap
    support multi-target panels.

Benefits

  • Ultra-low background when unbound → clean baselines.
  • Single-nucleotide discrimination from stem-loop energetics.
  • Isothermal compatibility (LAMP/RPA) and real-time qPCR/dPCR.
  • Non-destructive (no probe cleavage), enabling post-run melt analysis.
  • Reproducible manufacturing with HPLC + MS QC.

We tune stem strength, loop length, and backbone chemistry to your temperature and matrix.

Design Guide

  • Loop (target region): ~18–30 nt (miRNA: 15–22 nt with LNA/BNA).
  • Stem: 5–7 bp, GC-rich; avoid complementarity within the loop.
  • Tm rules: Tm(stem) ≈ assay temp + 5–10 °C; Tm(loop–target) ≥ assay temp + 7–10 °C.
  • Placement: Avoid terminal 5′-G (can quench some dyes); minimize secondary structure in the target.
  • Concentration: Start 100–250 nM (optimize 50–400 nM).
  • Chemistry: Consider 2′-OMe/2′-F or LNA/BNA mix for short targets and SNPs.
  • Multiplexing: Separate dye emissions by ≥40–60 nm; balance brightness.
  • Controls: NTC and mismatch controls; verify closing/opening by melt.
  • Imaging: Add PEG/AEEA spacers to reduce self-quenching near dyes.

Dyes & Quenchers

Dye (typical Ex/Em, nm) Suggested Quencher Notes / Use
FAM (~495/520) Dark quencher 480–580 Green channel; avoid 5′-G
HEX / VIC-like (~535/555) Dark quencher 520–600 Pairs well with FAM
TAMRA / ROX (~560–585/580–610) Dark quencher 560–650 Orange–red; check reference settings
Texas Red (~595/615) Dark quencher 580–670 Red channel; strong multiplex with FAM+HEX
Cy5 (~650/670) Dark quencher 640–700 Far-red; minimal crosstalk with FAM/HEX
Cy5.5 / Alexa 700 (≥690/710+) Dark quencher 690–760 Near-IR; confirm filters

ATTO® and Alexa Fluor® families, internal quenchers for longer loops, and custom FRET schemes available on request.

We offer much more than listed! - get in touch   
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Use Cases

Use Case Why Beacons Help Typical Setup
Isothermal detection (LAMP/RPA) Signal-on hairpin enables clean, real-time readout Beacon tuned to reaction temp; 100–250 nM start
SNP / miRNA discrimination Sharp single-base specificity from stem–loop energetics Short LNA/BNA mix loops; mismatch control
qPCR/dPCR monitoring Very low baseline; reversible (no probe cleavage) Hairpin with dye/quencher; melt analysis post-run
Imaging / in situ Signal only on perfect hybridization; low background Dye-beacon with PEG/AEEA spacer; matched filters
Plan My Beacon

Custom Synthesis Options

Parameter Options
Format Standard hairpin; short LNA/BNA beacons; internal-quencher (long loops); FRET beacons
Backbone DNA; 2′-OMe/2′-F; LNA/BNA mixmers
Labels FAM, HEX/VIC-like, TAMRA/ROX, Texas Red, Cy3/Cy5, ATTO/Alexa
Quenchers Dark quenchers spanning 480–730+ nm
Purification RP-HPLC (standard), PAGE on request
Scale 1 mg to grams; tubes or 96-well plates
Deliverables Lyophilized or in buffer; MS & analytical HPLC report

Quality Assurance

  • Mass spectrometry for identity confirmation
  • Analytical HPLC/PAGE for purity and integrity
  • Optional functional checks (melt curves, baseline, ΔRn)
  • RUO standard; GLP/cGMP support available

Typical Timelines

Known designs: 1–2 weeks. Complex multiplex sets or special labels: 2–3 weeks depending on dyes and purification.

Lead time varies with label availability and QC scope. Rush options may be available.

Oligo-get-started

How to Order

  1. Share target sequence/region and assay platform (qPCR, LAMP, imaging).
  2. Choose backbone (DNA/2′-mod/LNA/BNA), dyes, and quenchers.
  3. Select scale, purification, and any functional QC.
  4. Receive a same-day quote and timeline.

Specs Checklist

  • Target coordinates/sequence; assay temperature
  • Desired loop length and stem strength (or “recommend”)
  • Dye/quencher set; multiplex plan (if any)
  • Scale, purification, buffer, and documentation needs
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FAQ

Molecular beacon or hydrolysis probe?
Beacons offer **ultra-low background** and **reversible**, non-cleaving detection—great for isothermal assays and melt analysis. Hydrolysis probes are preferred for standard qPCR/dPCR when proportional, cleavage-based signal is desired.
How do I pick stem length and Tm?
Use **5–7 bp** GC-rich stems with Tm(stem) ≈ assay temp + 5–10 °C. Ensure Tm(loop–target) is above assay temp so the beacon opens on perfect match but remains closed otherwise.
Can beacons discriminate single-nucleotide variants?
Yes—beacons excel at **SNP and miRNA** detection. Shorter loops and LNA/BNA mixmers sharpen mismatch discrimination.
Are beacons suitable for multiplexing?
Yes. Choose **non-overlapping dyes**, balance brightness across channels, and validate NTC/no-probe controls for flat baselines.
What purification and QC do you provide?
RP-HPLC with **MS confirmation** is standard. PAGE and functional checks (melt, baseline, ΔRn) are available on request.
More FAQ'sAll FAQ's

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