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Affinity-Enhanced Probe Synthesis

We engineer high-Tm hydrolysis and hybridization probes using LNA/cEt, ZNA®, and MGB, PNA chemistries for qPCR, dPCR, SNP genotyping, and FISH.

LNA / cEt ZNA® (spermine) MGB 3′ caps qPCR • dPCR • FISH RUO → GMP-like

Shorter probes. Stronger binding. Cleaner baselines.

Overview Design Notes Reporters & Quenchers Chemistries & Options Specs & QC Ordering FAQ References Speak to a Scientist

Overview

Bio-Synthesis, Inc. is a trusted U.S. manufacturer of custom oligonucleotides and probes with over 45 years of expertise in advanced modification chemistry, conjugation, and large-scale synthesis. Our Affinity-Enhanced Probe Synthesis Services are designed for researchers and diagnostic developers who require short, high-Tm probes that deliver precise hybridization, robust signal-to-noise ratios, and batch-to-batch consistency.

We employ LNA/cEt monomers, ZNA® polyamine backbones, and 3′ Minor Groove Binder (MGB) caps— individually or in combination—to raise duplex stability and SNP discrimination without increasing probe length. Every project is managed through our in-house LIMS-integrated workflow and produced under ISO 9001 / 13485-aligned quality systems, with complete HPLC purification, ESI-/MALDI-MS verification, and comprehensive documentation (COA, purity, yield, and identity data).

From discovery assays to clinical-grade validation, Bio-Synthesis offers a full continuum of RUO → GMP-like manufacturing, supported by design consultation, formulation, pre-plating, and multiplex balancing. Our Texas and North Carolina facilities enable flexible turnaround times and scalable production from nanomole to multigram levels.

What You Get with Bio-Synthesis
  • Design Consultation & Modeling: Tm optimization, dye/quencher pairing, and multiplex layout support.
  • Advanced Chemistries: LNA / cEt, ZNA®, MGB, 2′-OMe/2′-F, and PNA options for high-affinity probe design.
  • QC & Traceability: Analytical HPLC, mass-spectrometry confirmation, UV quantitation, and COA per lot.
  • Flexible Formats: Tubes, 96-well plates, or custom aliquoting; ready-to-use probe mixes available.
  • Scalable Production: From 50 nmol research probes to multi-gram GMP-like manufacturing.
  • Dedicated Technical Team: Direct access to scientists for assay troubleshooting and sequence refinement.
Supply
RUO → GMP‑like
QC
HPLC • ESI-/MALDI-MS
Turnaround
Priority lanes
Applications
qPCR • dPCR • FISH • SNP

Also inquire for LNA probe synthesis, MGB probe design, ZNA qPCR probe, high-Tm hydrolysis probe, affinity-enhanced oligonucleotide, custom qPCR/dPCR probe manufacturer.

Design Notes

Quick Rules
  • Tm targets: Probe 68–72 °C; primers 60–64 °C (probe ≈6–10 °C higher).
  • LNA/cEt: Start with 2–6 inserts; place near weak AT stretches or SNP; avoid long LNA runs and 5′ adjacency to the reporter.
  • ZNA® charge: Select +2 to +6 to lift Tm uniformly without changing bases; useful when you want native sequence preserved.
  • MGB 3′ cap: Enables the shortest spans with high Tm; pair with NFQ dark quencher for low background.
  • Structure: hairpin/dimer ΔG > −2 kcal/mol; avoid 5′ G next to dye; amplicon 70–150 bp.
SNP & High-GC Targets

Center the SNP (positions 6–12 for 14–20-mers). In GC-rich regions, distribute LNA/cEt sparingly to prevent over-stabilization; consider ZNA® for a smoother Tm lift.

Multiplex Planning

Use spectrally separated reporters (FAM/HEX/Cy3/ROX/Cy5). Balance probe concentrations per channel and verify color compensation on your instrument. Pre-balanced 2–4-plex sets available.

Reporters & Quenchers

Standard hydrolysis layout: 5′ Reporter — probe — 3′ Quencher (3′ blocked). MGB can be combined with an NFQ; LNA/ZNA® designs pair with BHQ/QSY/BBQ classes or double-quenched layouts.

We offer much more than listed below - get in touch!

Reporter Channel Typical Quencher Notes
FAM ~520 nm BHQ-1 / QSY7 / Iowa Black FQ Workhorse green; avoid 5′ G adjacent to dye.
HEX / VIC-compatible ~555 nm BHQ-1 / QSY7 Great for duplex with FAM.
Cy3 / ATTO 550 ~560–570 nm BHQ-2 / QSY9 Orange channel; robust in multiplex.
ROX / TAMRA ~580–610 nm BHQ-2 / QSY21 Use when instrument supports ROX as reporter.
Cy5 / ATTO 647N ~650–670 nm BHQ-3 / QSY35 / BBQ-650 Far-red; low bleed-through.

Chemistries & Options

Select one or combine to meet performance goals (shortest span, highest ΔTm, lowest background, native sequence preservation).

Chemistry How it boosts affinity Best for Notes
LNA / cEt Locked ribose (C3′-endo) increases base-stacking; ~+2–8 °C per insert (context-dependent) SNP discrimination; compact qPCR/dPCR probes Use 2–6 inserts; avoid long LNA runs and 5′ adjacency to reporter.
ZNA® (spermine) Backbone polycations reduce electrostatic repulsion Uniform Tm lift without base substitutions Select +2 to +6 charge equivalent; improves salt tolerance.
MGB 3′ cap Minor-groove binding stabilizes duplex terminus Shortest spans with high Tm; allele assays Pair with NFQ dark quencher; keep amplicons 70–150 bp.
2′-OMe / 2′-F patterning Sugar modifications raise Tm and nuclease resistance Harsh matrices; improved stability Mix with LNA/cEt for balanced kinetics; avoid over-stabilization.
PNA (optional) Charge-neutral backbone; very high affinity & mismatch discrimination FISH, PCR clamping Typically used as probes/clamps; custom labeling options available.

Specs & QC

Build

  • Length: 14–22 nt typical (depends on chemistry & GC).
  • Chemistries: LNA/cEt, ZNA®, MGB, optional 2′-OMe/2′-F.
  • Purification: HPLC (dual-HPLC/UPLC on request).
  • Scales: 50 nmol → 5 µmol+; tubes or plates; custom aliquots.

QC (included)

  • Analytical HPLC trace
  • ESI-/MALDI-MS (m/z)
  • UV/OD yield (A260, nmol)
  • COA with sequence coding & chemistry map

Optional: Endotoxin (LAL), bioburden, sterility, ds%/UPLC purity, functional qPCR/dPCR.

Ordering Checklist (Affinity-Enhanced)

  • ✅ Assay & platform — qPCR/dPCR/FISH; instrument & channels.
  • ✅ Chemistry — LNA/cEt, ZNA® charge (+2 to +6), and/or 3′ MGB-NFQ.
  • ✅ Probe stats — 14–22 nt target; SNP near center; GC 40–60%.
  • ✅ Reporter/Quencher — FAM/HEX/Cy3/ROX/Cy5 + dark quencher (single or double-quenched).
  • ✅ Scale & QC — 50 nmol → 5 µmol; HPLC; optional endotoxin/bioburden.
  • ✅ Format — Tubes or plate; buffer/concentration; barcodes
Speak to a Scientist

Related Probe & qPCR Services

LNA qPCR Probes

Locked nucleic acid designs for compact, high-Tm hydrolysis probes.

MGB Probes

Minor groove binder 3′ caps for shorter spans and tight discrimination.

TaqMan® Probes

Standard hydrolysis probes matched to your affinity-enhanced designs.

FAQ

When should I use LNA/cEt vs. ZNA® vs. MGB?

Use LNA/cEt to locally boost Tm at a SNP or weak region; ZNA® when you want a uniform Tm lift without changing base identity; MGB when the goal is the shortest possible span with high Tm and low background.

How many LNA/cEt positions should I add?

Typically 2–6 per probe, spaced to avoid clusters and 5′ adjacency to the reporter. Model Tm after each addition.

Do these chemistries work in multiplex dPCR?

Yes. Use spectrally separated reporters and balance concentrations per channel. We can supply pre-mixed, pre-balanced sets.

What storage conditions are recommended?

Aliquot at 100 µM in water/TE, store at −20 °C protected from light. Working dilutions (e.g., 10 µM) at 4 °C for ≤2 weeks.

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References

  1. LNA monomers increase hybridization affinity (1998). PubMed
  2. cEt nucleic acids for high-affinity probes (2008). PubMed
  3. ZNA®: polycation-conjugated oligos for affinity boost (2010). PubMed
  4. MGB Eclipse probes for qPCR (2002). PubMed
  5. Quenchers & FRET in real-time probes (2009). PubMed

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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