Login / Register Order My Items
Contact Us
800.227.0627
Contact Us Quote Order Login / Register

Custom MGB Probe Synthesis

Short probes. High Tm. Real discrimination. Minor Groove Binder (MGB) hydrolysis probes engineered for qPCR and SNP genotyping with lower background and cleaner ΔRn.

qPCR hydrolysis SNP genotyping Multiplex panels RUO → GMP‑like
Overview Design Notes Dyes & Quenchers Specs & QC Ordering FAQ References Speak to a Scientist

Overview

MGB probes incorporate a minor‑groove‑binding moiety at the 3′ end alongside a non‑fluorescent dark quencher. The MGB raises duplex stability so you can use shorter probes (≈14–18 nt) without losing Tm, tightening SNP discrimination and reducing leak.

  • Higher Tm per base: Short probes with robust binding and cleaner baselines.
  • SNP power: Central mismatches resolve better with compact, high‑Tm designs.
  • Lower noise: 3′ MGB‑NFQ keeps off‑state dark for earlier Ct/Cq.
Supply
RUO → GMP‑like
QC
HPLC • ESI‑MS
Turnaround
Priority lanes
Formats
Single / Multiplex

Dyes & Quenchers

Standard layout: 5′ Reporter — probe — 3′ MGB‑NFQ (3′ blocked). Multiplex with spectrally separated reporters.

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

Specs & QC

Build

  • Length: 13–24 nt (sweet spot 14–18 nt)
  • Purification: HPLC (dual‑HPLC available)
  • Formulation: dry or resuspended; custom aliquots & plates
  • Scales: 50 nmol, 200 nmol, 1 µmol, 5 µmol+

QC (included)

  • Analytical HPLC trace
  • ESI‑/MALDI‑MS (m/z)
  • UV/OD yield (nmol, A260)
  • COA with sequence coding & dye/quencher IDs

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

Design Notes

Quick Rules
  • Place the SNP near center (positions 6–12 for 14–18‑mer).
  • Tm targets: Probe 68–72 °C; primers 60–64 °C (probe ~6–10 °C higher).
  • GC: 40–60% (30–65% tolerable with MGB).
  • Avoid runs (>3 identical bases) and avoid 5′ G next to dye.
  • Structure: hairpin/dimer ΔG > −2 kcal/mol preferred.
Thermal Budget & Amplicon

MGB enables shorter probes; keep amplicons 70–150 bp for crisp kinetics. Maintain probe Tm ~10 °C above anneal/extend to minimize breathing.

Multiplex Planning

Map dyes to non‑overlapping channels, verify your instrument’s color compensation, and balance probe concentrations per channel. We can deliver a pre‑balanced 2–4‑plex kit on request.

Ordering Checklist (MGB)

  • ✅ Assay & platform — qPCR (hydrolysis); instrument & channels.
  • ✅ Reporter & quencher — FAM/HEX/Cy3/ROX/Cy5 + 3′ MGB‑NFQ.
  • ✅ Probe stats — 14–18 nt target, SNP near center, GC 40–60%.
  • ✅ Scale & QC — 50 nmol → 5 µmol; HPLC; optional endotoxin/bioburden.
  • ✅ Format — Tubes or 96‑well plate; buffer/concentration; aliquoting.
Speak to a Scientist

FAQ

How do MGB probes compare to LNA probes?
Both boost Tm and mismatch discrimination. MGB stabilizes via minor‑groove binding at the 3′ end, enabling short probes with a dark quencher. LNA increases Tm through locked sugar chemistry across the sequence. Choose MGB for compact hydrolysis probes; choose LNA when you need localized Tm boosts within the span.
What are common design pitfalls?
Avoid 5′‑G adjacent to the dye; keep GC 40–60%; center the SNP; avoid >3 identical bases; check for hairpins/duplexes (ΔG < −2 kcal/mol); maintain probe Tm 6–10 °C above primer Tm; keep amplicon 70–150 bp.
Multiplex guidance?
Map dyes to well‑separated channels (e.g., FAM/HEX/Cy3/ROX/Cy5), verify color compensation on your instrument, and titrate each probe for balanced ΔRn. We can deliver pre‑balanced mixes.
What polymerases/master mixes are compatible?
Any 5′‑nuclease‑competent real‑time PCR mix (Taq‑based) works. Hot‑start Taq mixes with ROX reference are fine; ensure your reporter is not reserved as a passive reference on your platform.
High‑GC templates—any adjustments?
Use slightly longer probes (17–20 nt) or shift to a reporter with strong signal (e.g., FAM/Cy3). Optimize Mg²⁺ and anneal temperature; consider DMSO/Betain per platform guidelines.
What QC upgrades are available?
Dual‑HPLC, UPLC purity %, ds‑content, endotoxin (LAL), bioburden, sterility, and functional qPCR on request.
How should I reconstitute and store the probe?
Spin down, reconstitute in nuclease‑free water or TE to 100 µM, aliquot to avoid repeated freeze–thaws, store −20 °C protected from light. Working dilutions (e.g., 10 µM) should be kept at 4 °C for <2 weeks.
Do you offer plate formatting or barcoding?
Yes—96‑well or 384‑well plates with custom layout and barcodes; include your pick list with desired concentrations/volumes.
Can you match an existing supplier’s dye/quencher?
Often yes—we support common FAM/HEX/Cy3/ROX/Cy5 families and MGB‑class dark quenchers. Provide your current catalog code or excitation/emission requirements.
What information do you need to start design?
Target sequence or accession with ±100 bp context, platform/instrument, desired reporter channels, and any constraints (GC window, SNP position, amplicon length).

Speak to a Scientist

Email Instead

References

  1. MGB Eclipse probes (2002). PubMed
  2. Real‑time qPCR 5′‑nuclease assay (1996). PubMed
  3. Practical evaluation of dark quenchers (2009). PubMed
  4. General FRET/quenching principles (2002). Article

Why Choose BioSynthesis

Trusted by biotech leaders worldwide for over 45 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.

Quick Links