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Custom LightCycler® FRET Probes

Anchor/Sensor pair • FRET readout • Melt-curve genotyping • Real-time monitoring

Overview Technology & Benefits Design Guide Donor–Acceptor Pairs Models & Channels Synthesis Options FAQ Contact

Overview

Bio-Synthesis designs and manufactures LightCycler®-style FRET hybridization probes (anchor/sensor pairs) for melt-curve genotyping, pathogen typing, and real-time detection. Two probes hybridize adjacently on the amplicon: a donor-labeled anchor and an acceptor-labeled sensor. When both are bound, FRET occurs and acceptor fluorescence reports perfect hybridization and enables precise melt analysis.

Defaults here are tailored to LightCycler® 480/96 channels. For carousel-based LightCycler® 1.5/2.0, acceptor 705 nm is also common—just tell us your model.

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At-a-Glance

  • Format: Anchor (donor) + Sensor (acceptor) hybridization pair
  • Readout: FRET during anneal/extend; melt-curve for SNP discrimination
  • Applications: Genotyping, mutation scanning, pathogen differentiation, gene expression
  • Purification/QC: HPLC (standard), PAGE optional; MS confirmation
  • Instruments: LightCycler® family and compatible thermocyclers with donor/acceptor channels

Technology & Benefits

Technology

  • FRET mechanism: Donor dye is excited; energy transfers to a nearby acceptor only when both probes are hybridized adjacently on-target.
  • Anchor/Sensor roles: The longer anchor ensures stable binding; the shorter sensor spans the variant and drives mismatch-dependent Tm shifts.
  • Orientation: Typically donor at the anchor 3′ end and acceptor at the sensor 5′ end with a 1–5 nt gap between probes.
  • Non-cleaving: Probes remain intact—ideal for post-amplification melt-curve analysis.
  • Blocking: 3′ end of any probe susceptible to extension is blocked (e.g., 3′-phosphate/C3 spacer).

Benefits

  • Ultra-clean specificity—signal requires both probes correctly bound.
  • Sharp SNP resolution via Tm differences in melt curves.
  • Flexible multiplexing with distinct acceptor channels.
  • Reproducible manufacturing with HPLC + MS QC.
  • Instrument-aligned design for available filter sets.

We tune probe lengths, Tm, inter-probe spacing, and dye selection to your LightCycler® model and assay temperature.

Recommended Donor–Acceptor Pairs

Donor (Ex/Em ~nm) Acceptors (Em ~nm) Notes
FAM / Fluorescein (Ex ~465 / Em ~520) LC Red 640 (Em ~640), Cy5 (Em ~670) Standard pairings for LC 480/96 (465→640/660).
ATTO 488 / Alexa 488 (Ex ~488 / Em ~520) Cy5 / Alexa 647 (Em ~670) Alternative FRET combo—confirm filter compatibility for your model.
HEX / VIC-like (Ex ~533 / Em ~555) ROX / Texas Red (Em ~605–615) Requires 533 nm excitation channel; FRET spacing must be validated.
For LC 1.5/2.0 (carousel) LC Red 640 or LC Red 705/Cy5.5 Classic HybProbe FRET with 470 nm excitation and emissions at 640 or 705 nm.

Tell us your exact model (LC 480 II, LC 96, LC 2.0/1.5) and we’ll match dye sets to available channels.

We offer much more modification than listed!   
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LightCycler® Models & Channels

Lightcycler in Action
Model Excitation Detection / Channels Notes
LightCycler® 480 Instrument II 440, 465, 498, 533, 618 nm 488, 510, 580, 610, 640, 660 nm Common FRET: 465→640 (or 465→660)
LightCycler® 96 Fixed pairs 470/514, 533/572, 577/620, 645/697 nm Choose donor/acceptor to fit these channel pairs
LightCycler® 2.0 (carousel) ~470 nm 530, 560, 610, 640, 670, 705 nm Classic HybProbe: 470→640 or 470→705
LightCycler® 1.5 (carousel) ~470 nm 530, 640, 705 nm Legacy optics; confirm availability

Design Guide

  • Anchor (donor): ~25–35 nt; Tm ≈ assay temp + 7–12 °C
  • Sensor (acceptor): ~15–25 nt; spans variant/SNP; Tm ≈ assay temp + 2–7 °C
  • Gap: 1–5 nt between probe ends when bound
  • Amplicon: 60–200 bp typical; place pair centrally
  • GC content: 40–60%; avoid long homopolymers
  • Blocking: 3′-phosphate or C3 to prevent extension
  • Concentration: Start 100–250 nM each (opt. 50–400 nM)
  • Controls: NTC, known WT/variant controls
  • Multiplex: Use distinct acceptors; balance brightness

Use Cases

Application Why FRET Helps Typical Setup
SNP genotyping Melt-curve Tm shift distinguishes alleles Anchor (donor) + short sensor (acceptor) spanning SNP
Pathogen typing High specificity from dual hybridization Species-specific pair; acceptor per channel
Mutation scanning Detects single-base changes without probe cleavage Sensor designed over hotspot; analyze melt peaks
Gene expression Proportional signal during amplification Validated primer + FRET pair
Design My FRET Pair

Custom Synthesis Options

Parameter Options
Format Anchor/Sensor hybridization pair (donor + acceptor)
Backbone DNA; optional 2′-OMe/2′-F/LNA/BNA mix for tuning
Labels FAM/fluorescein donors; LC Red 640, Cy5/Alexa 647 acceptors; others on request
Blocking 3′-phosphate or C3 spacer to prevent extension
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 labeling integrity
  • Optional functional checks (melt-curve, baseline, ΔRn)
  • RUO standard; GLP/cGMP support available

Typical Timelines

Known designs: 1–2 weeks. Complex multiplex or special dyes: 2–3 weeks.

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

How to Order

  1. Share target region and instrument model (e.g., LightCycler® 480 II).
  2. Choose donor–acceptor pair and channels.
  3. Select scale, purification, and any functional QC.
  4. Receive a same-day quote and timeline.

Include organism/cell line and delivery method (e.g., microinjection, electroporation, CPP) if known.

Design Checklist

  • Primer sequences & amplicon length
  • Probe coordinates (anchor/sensor) & desired Tm
  • Preferred donor/acceptor dyes; multiplex plan
  • Scale, purification, buffer, and documentation needs
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FAQ

How do FRET probes differ from dual-labeled hydrolysis probes?

FRET probes are non-cleaving; two probes must hybridize adjacently to generate acceptor signal, enabling melt-curve genotyping. Hydrolysis probes are single probes cleaved by polymerase nuclease during extension.

What’s the difference between the anchor and sensor?

The anchor is longer (stability), carries the donor; the sensor is shorter, spans the variant, and carries the acceptor—its mismatch sensitivity drives Tm shifts.

Can I multiplex FRET assays?

Yes—use distinct acceptor channels and balance brightness. We’ll select donor/acceptor pairs compatible with your LightCycler® filters.

Do I need to block probe extension?

Yes—block any 3′ end that could be extended (e.g., 3′-phosphate/C3). This preserves probe integrity during PCR.

What purification and QC do you provide?

RP-HPLC with MS confirmation is standard. PAGE and functional checks (melt-curve, baseline, ΔRn) are available on request.

Ready to design your FRET probe?

Tell us your sequence, modification, linker requirments - our team will send a tailored plan and quote.

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We’ll propose donor–acceptor pairs, Tm targets, and melt protocols tuned to your LightCycler® model (465→640/660 defaults for LC 480/96; 470→640/705 for LC 1.5/2.0).
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Why Choose BioSynthesis

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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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