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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
ISO 9001:2015 / ISO 13485:2016 45+ Years of Expertise U.S.A. Facilities-Texas GLP/GMP-Aligned

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.

Speak to a Scientist See Design Guide

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

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

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.
More FAQ'sAll FAQ's

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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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