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LightCycler probes are highly sensitive and sequence-specific hybridization FRET probes with two different labeled probes. The donor and acceptor probes are designed to hybridize specific to two adjacent sequence in the target DNA used for quantitative Real-Time PCR reaction with the Roche LightCycler Instruments.

Lightcycyler Hybridization Probes from Bio-Synthesis include a pair of single-stranded fluorescent-labeled oligonucleotide. The donor probe is labeled at 3' end with fluorescein such as FAM. The acceptor probe is labeled at its 5' end with LightCycer dye, ROX or Cy5. In addition, a 3’ OH is capped by a phosphate group to prevent extension of the reporter probe by Taq DNA polymerase during the PCR cycles.

All probes are HPLC purified and controlled by MALDI-TOF Mass Spectrometry. LC hybridization probes are provided lyophilized in individual tubes.

All Bio-Synthesis's Lightcycler Hybridization Probes are available at a fixed price per oligonucleotide, within the following parameters:

  • DNA up 40 bases in length
  • BNA, 2' O-Methyl RNA molecular beacon available
  • 3' donor probe, 5' acceptor probe and capped by phosphate
  • All dual HPLC purified
  • 100% QC by MALDI-TOF MS, analytical HPLC
  • Deliver lyophilized in tube
  • TAT of 4-6 working days

LC Hybridization Probes

LC hybridization probes is designed for use with the Idaho Technology/Roche capillary-based instruments, but can also be used with other real-time PCR instruments.

How Does LightCycler LC Hybridization Probe Work

LC probes contain two probes that are designed to bind adjacent to one another on the amplicon. Probe 1 has a donor dye at its 3' end such as FAM. Probe 2 has acceptor dye on its 5' end, such as LightCycler® Red 640, and 3' end is capped with phosphate to prevent extension during the annealing step. Both probes hybridize to the target sequence in a head-to-tail arrangement during the annealing step. The reporter is excited and passes its energy to the acceptor dye through FRET and the intensity of the light emitted is measured by the 2nd probe. Since the probes are not hydrolyzed, fluorescence of LC probes are reversible and allows for the generation of melting curves in Genotyping application. This technique of genotyping is more precise than the analysis through TaqMan® probes, because unknown variants can be detected. TaqMan® probes also emit fluorescence in the absence of a variant and can, therefore, only be used for biallelic systems.

LightCycler FRET Probe

Choosing LC Hybridization Probes for:

LightCycler probes can be used for SNP/mutation detection, where one probe is positioned over the polymorphic site and the mismatch causes the probe to dissociate at a different temperature to the fully complementary amplicon. They can also be used to generate melting curves.

  • SNP detection
  • Allele discrimination
  • Multiplexing
  • Gene expression analysis
  • Gene copy determination
  • Pathogen detection
  • Viral load quantification

Design of LightCycler Probes:

To avoid sterical problems between the 3' Fluo and 5' LC® Red end labels of the probes there should be a spacer of 1 to 5 nucleotides (4 to 25 Å distance) to separate the two probes from each other.

Dye Abs (nm) Em (nm) Color RotoGene Instruments
LC® CYAN 500 450 500
LC® Fluorescein 495 520
Texas Red 583 603 LC 2.0, LC480 Und Rotor Gene
LC® Red 610 590 610 LC 2.0, LC480 Und Rotor Gene
LC® Red 640 625 640 Alle Geräte
CY5 649 670 LC 2.0, LC480 und RotorGene
LC® Red 670 650 670 Alle Geräte
CY5.5 675 694 LC 1.x und 2.1
LC® Red 705 685 705 LC 1.x und 2.0

Purification

In order to ensure that there is no background fluorescence, 100% of the dual labeled probes are purified by single or dual rp-HPLC and ie-HPLC as a standard protocol for all dual–labeled probes which give 90-97% purity. Depending on the type of the probes, one or two purification may performed to ensure the highest purity level

Quality Control

All dual labeled probes are quality checked by MALDI-TOP Mass spectrometry , analytical HPLC and analyze by Fluorometric Scan (ABS/EM) . In addition, the signal-to-background ratio is measured against a target sequence for all molecular beacons.

Delivery times

3-5 Working days

Packaging

Lyophilised

Shipping conditions

Room temperature

Storage conditions  

-20 oC to -70 oC

Oligonucleotides are stable in solution at 4oC for up to 2 weeks. Properly reconstituted material stored at -20oC should be stable for at least 6 months. Lyophilized DNA (when kept at -20oC) in a nuclease-free environment should be stable for year

References:

  1. Pals, G., et al., Detection of a single base substitution in a single cell using the LightCycler. J. Biochem. Biophys. Methods, 47, 121-129 (2001)

Enhanced your Detection with BNA LightCycler Hybridization Probe for

Incorporate modified base such as modified base such as BNA to your to RNA LightCylcer probes not only have stronger nuclease resistant, but also possess faster hybridization kinetics, and superior binding affinity and specificity toward targets compare to DNA oligonucleotide.

  • Increased thermal stability and hybridization specificity
  • Greater accuracy in SNP detection and allele discrimination
  • Easier and more sensitive probe designs for problematic target sequences

Learn more about Bridged Nucleic Acids (BNAs).

LightCycler FRET Probe for Real-time PCR

Donor Probe 1
3' Fluorescein LC610, LC640, LC670, LC705, LC Fluro, Cy® 5, Cy® 5.5 3' FAM
Acceptor Probe 2*
3' end Phosphate
5' end LC® Cyan 500, LC® Fluo, Texas Red, LC® Red 610, LC® Red 640, LC® Red 670, ROX
BNA can be incorporated into acceptor probe to enhance discrimination.

Delivered quantity (nmol)*
OD (nmol)* No. of µg Estimate No. of Reaction max. Length
0.5 2 16 400 40 mer
1.25 5 40 1,000 40 mer
2.5 10 80 2,000 40 mer
5.5 20 1600 4,000 40 mer
* including dual HPLC and Mass Check
For probe design, larger molar amounts, large volume orders or other special requests, please contact us or email at info@biosyn.com

*   Values calculated for 3 pmol of probe per assay (range of optimal amount 2-10 pmol).

**  LightCycler probe is sold under license from Hoffmann La-Roche Ltd. Information on purchasing licenses to practice the PCR process may be obtained by contacting the Director of Licensing at Roche Molecular Systems, Inc., Custom synthesis of LC probes and oligonucleotides containing LCRed dyes under licence of Roche Diagnostics. LightCycler® is a trademark of Idaho Technology Inc. All products are for research only.

Purification

In order to ensure that there is no background fluorescence, 100% of the dual labeled probes are purified by single or dual rp-HPLC and ie-HPLC as a standard protocol for all dual–labeled probes which give 90-97% purity. Depending on the type of the probes, one or two purification may performed to ensure the highest purity level

Quality Control

All dual labeled probes are quality checked by MALDI-TOP Mass spectrometry , analytical HPLC and analyze by Fluorometric Scan (ABS/EM) . In addition, the signal-to-background ratio is measured against a target sequence for all molecular beacons.

Delivery times

3-5 Working days

Packaging

Lyophilised

Shipping conditions

Room temperature

Storage conditions  

-20 oC to -70 oC

Oligonucleotides are stable in solution at 4oC for up to 2 weeks. Properly reconstituted material stored at -20oC should be stable for at least 6 months. Lyophilized DNA (when kept at -20oC) in a nuclease-free environment should be stable for year

Specifications

Quality control:

Every oligo is quality checked by MALDI-TOF mass spectrometry, electrospray ionization mass spectrometry (ESI-MS), capillary electrophoresis (CE), and/or polyacrylamide gel electrophoresis (PAGE).

Purification

Fully deprotected and desalted. Purified by PAGE or RP-HPLC

Length

Length
LightCycler probes 15 to 40 mers (optimal length: 20 to 30 mers)
Dual-labeled fluorogenic probes: 15 to 40 mers
Molecular Beacons 15 to 40 mers
Scorpions probes 30 to 60 mers (uni-probe)
15 to 45 mers (bi-probe)
Plexor up to 30 mers

Backbone

Phosphodiester bond

Format

Delivered as dried-down product in opaque tubes

Turn-around time:

Turn-around time
LightCycler probes 4 to 5 working days
Dual-labeled fluorogenic probes: 5 to 6 working days
Molecular Beacons 5 to 6 working days
Scorpions probes 7 to 10 working days

Turn-around time is dependent upon successful QC validation and does not include shipping time.

Storage and stability:

See information on our oligos: storage recommendations

Shipment:

Shipped by mail or express delivery, dry, in individual, opaque tubes

Technical Documentation:

Oligonucleotides are delivered with an OligonucleotideTechnical Data Sheet, which includes oligonucleotide name, sequence, concentration, precise quantity in OD and nmols, Tm, MW, length, extinction coefficient and purification data.

Additional Services:

  • Probe design services
  • High-throughput screening formats (microplates, etc)
  • Aliquoting

Additional services may increase turn-around time

Pricing:

Please contact your local Bio-Synthesis representative

Ordering:

On-line (contact us), by email (info@biosyn.com) or fax

Ordering & Contact Information

  • Please contact us for additional information or send an email to info@biosyn.com.
  • You may also request an online quote.
  • To contact us by phone, please call 1-972-420-8505 or Fax at 1-972-420-0442
  • Orders may be placed using a purchase order (PO) or by credit card through our secure online ordering system.
  • When using a credit card ( creditcard ) it will be billed under "Bio-Synthesis, Inc."