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 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. As the reporter is excited and passes its energy to the acceptor dye through FRET, the intensity of the light emitted is measured by the 2nd probe. Since the probes are not hydrolyzed, the LC probes’ fluorescence is reversible and allows for the generation of melting curves in Genotyping application. This genotyping technique is more precise than analyzing 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.
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.
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. 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 probe types, one or two purifications may performed to ensure the highest purity level
Quality Control
All dual labeled probes are quality checked and analyzed by MALDI-TOP Mass spectrometry , analytical HPLC and 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:
- 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 bases such as BNA to RNA LightCylcer probes not only to have stronger nuclease resistance, but also possess faster hybridization kinetics, 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.
|
* Values calculated for 3 pmol of probe per assay (range of optimal amount 2-10 pmol).
** LightCycler probe is sold under Hoffmann La-Roche Ltd. license. Information on purchasing licenses for practicing the PCR process may be obtained by contacting Roche Molecular Systems Inc. Director of Licensing. Custom synthesis of LC probes and oligonucleotides containing LCRed dyes are licensed under 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 probe types, one or two purifications may performed to ensure the highest purity level
Quality Control
All dual labeled probes are quality checked and analyzed by MALDI-TOP Mass spectrometry , analytical HPLC and 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