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qPCR Total Solution Seminar Series
Seminars begin February 7th,
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Attention: Over the next few weeks, we will be updating our online order system and streamlining product specifications.
USA and Europe, Click Here
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LightCycler® probes are highly sensitive and sequence–specific fluorescent probes1-3 designed for use with the Roche™ LightCycler instrument. Several fluorophores are available, and are suitable for multiplex analysis. Sigma-Proligo produces LightCycler probes under license from Roche Molecular Diagnostics Inc. |
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- Deprotected, desalted and purified by RP-HPLC
- Available in lengths of 15 to 40mers (optimal length: 20 to 30mers)
- Delivered dried in individual, opaque tubes
- Shipped within 4 to 5 working days of receiving your order, pending successful QC validation
Guaranteed yields of LightCycler probes
Guaranteed yield
(OD) |
Approximate yield (nmols) |
Estimated assays (reactions) |
| 0.1 |
0.3 |
100 |
| 0.25 |
1 |
300 |
| 1.5 |
3 |
1,000 |
| 15 |
30 |
10,000 |
Please enquire for alternative quantities
Labels and modifications for LightCycler Probes
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Probe 1 |
Probe 2 |
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3'end donor fluorophore |
3'end |
5'end acceptor fluorophore |
| Labels and modifications |
Fluorescein |
Phosphate |
LightCycler Red 610, 640, 670 or 705 |
Probe 2 may also be used as a primer, labeled with an internal LightCycler Red dye. In this case, the 3'Fluorescein Probe 1 must be complementary to the newly-synthesized strand, labeled with the internal LightCycler Red dye.
Click here for a complete table of product specifications.
How do LightCycler probes work?
A LightCycler FRET probe system is a pair of single-stranded fluorescently-labeled oligonucleotides. Probe 1(the donor probe) is labeled at its 3'end with a donor fluorophore (generally fluorescein) and Probe 2(the acceptor probe) is labeled at its 5'end with one of four available fluorophores (red 610, 640, 670 or 705). The free 3' hydroxyl group of Probe 2 must be blocked with a phosphate group (P) to prevent Taq DNA polymerase extension. To avoid any steric problems between the donor and the acceptor fluorophores on both probes, there should be a spacer of 1 to 5 nt (4 to 25Å distance) to separate the two probes from each other. Before any real-time quantitative PCR reaction takes place, fluorescence background may be observed inside the tube.
During the annealing step of real-time quantitative PCR, the PCR primers and the LightCycler probes hybridize to their specific target regions causing the donor dye to come into close proximity to the acceptor dye. When the donor dye is excited by light from the LightCycler instrument (hγ1), energy is transferred by Fluorescence Resonance Energy Transfer (FRET) from the donor to the acceptor dye. The energy transfer causes the acceptor dye to emit light (hγ2) at a longer wavelength than the light emitted from the LightCycler instrument (hγ1). The acceptor fluorophore's emission wavelength is detected by the LightCycler instrument's optical unit. The increase in measured fluorescent signal is directly proportional to the amount of accumulating target DNA.
Click here for reporters & quenchers available for real-time QPCR probes.
References
1. Kreuzer, K.A., et al. LightCycler technology for the quantitation of brc/abl fusion transcripts. Cancer Research 59(13), 3171-74, 1999.
2. Nauck, M.S., et al. Rapid genotyping of human platelet antigen 1 (HPA-1) with fluorophore-labelled hybridization probes on the LightCycler.
Br. J. Haematol. 105(3), 803-10, 1999.
3. Lachnik, J., et al. Rapid-cycle PCR and fluorimetry for detection of mycobacteria. J. Clin. Microbiol. 40(9), 3364-73, 2002
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