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Attention: The following specifications apply to the USA and Europe. All other countries, 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 inquire for alternative quantities
Labels and modifications for LightCycler Probes
|
Probe 1 |
Probe 2 |
|
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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