Detection
LightCycler™ hybridization probes allow a specific and quantitative real-time detection of the amplification of a nucleic acid template(1-3).
Structure
LightCycler™ hybridization probes are comprised of a pair of fluorescently monolabeled oligonucleotides that anneal closely to each other on the target DNA.
Bilabeling
A donor fluorophore (fluorescein) is attached on the 3' end of the first oligo (Oligo Probe 1), while an acceptor fluorophore (Red 640 or Red 705) is attached on the 5' end of the second oligo (Oligo Probe 2). Oligo Probe 2 must be phosphorylated on its 3' end to prevent extension by the polymerase during amplification.
Hybridization
Once excited by the light-emitting diode (LED), the donor fluorophore of Oligo Probe 1 excites the acceptor fluorophore of Oligo Probe 2 through fluorescence resonance energy transfer (FRET). The acceptor fluorophore emits a specific wavelength which is detected, analyzed and quantified during amplification.
Applications
The LightCycler™ system is ideally suited for applications requiring repetitive and reliable PCR* techniques, such as(4-6):
- allelic discrimination studies
- genotyping
- automatic mutation detection
- gene expression studies
Sequence
Oligo Probe 2 must be phosphorylated on the 3' end.
Probes must not contain secondary structure (dimerization, hairpin).
The two oligonucleotide probes must anneal between 1 and 5 bases away from each other on the DNA target.
Quality Entirely deprotected and desalted. Purified by reverse phase HPLC.
Length 15 to 40 bases (optimal length: 20-30 bases).
Tm From 5° C to 10° C above primer Tm.
Quantity (in nmoles)
- 0.3 : 100 reactions
- 1 : 300 reactions
- 3 : 1000 reactions
- 30 : 10 000 reactions
Quality Controls
Length and quality by polyacrylamide gel electrophoresis (PAGE).
Purified by reverse phase HPLC.
Absorbance measurements taken at 495 nm and at 625 nm or 685 nm.
Quantity validated by UV absorbance at 260nm.
Validation of probe design integrity.
Form
Delivered in dry form.
Guarantees
Conformity of the sequence.
Minimum final quantity.
Quality according to above specifications.
Documentation
1 - Oligonucleotide Technical Data Sheet
- Concentration (uM and ug/ul), quantity (OD and nmoles), Tm, MW, size and molecular extinction coefficient.
2 - Analysis Certificate
- Reverse phase HPLC profile at 260 nm
- Absorbance profiles at 495 nm and at 625 nm or 685 nm.
Turn-around time
5 working days.
* PCR is patented by Hoffman La Roche Ltd.
* LightCycler™ is a trademark of Idaho Technology, Inc.
* Red 640, Red 705 and LightCycler™ hybridization probes are licensed from Hoffman La Roche Ltd.
* LightCycler™ is licensed by Roche Diagnostics from Idaho Technology, Inc.
* LightCycler™ logo is used with the permission of Roche Diagnostics.
* For research use only.
References
1. Rasmussen R., et al. (1998). Quantitative PCR by continuous fluorescence monitoring of a double
strand DNA specific binding dye. Biochemica. 2: 8-11.
2. Wittwer, C.T. et al. (1997). The LightCycler™ : A microvolume Multisample Fluorimeter with Rapid
Temperature Control. Biotechniques. 22(1): 176-181.
3. (1998). The LightCycler - the smartest innovation for more efficient PCR. Biochemica. 2: 4-7.
4. De Silva, D., et al. (1998). Rapid genotyping and quantification on the LightCycler with hybridization
probes. Biochemica. 2: 12-15.
5. Kreuzer, K.A., et al. (1999). LightCycler technology for the quantitation of brc/abl fusion transcripts
Cancer Res. 59(13) : 3171-4.
6. Nauck, M.S., et al. (1999). Rapid genotyping of human platelet antigen 1 (HPA-1) with fluorophore
-labelled hybridization probes on the LightCycler.
Br J Haematol. 105(3): 803-10.
