How Does Molecular Beacon Probe Work
Molecular Beacons are hairpin-loop shaped oligonucleotides that contain a probe sequence complementary at the 5' and 3' ends, which are modified with a fluorophore and quencher. This key feature of constrained structures is designed so close together that the fluorophore and the quencher contact quench to form a non-fluorescent, transient "ground-state" heterodimer. Upon hybridization with the target sequence, the hairpin-loop structure unfolds and separates the 5' end reporter dye from the 3'-end quencher. The quencher is no longer in close proximity to absorb the emission from the reporter dye. The increased emitted flourescence signal is detected by PCR instruments, which are directly proportional to the amount of target DNA. If the target DNA sequence does not exactly match the Molecular Beacon probe sequence, hybridization and fluorescence does not occur.
Molecular beacon3 offers greater specificity for mismatch discrimination due to structural constraints. These beacon-based assays are fast, simple, inexpensive, and enable real-time monitoring of nucleic acid reactions both in vivo and in vitro in real-time monitoring of nucleic detection. These stem-loop structure probes have become popular for standard analyses such as quantification of DNA and RNA4. They have also been used for monitoring intracellular mRNA hybridiza-tion,5 RNA processing, 6 and transcription7 in living cells in real-time. They are also ideally suited to SNP/mutation analysis 8 as they can readily detect single nucleotide differences 9,10 and have been reported to be reliable for analysis of G/C-rich sequences. 11 The sensitivity of Molecular Beacons probes permits their use for the accurate detection of mRNA from single cells. 12 They have been used in fourplex assays to discriminate between as few as 10 copies of one retrovirus in the presence of 1 × 105 copies of another retrovirus.13
Choosing Molecular Beacons for:
- DNA-protein interaction
- Microarray technology
- Pathogen Detection
- Multiplexing
- SNP detection
- Allele discrimination
- Gene expression analysis
- Viral load quantification
- Genotyping
- In vitro quantification or detection
- Aptamers and biosensors
Quality Control:
All our molecular beacon probes undergo stringent monitoring and strict quality control. Length and labeling is systematically controlled by PAGE, HPLC or MALDI-TOF mass spectrometry analysis. Quantity is determined and validated by UV Absorbance at 260 nm. Fluorescent probes are quality-checked by analytical ie-HPLC.
Purification
All molecular beacon probes are dual HPLC purified
Bases Assignment
DNA/BNA Chimeras:
- BNA base (+A, +C, +T, +G) (+ denotes the BNA bases)
- DNA (A,T,C,G)
- RNA (rA, rT, rC, rG)
BNA LightCycler FRET Probes can contain up to 6 BNA bases
BNA Molecular beacon can contain up to 8 BNA bases
Molecular Beacons are sold under license from the Public Health Research Institute, Newark, NJ. For information on designing Molecular Beacons please refer to The Public Health Research Institute's Molecular Beacons..
Reference:
- Bonnet, G., et al., Thermodynamic basis of the enhanced specificity of structured DNA probes. Proc. Natl. Acad. Sci. USA, 96, 6171-6176 (1999).
- 2. Broude, N.E., Stem-loop oligonucleotides: A robust tool for molecular biol-ogy and biotechnology. Trends Biotechnol., 20, 249-256 (2002).
- Tyagi, S. And Kramer, F.R., , Molecular Beacons: probes that fluoresce upon hybridization. Nat. Biotechnol., 14, 303-308 (1996).
- 8. Antony, T. and Subramaniam, V., Molecular Beacons: nucleic acid hybridiza-tion and emerging applications. J. Biomol. Struct. Dyn., 19, 497-504 (2001).
- Perlette, J. and Tan, W., Real-time monitoring of intracellular mRNA hybrid-ization inside single living cells. Anal. Chem., 73, 5544-5550 (2001)
- Dirks, R.W., et al., Methods for visualizing RNA processing and transport pathways in living cells. Histochem. Cell Biol., 115, 3-11 (2001).
- Liu, J., et al.,Real-time monitoring in vitro transcription using Molecular Beacons. Anal. Biochem., 300, 40-45 (2002).
- Mhlanga, M.M. and Malmberg, L., Using Molecular Beacons to detect single-nucleotide polymorphisms with real-time PCR. Methods, 25, 463-471 (2001).
- Giesendorf, B.A., et al., Molecular Beacons: a new approach for semi-automated mutation analysis. Clin. Chem., 44, 482-486 (1998).
- Marras, S.A., et al., Multiplex detection of single-nucleotide variations using Molecular Beacons. Genet. Anal., 14, 151-156 (1999).
- Tapp, I., et al., Scoring of single-nucleotide polymorphisms: comparison of the 5’-nuclease TaqMan assay and Molecular Beacon probes. Biotechniques, 28, 732-738 (2000).
- Steuerwald, N., et al., Analysis of gene expression in single oocytes and embryos by real-time rapid cycle fluorescence monitored RT-PCR. Mol. Hum. Reprod., 5,1034-1039 (1999).
- Vet, J.A., et al., Multiplex detection of four pathogenic retroviruses using Molecular Beacons. Proc. Natl. Acad. Sci. USA, 96, 6394-6399 (1999).
Enhanced your Detection with BNA Molecular Beacons
Benefits of
adding a BNA base to molecular Beacons:
- 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).
Molecular Beacon Probe for Real-time PCR
All Molecular Beacons dual labeled probes include 5'-reporter dye, 3'-quencher and dual HPLC purified. Please submit your order or quotation request to the client relations department at info@biosyn.com, fax (972) 584-0477, or you can use our online ordering system.
Please browse our selection below to find a suitable dye and quencher combination, or you may contact us
3' BHQ Modified Molecular Beacon
3' DABCY Modified Molecular Beacon
3' Wavelength-shifting molecular beacons
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 yields up to 97% purity. Depending on the type of probes, one or two purifications may be 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 °C to -70 °C
Oligonucleotides are stable in solution at 4°C for up to 2 weeks. Properly reconstituted material stored at -20°C should be stable for at least 6 months. Lyophilized DNA (when kept at -20°C) 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