Dual-Labeled DNA Probes

Molecular Beacons

  Molecular Beacons are highly sensitive, sequence–specific, fluorescent probes designed for real-time quantitative PCR1-3. Sigma-Proligo produces Molecular
Beacons and Wavelength-Shifting Molecular Beacons under license from the Public Health Research Institute.
 

  • Deprotected, desalted and purified by PAGE or RP-HPLC
  • Available in lengths of 15 to 40 mers
  • Delivered dried in individual, opaque tubes
  • Shipped within 5 to 6 working days of receiving your order, pending successful QC validation.

Guaranteed yields of Molecular Beacons and Wavelength-Shifting Molecular Beacons

Guaranteed yield (OD) Approximate yield (nmols*)
1 5
5 25
10 50
50 250
100 500

*Estimate 1 OD = 5 nmols = 30 µg, for a 20 mer oligo
Please enquire for alternative quantities.


Labels for Molecular Beacons and Wavelength-Shifting Molecular Beacons

Molecular Beacons Wavelength-shifting Molecular Beacons
5'end reporter 3'end quencher 5'end reporter Harvester fluorophore (internal) 3'end quencher
6-FAM, HEX, JOE, TET, ROX, TAMRA, Fluorescein, Cy3, Cy5, Cy5.5, Texas Red, Rhodamine, Rhodamine Green, Rhodamine Red, 6-Carboxyrhodamine 6G, Oregon Green 488, Oregon Green 500 or Oregon Green 514 TAMRA, DABCYL, BHQ-1 or BHQ-2 6-Carboxyrhodamine 6G, TAMRA or Texas Red Fluorescein DABCYL, BHQ-1™ or BHQ-2™

Note: Wavelenth-shifting Molecular Beacons are non-standard products. Available only on request.

Click here for a complete table of product specifications.

How do Molecular Beacons work?
Molecular Beacons is a single-stranded bi-labeled fluorescent probe held in a hairpin-loop conformation (around 20 to 25 nt) by complementary stem sequences (around 4 to 7 nt) at both ends of the probe. The 5' and 3'ends of the probe contain a reporter dye and a quencher moeity, respectively. The loop contains a probe sequence complementary to the target sequence and the stem is formed by annealing of the complementary arm sequences.. The close proximity of the reporter dye and quencher therefore eliminates the ability of the dye to fluorescence.

During the annealing step of PCR, the probe is excited by light from the PCR instrument (hγ1). Molecular Beacons hybridize to their target sequence causing the hairpin-loop structure to open and separate the 5'end reporter dye from the 3'end quencher. As the quencher is no longer close enough to absorb the emission from the reporter dye, the dye is allowed to fluoresce and the PCR instrument detects the increase of emitted energy (hγ2). The measured fluorescent signal is directly proportional to the amount of target DNA.

 

Why use Wavelength-Shifting Molecular Beacons?

Molecular Beacons provide a powerful and sensitive design but signal strength can be low. This can be overcome by using Wavelength-shifting Molecular Beacons. These are conventional Molecular Beacons that contain an additional internal harvester fluorophore which enables the 5'end reporter dye to emit a stronger fluorescent signal.

During the annealing step of PCR, the 5'end reporter is separated from the 3'end quencher. The harvester fluorophore is excited by light (hγ) from the real-time quantitative PCR instrument and transfers absorbed energy to the 5'end reporter by Fluorescence Resonance Energy Transfer (FRET). This shift of energy only takes place when the probe hybridizes to its specific target sequence and if the fluorophores are separated by 6-7 nt. The 5'end reporter emits fluorescence (hγ2) to be detected by the PCR instrument according to its own spectral properties. Increase of measured fluorescence signal is directly proportional to the amount of target DNA.

Click here for reporters & quenchers available for bi-fluorescent probes.

References
1. Tyagi, S., and F. R. Kramer. Molecular beacons probes that fluoresce upon hybridization. Nature Biotechnology 14(3), 303-8, 1996.

2. Tyagi S., et al. Wavelength-shifting molecular beacons. Nature Biotechnology 18(11), 1191-96, 2000.

3. Park S., et al. Rapid identification of Candida Dubliniensis using a species-specific molecular beacons.
J. Clin. Microbiol. 38(8), 2829-36, 2000.