Molecular Beacons are probes which contain a stem-loop structure, a fluorophore and a quencher at their 5’ and 3’ ends, respectively.
Eurogentec is a licensed supplier of Molecular Beacons, and offers a large number of fluorescent reporters and quenchers. All Molecular Beacons are provided double-HPLC purified and controlled by analytical HPLC and MALDI-TOF MS. The maximum length of standard Molecular Beacons is 45 bases. Molecular Beacons are provided lyophilized in individual tubes.
Eurogentec determines upon request the signal-to-noise ratio (STNR) for each Molecular Beacons by hybridizing it to its complementary sequence and comparing fluorescence to background. For FAM labelled Molecular Beacons, the STNR should be > 15. All other dyes require a signal-to-noise ratio > 10.
How do the Molecular Beacons work?
The ‘stem’ sequence keeps the fluorophore and the quencher together, but only in the absence of a sequence complementary to the ‘loop’ sequence. As long as the fluorophore and the quencher are in close vicinity, any photons emitted by the fluorophore are absorbed by the quencher. This phenomenon is called collisional (or proximal) quenching.
In the presence of a complementary sequence, the probe unfolds and hybridizes to the target, the fluorophore is displaced from the quencher, which can no longer absorb the photons emitted by the fluorophore, and the probe starts to fluoresce. The amount of signal is proportional to the amount of target sequence, and is measured in Real-Time to allow quantification of the amount of target sequence.
Alexa® 350 - Absorbance = 346 nm, Emission = 442 nm
Alexa® 430 - Absorbance = 434 nm, Emission = 541 nm
Alexa® 488 - Absorbance = 495 nm, Emission = 519 nm
Alexa® 514 - Absorbance = 517 nm, Emission = 542 nm
Alexa® 532 - Absorbance = 532 nm, Emission = 554 nm
Alexa® 546 - Absorbance = 556 nm, Emission = 573 nm
Alexa® 555 - Absorbance = 555 nm, Emission = 565 nm
Alexa® 568 - Absorbance = 578 nm, Emission = 603 nm
Alexa® 594 - Absorbance = 590 nm, Emission = 617 nm
Alexa® 610 - Absorbance = 612 nm, Emission = 628 nm
Alexa® 633 - Absorbance = 632 nm, Emission = 647 nm
Alexa® 647 - Absorbance = 650 nm, Emission = 665 nm
Alexa® 660 - Absorbance = 663 nm, Emission = 690 nm
Alexa® 680 - Absorbance = 679 nm, Emission = 702 nm
Alexa® 700 - Absorbance = 702 nm, Emission = 723 nm
Alexa® 750 - Absorbance = 749 nm, Emission = 775 nm
DABCYL : Quenching range [390-510]; Max 479 nm