Fluorescence quenching, molecular beacons

Because of the hairpin formation, these dyes are in such a close proximity that their fluorescence is quenched (molecular beacon Box 18) unless the structure is unfolded in the course of second-strand synthesis (Figure 4.3.4b). Thus, detection of a fluorescence signal from one of both dyes is a direct measure of the progress of the reaction. These researchers also showed that primer extension reactions can be monitored directly in cleared lysates of cells overexpressing the Klenow fragment of E. coli DNA polymerase I. Thus, the molecular beacon assay might supersede extensive purification. 

Figure 27 AuNP-quenched molecular beacon for DNA sensing. The molecular beacon brings a fluorophore and AuNP close enough to facilitate the fluorescence quenching. After the complementary target binds the DNA on the molecular beacon, the fluorophore is far away from the quencher and the fluorescence is recovered.

Fig. 5 Two RET"based sensing schemes. A molecular beacon structure with a fluorescent donor (D) and a quenches (Q) enhances emission upon target hybridization. A protease substrate with donor and acceptor conjugated to an amino acid recognition sequence (DEVD) enhances emission upon enzymatic cleavage. (View this art xi color at www.dekker.com.)...

Figure 5.1 Sketch of the DNA molecular beacon. The five bases at the two ends of the beacon are complementary to each other. The size of the loop and its content are varied. The beacon flips between open and closed states with the characteristic rates and k+.Thefluorophore(F)and the quencher (Q) are covalently linked to the two arms of the beacon. In the open state the beacon fluoresces, in the closed state the fluorescence is quenched. Reprinted with permission from Bonnet etal., Kinetics of conformational fluctuations in DNA hairpin-loops. Proceedings of the National Academy of Sciences of the United States of America 95 (1998) 8602-8606. Copyright 1998 NationalAcademy of Sciences, USA.

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