Design of molecular beacons: 3′ couple quenchers improve fluorogenic properties of a probe in real-time PCR assay

Ryazantsev D. Y. , Kvach M. V., Tsybulsky D. A., Prokhorenko I. A., Stepanova I. A., Martynenko Y. V., Gontarev S. V., Shmanai V. V., Zavriev S. K., Korshun V. A.

Convenient preparation of fluorogenic hairpin DNA probes (molecular beacons) carrying a pair of FAM fluorophores (located close to 5′-terminus of the probe) or a pair of BHQ1 quenchers on 3′-terminus (with (BHQ1)2 or BHQ1–BHQ1 composition) is reported. These probes were used for the first time in a real-time PCR assay and showed considerable improvements in fluorogenic properties (the total fluorescence increase or signal-to-background ratio) in assay conditions vs. conventional one-FAM-one-BHQ1 molecular beacon probes as well as vs. hydrolyzable one-FAM-one-BHQ1 TaqMan probes. At the same time, such multiple modifications of the probe do not influence its Cq (a fractional PCR cycle used for quantification). The probe MB14 containing a BHQ1–BHQ1 pair showed a PCR fluorescence/background value of 9.6 which is more than two times higher than that of a regular probe MB2 (4.6). This study demonstrates prospects for the design of highly fluorogenic molecular beacon probes suitable for quantitative real-time PCR and for other potential applications (e.g. intracellular RNA detection and SNP/mutation analysis).

Analyst 2014, 139, 2867–2872


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