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Issue 11, 2014
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Design of molecular beacons: 3′ couple quenchers improve fluorogenic properties of a probe in real-time PCR assay

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Abstract

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).

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

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Supplementary files

Article information


Submitted
12 Jan 2014
Accepted
19 Mar 2014
First published
19 Mar 2014

Analyst, 2014,139, 2867-2872
Article type
Paper
Author version available

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

D. Y. Ryazantsev, M. V. Kvach, D. A. Tsybulsky, I. A. Prokhorenko, I. A. Stepanova, Y. V. Martynenko, S. V. Gontarev, V. V. Shmanai, S. K. Zavriev and V. A. Korshun, Analyst, 2014, 139, 2867
DOI: 10.1039/C4AN00081A

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