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Analyst. 2014 Jun 7;139(11):2867-72. doi: 10.1039/c4an00081a.

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

Author information

1
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, 117997 Moscow, Russia. szavriev@ibch.ru korshun@ibch.ru.

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).

PMID:
24736939
DOI:
10.1039/c4an00081a
[Indexed for MEDLINE]

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