title: Design of molecular beacons: 3' couple quenchers improve fluorogenic properties of a probe in real-time PCR assay.
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).
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, 117997 Moscow, Russia. szavriev@ibch.ru korshun@ibch.ru.
authors.forename: Dmitry Y
authors.initials: DY
authors.lastname: Ryazantsev
authors.forename: Maksim V
authors.initials: MV
authors.lastname: Kvach
authors.forename: Dmitry A
authors.initials: DA
authors.lastname: Tsybulsky
authors.forename: Igor A
authors.initials: IA
authors.lastname: Prokhorenko
authors.forename: Irina A
authors.initials: IA
authors.lastname: Stepanova
authors.forename: Yury V
authors.initials: YV
authors.lastname: Martynenko
authors.forename: Sergey V
authors.initials: SV
authors.lastname: Gontarev
authors.forename: Vadim V
authors.initials: VV
authors.lastname: Shmanai
authors.forename: Sergey K
authors.initials: SK
authors.lastname: Zavriev
authors.forename: Vladimir A
authors.initials: VA
authors.lastname: Korshun
mesh_terms: D001483:Base Sequence; D017931:DNA Primers; D005456:Fluorescent Dyes / Q000737:chemistry*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*
chemical_list: D017931:DNA Primers; D005456:Fluorescent Dyes