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).

Molecular Beacon DNA Probes with Fluorescein Bifluorophore

“…It can be seen that the structure of the modification of the 5'-end region (linker and the number of 5-FAM residues) has almost no effect on the melting temperature of the probe. On the contrary, introduction of an additional quencher residue BHQ1 (Q) into the 3'-end region increases the melting point of the hairpin by 4–5°C, which is consistent with previously published data [ 14 ]. It is difficult to infer any regularities in fluorescence enhancement of the probes during melting; it can only be noted that probes with short linkers give a greater increase of emission intensity ( Table 1 ).…”

“…In this work, we used an optimized hairpin probe complementary to the region of the amplified fragment of the fungal translation factor 1α gene Fusarium avenaceum [ 14 ]. The nucleotide sequence of the probe, the structures of its chemical modifications, and approaches to the synthesis of 5-FAM-bifluorophore probes are shown in Fig.…”

Development of a Universal RNA Beacon for Exogenous Gene Detection

“…No detectable I F was expressed in the control cells (data not shown). Robust expression of I F was detected in cMSCs transfected with hHCN2-UB with a V h at 79.0 6 0.3 mV and a K at 6.7 60.3 mV (n = 6 cells), respectively [6,10].…”

“…Promising studies of this technology were recently reported by Ban et al [9] who used five molecular beacons to separate cardiomyocytes to 99% purity. The implanted tissue did not show tumors for up to 4 weeks [10].…”

Protected DNA strand displacement for enhanced single nucleotide discrimination in double-stranded DNA

“…1). Among these attempts are the developments of molecular beacons2345678, conformationally constrained binary probes91011, probes modified with artificial nucleotides such as peptide nucleic acids (PNAs)8121314 and locked nucleic acids (LNAs)14151617 etc. Thermodynamic approaches such as an elevated hybridization temperature (close to the duplex melting point)1819 or high concentrations of chaotropic agents in hybridization buffers192021 are often used to weaken non-complementary strand interactions in order to increase specificity.…”