Cy5/BHQ dye–quencher pairs in fluorogenic qPCR probes: effects of charge and hydrophobicity

Abstract

For the first time we used CuAAC click reaction for the synthesis of cyanine labeled qPCR probes. We performed the comparison of dyes and quenchers (Cy5 vs disulfo-Cy5 and BHQ2 vs BHQ3) in several TaqMan probes with different stability of the secondary structure. Based on the studies of thermal quenching of dyes, increase of fluorescence during probe melting and in the course of qPCR, we suggest that disulfo-Cy5-BHQ2 pair is a preferable one for molecular beacons due to the highest increase of fluorescence during melting. As Cy5-BHQ3 pair gave better Cq and maximal relative increase of fluorescence in qPCR in comparison to others we recommend this pair for Taqman style probe design.

Ultra‐fast Cycling for Multiplexed Cellular Fluorescence Imaging

“…Intramolecular contact quenching for fluorophore–BHQ pairs is well known for dual‐labeled oligonucleotide and peptide cleavage probes. In the oligonucleotide context for which this has been studied more quantitatively, fluorophore–quencher interactions subtly perturb melting temperatures and can promote intramolecular dimer formation with an affinity sufficient to form a stemless hairpin .…”

Ultra‐fast Cycling for Multiplexed Cellular Fluorescence Imaging

“…Click/quenching rates remained profoundly accelerated, with only subtle changes in the temporal profile (Supporting Information, Figure S7). Intramolecular contact quenching for fluorophore-BHQ pairs is well known for dual-labeled oligonucleotide [20] and peptide [21] cleavage probes.Inthe oligonucleotide context for which this has been studied more quantitatively,fluorophorequencher interactions subtly perturb melting temperatures [19] and can promote intramolecular dimer formation with an affinity sufficient to form as temless hairpin. [22] We therefore hypothesize that dye-dependent fluorophore-BHQ3 interactions drive ar eversible molecular association of sufficient affinity/duration to promote Tz-TCO ligation, even at nanomolar concentrations.T he temporal dynamics of this process are intriguing, given that no fluorescence quenching (a process occurring on the nanosecond timescale) is observed in the absence of the click reaction.…”

Molecular Beacon DNA Probes with Fluorescein Bifluorophore

“…In RT-PCR, various types of fluorogenic DNA probes are used, which are capable of increasing fluorescence when interacting with the accumulating PCR product; the fluorogenic effect is achieved as a result of the interaction of two dyes, one of which can be nonfluorescent (quencher) [ 5 , 8 ]. For fluorogenic probes, the relationship between the type of dye and the structure of the probe is being studied [ 9 ], new dyes are being developed [ 10 – 12 ], and probes with two residues of a fluorescent dye and/or a fluorescence quencher are being investigated [ 13 – 15 ]. The most popular dye for DNA probes is fluorescein, which is attached in the form of a carboxyl derivative at the amino group of a linker; such fluoresceinamide is abbreviated as FAM.…”