Two-dye and one- or two-quencher DNA probes for real-time PCR assay: synthesis and comparison with a TaqMan™ probe

Abstract

A typical TaqMan™ real-time PCR probe contains a 5'-fluorescent dye and a 3'-quencher. In the course of the amplification, the probe is degraded starting from the 5'-end, thus releasing fluorescent dye. Some fluorophores (including fluorescein) are known to be prone to self-quenching when located near each other. This work is aimed at studying dye-dye and dye-quencher interactions in multiply modified DNA probes. Twenty-one fluorogenic probes containing one and two fluoresceins (FAM), or a FAM-JOE pair, and one or two BHQ1 quenchers were synthesized using non-nucleoside reagents and "click chemistry" post-modification on solid phase and in solution. The probes were tested in real-time PCR using an ~300-bp-long natural DNA fragment as a template. The structural prerequisites for lowering the probe background fluorescence and increasing the end-plateau fluorescence intensity were evaluated and discussed.

Improved multiplex TaqMan qPCR assay with universal internal control offers reliable and accurate detection of Clavibacter michiganensis

“…Additionally, in this study, we used the dual-probe strategy by adding another quencher to the probe that reduces the distance between the reporter and the quencher. This reduces the background signal and increases the signal strength of the qPCR assays (Ryazantsev et al 2012;Ouyang et al 2013;Arif et al 2014;Atkinson et al 2014). The developed assay is highly specific for Cm.…”

Enzyme-linked DNA dendrimer nanosensors for acetylcholine

“…1 ) 26 . This structural arrangement mitigates fluorophore self-quenching 27 28 and tunes the sensitivity and dynamic range to the physiological concentrations of acetylcholine. The combined spatial control and simple assembly make the DNA dendrimers an excellent platform for developing tailored nanosensors based on enzymatic small molecule recognition.…”

Molecular Beacon DNA Probes with Fluorescein Bifluorophore

“…Terminal alkyne was introduced into oligonucleotides using a phosphamidite reagent [ 24 ]. The doubled quencher BHQ1 (Q 2 ) was introduced into oligonucleotides as described earlier [ 13 ]. All probe components are shown in Fig.…”

“…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.…”