Abstract
—An azido-derivative of a fluorescein bifluorophore was obtained and used for the synthesis of “molecular beacon”-type oligonucleotide fluorogenic probes for RT-PCR. Eight probe variants were synthesized based on an optimized sequence: with one or two quencher residues at the 3'-end, with a single or bifluorophore fluorescein label attached to 5'-end using modifying phosphoramidites (short linker) or “click reaction” (long linker). Comparison of probes in RT-PCR showed that probes with a doubled quencher (single fluorescein on a short linker) and doubled dye on a short linker (single dye) are somewhat superior in sensitivity to a standard probe (single quencher, single dye on a short linker) by the value of ΔCt = 1–2.
Highlights
Fluorescent DNA probes, which are part of reagent kits for qualitative and quantitative detection of DNA and RNA, continue to be a powerful research and diagnostic tool [1,2,3,4]
In real-time polymerase chain reaction (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 [5, 8]
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
Summary
Fluorescent DNA probes, which are part of reagent kits for qualitative and quantitative detection of DNA and RNA, continue to be a powerful research and diagnostic tool [1,2,3,4]. One of the most important areas of application of such probes is the real-time polymerase chain reaction (RT-PCR) [5]. This method is used for express detection and semiquantitative (to the order of magnitude) analysis of genetic material; the most common uses of RT-PCR are molecular diagnostics of inherited diseases, genetically modified organisms, microbial and viral pathogens, for example, HIV [6] and SARS-CoV-2 [7]. The purpose of this work was to study FAM-bifluorophore on various linkers compared to a single fluorescein label as part of fluorogenic molecular beacon oligonucleotide probes for RT-PCR
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