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• W4386860593 abstract "The 2'-deoxyuridine-5'-triphosphates modified with fluorescein (dUTP-Fl) or rhodamine (dUTP-Rh) were tested as bearers of electroactive labels and as a proper substrate for polymerases used in polymerase chain reaction (PCR) and isothermal recombinase polymerase amplification (RPA) with the aim of electrochemical detection of double-stranded DNA (dsDNA) amplification products. For this purpose, electrochemical behavior of free fluorescein and rhodamine as well as the modified nucleotides, dUTP-Fl and dUTP-Rh, was studied by cyclic (CV) and square wave (SWV) voltammetry on carbon screen printed electrodes. Both free fluorescein and dUTP-Fl underwent a two-step oxidation at the peak potentials (Ep) of 0.6–0.7 V and 0.8–0.9 V (phosphate buffer, pH 7.4). The reduction peaks of fluorescein and dUTP-Fl were registered between –0.9 and –1 V. The free rhodamine and dUTP-Rh have demonstrated the well-defined oxidation peak at 0.8–0.9 V. In addition, the distinct reduction peaks at Ep between –0.8 and –0.9 V were registered for both rhodamine and dUTP-Rh. The dUTP-Fl and dUTP-Rh were further tested as substrates to incorporate an electroactive label into 210 or 206 base pair long dsDNA amplicons generated either by PCR or RPA. Among two dUTP derivatives tested, dUTP-Fl revealed significantly better compatibility with PCR and RPA, producing the full-size amplicons at 50–90% substitution of dTTP in the reaction mixture. In the PCR, the best compromise between amplicon yield and labeling was achieved at the dUTP-Fl: dTTP and dUTP-Rh: dTTP molar ratios of 70: 30% and 20: 80% in the PCR mixture, respectively, allowing in the case of fluorescein the direct electrochemical detection of amplicons at micromolar concentrations. Alongside with fluorescence DNA assays, the fluorescein-modified dUTP appears as a promising electroactive label to develop direct electrochemical DNA assays for detecting PCR and RPA products." @default.
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• W4386860593 date "2023-11-01" @default.
• W4386860593 modified "2023-10-14" @default.
• W4386860593 title "Polymerase incorporation of fluorescein or rhodamine modified 2'-deoxyuridine-5'-triphosphates into double-stranded DNA for direct electrochemical detection" @default.
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• W4386860593 doi "https://doi.org/10.1016/j.jpba.2023.115737" @default.
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