Designing a TaqMan probe for qPCR based on the principle of FRET using Bio-dot as a Reporter

Abstract

Introduction: DNA-based analytical assays have introduced an advantageous biosensing platform for diagnostics and bioimaging purposes. The present study aimed to design a dual-labeled probe based on FRET principle using DNA-derived bio-dots and Eclipse quencher. The feasibility of TaqMan qPCR using the designed FRET probe as TaqMan internal probe was evaluated. Methods: Bio-dots were composited from DNA by hydrothermal method. Bioconjugation of the synthesized bio-dot with Eclipse labeled DNA probes was carried out through carbodiimide chemistry. Characterization of physicochemical, properties of the synthesized bio-dot and labeled probes were investigated through FTIR spectroscopy, EDAX, NMR, TEM, DLS, zeta potential, and Ninhydrin colorimetric assay. TaqMan PCR reaction was performed using the designed dual-labeled FRET probe. Results: Prosperous functionalization of the bio-dot was confirmed. The presence of amine groups on bio-dot surface was approved and quantified for bioconjugation. FTIR, size, and zeta potential results verified prosperous functionalization of the bio-dot as well as bio-dot probe linking. A concentration-dependent quenching of fluorescent signal was ascertained after bioconjugation. The probes represented great stability in various ambient environments. No target amplification was observed in TaqMan PCR using the designed probe. Conclusion: We synthesized DNA-derived fluorescent bio-dots through the hydrothermal method and investigated their optical and physicochemical properties. We designed a dual-labeled probe based on the FRET principle using bio-dot as a reporter. We evaluated the feasibility of TaqMan PCR using the designed FRET probe as an internal probe. The results demonstrated that the dual-labeled probe could not be used for TaqMan PCR.