Figure 1

Position of oligonucleotides, oligofluorophores hybridization and mechanism of detection illustrated on the HCV 5′-NCR.

(A) Illustration of the positions of oligonucleotides and oligofluorophores used for sequence-specific detection on the 5′-NCR of the HCV genome. (B) Biomolecular mechanism of the bi-labeled Loop Reverse oligonucleotide (LRp) covalently linked with 6-FAM as reporter on the 5′ end and BHQ1 as quencher on the 3′end. In step 1, in the absence of hybridization to amplified product (target sequence), the reporter remains quenched, with no fluorescence emission due to the close proximity of the quencher to the fluorophore. In the presence of amplified product in steps 2 and 3, the LRp hybridizes to the specific target between the R1c and R2 segments of the gene sequence, thus fluorophore begins to fluoresce as it separates from the quencher. From step 3 to step 4 as Bst DNA Polymerase catalyzes the reaction, quenching is disrupted and fluorescence is increased due to the increased distance (separation) between the reporter and quencher when the fluorooligonucleotide hybridizes and also due to fluorescence resonance energy transfer (FRET). The emitted fluorescence which corresponds to the amount of amplified nucleic acid is measured by spectrofluorometer in relative fluorescence units (RFU) and visualized by the naked-eye under UV-illumination.