Fig. 1.

Fluorescence based real time analysis of uracil excision by MtuUng: [A] Schematic of the molecular beacon used for Ung assays. The action of MtuUng opens the hairpin structure resulting in recovery of fluorescence which was masked by the quencher in the closed state. [B] Real time detection of uracil excision by MtuUng as a measure of increasing fluorescence (purple). The assay was performed with 30 nM oligomeric substrate and 0.3 nM MtuUng. Addition of 2.1 nM Ugi or 80 mM NaOH in the reactions is as indicated. The reactions were carried out in 100 μl volumes in a buffer containing 50 mM Tris-Cl (pH 8), 1 mM Na₂EDTA and 37.59 nM BSA, n = 3 and p < 0.0001 with control v/s Ung containing group. [C] Gel electrophoretic analysis of the reactions. Lanes 1–5 depict the products generated in the fluorescence assay (Fig. 1B) at the end of 40 min incubation. Lanes 6–9 show further treatment of the reactions with 0.096 N NaOH and heating at 95 °C. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)