Figure 1.

Strand displacement DNA synthesis assay. (A) Assay design. DNA polymerase incorporates dNTP thereby extending the primer strand and displacing the downstream reporter strand labeled with a 3′-fluorophore donor (F), leading to an increased fluorescence signal. F and Q denote 6-TAMRA and Black Hole Quencher-2 (BHQ-2), respectively. Chemical structures of 6-TAMRA and BHQ-2 used in the present study are also provided. (B) and (C) Assay demonstration. (B) DNA polymerase β concentration effect on reporter strand-displacement assay signal was tested using substrate 0–17 (Table 1). Time courses were run at room temperature using 50 nM of 0–17 and 100 μM of dTTP without (open square) or with pol β at different concentrations: 10, 20, 40 and 80 nM (filled squares, triangles, rhombs and circles, respectively). (C) dTTP concentration effect on the signal evolution was tested using 20 nM of pol β and 50 nM of 0–17 at different concentrations of dTTP : 0 (open squares), 25, 50, 100, 200 and 400 μM (filled triangles, inverted triangles, rhombs, circles and squares, respectively). (D) and (E) Template strand titration. Time course studies were performed at 50 nM substrate 0–10 with (filled squares) or without (open squares) 10 nM pol β (D) or 20 nM pol ι (E), respectively. When the fluorescence signal reached near saturation, 100 nM of template-strand labeled with BHQ-2 was added to the reaction mixture (arrow) and the fluorescence monitoring was resumed. Signal quenching upon addition of excess template strand is indicative of re-annealing of the intact displaced reporter strand.