Figure 4.
DNase I footprinting assay for confirmation of triplex formation. 5′-FAM-labeled duplex DNA (100 nM) was incubated with TFO (0–250 nM) in buffer containing 20 mM Tris–HCl, 20 mM MgCl2 at 37°C and pH 7.5. The mixture was subsequently treated with DNase I (0.05 U) for 1 min at 37°C. The reaction was terminated by adding formamide containing 20 mM EDTA and heating at 90°C. M is the mixture of 5′-FAM-labeled 12-, 30- and 42-mer ODNs as makers. The products were separated by 20% denatured polyacrylamide gel containing 8 M urea and visualized by LAS-4000.