FIG. 9.

CDV and (S)-HPMPA are faithfully copied by vaccinia DNA polymerase. The four template strands were prepared containing dCMP/CDV (upper panel) or dAMP/(S)-HPMPA (lower panel) and annealed to ³²P-labeled primers P10 or P17 as indicated. These primer-template pairs were incubated with 2.5 ng of vaccinia DNA polymerase/μl at 37°C for 1 min in the presence of each of the indicated single dNTPs (50 μM), and the products were recovered using magnetic beads. The reaction products were then separated on a 10% polyacrylamide gel, and the radioactivity was visualized by phosphorimaging. CDV and (S)-HPMPA direct the incorporation of dGMP and dTMP, respectively. Each of the enzymatically prepared template strands was also separately labeled with terminal transferase to measure the length of the original extension products (template). Sequencing ladders were prepared using primer P10 annealed to template T14 (upper panel) or primer P17 annealed to template T18 (lower panel).