FIG. 3.
(S)-HPMPApp is a weak inhibitor of primer extension assays. The reactions contained 32P-labeled primer P1 annealed to template T11, four dNTPs (5 μM dATP, 10 μM dCTP, 10 μM dGTP, and 10 μM dTTP), 2.5 ng of vaccinia DNA/μl, and 0 μM (A) or 10 μM (B) (S)-HPMPApp. The mixture was incubated at 37°C, and samples were removed at the indicated time points and mixed with a formamide-containing stop/loading buffer. The reaction products were then separated on 10% denaturing polyacrylamide gels, and the radioactivity was detected by using a phosphorimager. The control reactions (lanes numbered 1) were incubated for 15 min with no added DNA polymerase. Size markers were prepared by using dideoxy sequencing reactions and Klenow DNA polymerase (lanes 8 to 11). The band corresponding to the (S)-HPMPA+1 extension product was seen (arrowed, panel B) but comprised ∼1.4% of the total label in each of lanes 3 to 7. The same band comprised ∼0.3% of the extension products in panel A.