FIG. 4.

(S)-HPMPA can be incorporated into DNA and extended by vaccinia DNA polymerase. (A) Primer P1 was labeled with ³²P, annealed to template T11, and incubated for 1 min with 2.5 ng of vaccinia DNA polymerase/μl plus 10 μM dATP or (S)-HPMPApp at 25°C. A sample of the product was removed from each reaction and added to formamide stop buffer. dTTP was then added to each of the remaining mixtures, to a final concentration of 10 μM, and the incubation continued for another minute. A second sample was removed, followed by the addition of dCTP, dGTP, and dTTP (all to 10 μM final concentration), and the incubation was continued with periodic sampling. The reaction products were then size fractionated and detected by phosphorimaging. A slight lag may be seen in the extension of molecules terminated by (S)-HPMPA+dGMP, but the majority of the primer chases into a series of extension products in less than a minute at 25°C. (B) Experiment similar to that in panel A except that template T12 directs the incorporation of two consecutive molecules of dAMP or (S)-HPMPA.