反应详情

Propidium iodide was tested for its effect on the replication of wild type RNA in a preparation of Qβ replicase. Propidium iodide in an amount of either 0, 1, or 3 μg/ml was added to a reaction buffer containing: 90 mM Tris HCl (pH7.5), 14 mM MgCl2, 0.4 mM each ATP, GTP, UTP, and CTP, and 10 μCi α- 32P-CTP. The Qβ replicase reaction was initiated in each sample by addition of 1.2 μg of Qβ replicase prepared according to the method of Eoyang and August, (1972), ibid. Aliquots of the reaction mixture were withdrawn every 5 minutes and spotted on DE81 filters (Schleicher & Schuell Inc., Keene, N.H.). The spotted filters were washed repeatedly with 0.5M NaPO4, (pH7.0) to remove unincorporated nucleotide. The filters were counted for Cerenkov radiation by standard procedures in a scintillation counter. The results are shown in FIG. 1. Addition of propidium iodide delayed the point in time at which levels of incorporation resulting from the replication of wild type MDV-1 template became detectable. One microgram per milliter delayed the appearance of replicated product between about 2-5 minutes, which corresponds to about 20-50% inhibition of the rate of replication in the geometric phase, and 3μg/ml delayed the appearance of product for about 20 minutes, which corresponds to approximately a 3-fold reduction in the rate of replication.

1. additionbuffer containing
2. customThe Qβ replicase reaction
3. additionwas initiated in each sample by addition of 1.2 μg of Qβ replicase
4. customprepared
5. customAliquots of the reaction mixture were withdrawn every 5 minutes
6. washThe spotted filters were washed repeatedly with 0.5M NaPO4, (pH7.0)
7. customto remove unincorporated nucleotide
8. additionAddition of propidium iodide