Analysis of the products of the RNA-directed DNA polymerase reaction using glycerol-substituted polyacrylamide gels has proven superior to the glycerol gradient method for quantitative assay of a series of nucleic acid samples. To overcome the phenomenon of a smearing of background radioactive counts in the gels caused by unincorporated 'H-thymidine triphosphate, an automatic washing procedure making use of activated charcoal has been devised for the gels. This washing virtually eliminates all background while leaving the "H-DNA/35S
RNA hybrid as well as the "H-DNA/"H-DNA molecules fixed in situ on the gels.
Recently a system has been described for the detection of oncogenic viruses by the identification of a 70 S RNA hybridized to a newly synthesized radioactive DNA indicating the presence of an RNA-directed DNA polymerase (1). In an effort to simplify this technique for extension to the screening of large numbers of samples, we sought to analyze the hybrid product formed by such a reaction by electrophoresis on glycerolsubstituted polyacrylamide gels inst.ead of glycerol gradients. We found, however, that even after two alcohol precipitations of the nucleic acids, sufficient SH-thymidine triphosphate remains to run as a smear on glycerol-substituted gels obscuring the "H-DNA/RNA peak. To obviate this difficulty we have devised a washing procedure in which unincorporated 3H-thymidine triphosphate is removed from the gel while the 3H-DNA/RNA hybrid remains fixed in situ. An alternate approach to this problem was taken by Bishop et ,al. (2) who used Sephadex column chromatography to remove unincorporated nucleotides. We have found that the washing method described here is simpler for multiple samples of small volume.
Methods
The preparation of virus and the DNA polymerase reaction were carried out as described by Schlom and Spiegelman (1). After extraction of t,he reaction mixture with phenol-cresol, 50 pg of high molecular weight 54