FIG. 5.
Amplification of viral DNA by Rta. Total DNA from 2 × 106 S11E cells transfected with pcDNA3 (lanes 3 to 5) or pcDNA3/MHV-68 rta (lanes 6 to 8) was harvested at 24 (lanes 3 and 6), 48 (lanes 4 and 7), or 72 (lanes 5 and 8) h posttransfection (p.t.) and subjected to digestion with HindIII. The digested DNAs were electrophoresed on a 0.7% agarose gel for Southern analysis. HindIII-digested DNA from 5 × 105 MHV-68-infected BHK-21 cells was loaded in lane 1 as a positive control (I), and digested DNA from 2 × 106 untransfected S11E cells was loaded in lane 2 as a negative control (UT). The probe used to detect the viral DNA was derived from the 6.2-kb PCR product (nt 51 to 6298) spanning the far left end of the viral genome. Based on the sequence (40), a 6.2-kb DNA fragment would be detected with the probe. Radioactivity was detected and quantitated with a STORM imaging system.