Figure 4. . . Nested PCR amplification of a subgenomic region of cDNA for baboon hepatitis B virus, South Africa. Reverse transcribed, DNase I–treated cDNA products were amplified by PCR, and amplicons were resolved by electrophoresis on a 1% agarose gel containing ethidium bromide. Non–reverse transcribed samples (in which diethyl pyrocarbonate [DEPC]–treated water was added instead of enzyme during reverse transcription) were included as negative controls. Top panel: Nested PCR 1: 255F–759R; PCR 2: 459F–710R. Bottom panel, 550-bp region of glyceraldehyde-3-phosphate dehydrogenase gene amplified to assess quality of mRNA. Lanes 1 and 8, 100 mg of baboon liver tissue used for RNA extraction; lanes 2 and 9, RNA extraction negative control; lanes 3 and 10, 200 mg of baboon liver tissue used for RNA extraction; lanes 4 and 11, RNA extraction negative control; lane 5, DNase I treatment negative control in which DEPC-treated water was added instead of RNA; lane 6, reverse transcription negative control in which DEPC-treated water was added instead of cDNA; lanes 7 and 12, double-round nested PCR negative control containing best-quality water instead of cDNA; lane 13, single-round PCR negative control containing best-quality water instead of cDNA; lane 14, PCR-positive control containing DNA extracted from liver tissue of baboon 9732 as template.