Detection of integration during active replication of hepatitis B virus in the liver

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Abstract
A method is described that enables the unequivocal detection of the integration of hepatitis B virus DNA (HBV-DNA) into the genomic DNA of the host cell, while at the same time the virus exhibits active replication. This detection was achieved by separating the low molecular weight replicating HBV-DNA from the high molecular weight genomic DNA of the host by electrophoresis of the total undigested cellular DNA through low melting agarose. The high molecular weight DNA was isolated from this gel and electrophoresed after digestion with restriction enzyme(s) on a second agarose gel. Transfer of the DNA content of both gels and hybridization of these blots with 32P-labelled HBV-DNA will reveal whether any integrated and/or actively replicating DNA is present. By using this method the presence of a single copy of HBV-DNA integrated in host DNA was demonstrated in hepatocellular carcinoma tissue of a patient with active HBV replication.