Inverse polymerase chain reaction for cloning cellular sequences adjacent to integrated hepatitis B virus DNA in...

Integration of hepatitis B virus (HBV) DNA is found in most HBV-related human hepatocellular carcinomas (HCCs). In the past, construction of genomic libraries was mainly employed to study the role of viral integration. However, large amounts of tissue DNA and a laborious screening procedures were required. Inverse polymerase chain reaction (IPCR) is based on the simple procedures of digestion of DNA with restriction enzymes and circularization of cleavage products before amplification using primers synthesized in the opposite orientations to those normally employed for PCR. This technique allows the in vitro amplification of DNA flanking a region of known sequence. By employing this method, starting from nanograms of hepatoma DNA, two adjacent cellular sequences were cloned from 11 HBV integrants in three HCCs. The original configurations in the chromosomes were further confirmed. One of the flanking cellular sequences was identified as the human 28S rRNA gene, the other was not found homologous to any known human sequences. This method appears to be practical and can be improved further to clone more flanking cellular sequences, especially in early and small HCCs.