title: Detection of Hepatocyte Clones Containing Integrated Hepatitis B Virus DNA Using Inverse Nested PCR.
abstract: Chronic hepatitis B virus (HBV) infection is a major cause of liver cirrhosis and hepatocellular carcinoma (HCC), leading to ~600,000 deaths per year worldwide. Many of the steps that occur during progression from the normal liver to cirrhosis and/or HCC are unknown. Integration of HBV DNA into random sites in the host cell genome occurs as a by-product of the HBV replication cycle and forms a unique junction between virus and cellular DNA. Analyses of integrated HBV DNA have revealed that HCCs are clonal and imply that they develop from the transformation of hepatocytes, the only liver cell known to be infected by HBV. Integrated HBV DNA has also been shown, at least in some tumors, to cause insertional mutagenesis in cancer driver genes, which may facilitate the development of HCC. Studies of HBV DNA integration in the histologically normal liver have provided additional insight into HBV-associated liver disease, suggesting that hepatocytes with a survival or growth advantage undergo high levels of clonal expansion even in the absence of oncogenic transformation. Here we describe inverse nested PCR (invPCR), a highly sensitive method that allows detection, sequencing, and enumeration of virus-cell DNA junctions formed by the integration of HBV DNA. The invPCR protocol is composed of two major steps: inversion of the virus-cell DNA junction and single-molecule nested PCR. The invPCR method is highly specific and inexpensive and can be tailored to DNA extracted from large or small amounts of liver. This procedure also allows detection of genome-wide random integration of any known DNA sequence and is therefore a useful technique for molecular biology, virology, and genetic research.
authors.affiliation: Liver Cell Biology Laboratory, Centenary Institute, Sydney, NSW, 2050, Australia.|Sydney Medical School, University of Sydney, Sydney, NSW, 2050, Australia.|Department of Molecular and Cellular Biology, School of Biological Sciences, University of Adelaide, Adelaide, SA, 5005, Australia.
authors.forename: Thomas
authors.initials: T
authors.lastname: Tu
authors.affiliation: Department of Molecular and Cellular Biology, School of Biological Sciences, University of Adelaide, Adelaide, SA, 5005, Australia. allison.jilbert@adelaide.edu.au.
authors.forename: Allison R
authors.initials: AR
authors.lastname: Jilbert
mesh_terms: D004279:DNA, Viral; D006515:Hepatitis B virus / Q000502:physiology*; D019694:Hepatitis B, Chronic / Q000821:virology*; D022781:Hepatocytes / Q000821:virology*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA; D016662:Virus Integration
chemical_list: D004279:DNA, Viral
keywords: Clonal expansion; Insertional mutagenesis; Integrated DNA; Inverse nested PCR; Virus–cell DNA junction