A PCR-based strategy for rapid mapping of hepadnavirus integrated sequences in hepatocellular carcinomas.

Abstract

A methodology based on polymerase chain reaction (PCR) and restriction analysis for rapid mapping of woodchuck hepatitis virus (WHV) integrations in hepatocellular carcinoma (HCC) tissues is described. Conventional PCR with viral primer pairs is not suitable for mapping WHV-integrated regions because the presence of minimum amounts of non-integrated (PCR amplifiable) WHV genome and replicative intermediates cannot be excluded. The first relevant part of the strategy is the identification of the cellular sequences flanking the WHV integration in order to select one (or more) integration-specific primer. The cellular flanking sequence can be rapidly obtained by means of inverse-PCR amplification of the viral/cellular junction and sequencing of the product. Mapping of the integrated regions is carried out by fixed flanking primer PCR (FFP-PCR) using the cellular primer as a 'fixed' primer in PCR association with each of an available set of WHV primers. Amplification of episomal WHV sequences is thus avoided. PCR products can also undergo restriction analysis. PCR-positive viral primers and specific WHV restriction sites are assembled into a map, based on the size and restriction pattern of the PCR products. The results of WHV integration mapping in a woodchuck HCC are described.