Bruni R, Argentini C, D'Ugo E, Giuseppetti R, Rapicetta M
Laboratory of Virology, Istituto Superiore di Sanità, Rome, Italy.
J Virol Methods. 1995 Apr;52(3):347-60. doi: 10.1016/0166-0934(94)00170-l.
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.
本文描述了一种基于聚合酶链反应（PCR）和限制性分析的方法，用于快速绘制土拨鼠肝癌（HCC）组织中旱獭肝炎病毒（WHV）的整合图谱。使用病毒引物对进行常规PCR不适用于绘制WHV整合区域，因为无法排除极少量非整合（可PCR扩增）的WHV基因组和复制中间体的存在。该策略的第一个相关部分是鉴定WHV整合侧翼的细胞序列，以便选择一个（或多个）整合特异性引物。通过病毒/细胞连接的反向PCR扩增和产物测序，可以快速获得细胞侧翼序列。整合区域的绘制通过固定侧翼引物PCR（FFP-PCR）进行，在PCR中使用细胞引物作为“固定”引物，并与一组可用的WHV引物中的每一个结合。从而避免了游离型WHV序列的扩增。PCR产物也可以进行限制性分析。基于PCR产物的大小和限制性图谱，将PCR阳性病毒引物和特定的WHV限制性位点组装成一张图谱。本文描述了土拨鼠肝癌中WHV整合图谱的结果。
