Human Papillomavirus Integration: Analysis by Molecular Combing and Fiber-FISH.

Human papillomaviruses (HPVs) are frequently integrated in HPV-associated cancers. HPV genomes can be integrated in three patterns: A single integrated HPV genome (type I), multiple, tandemly integrated HPV genomes (type II), and multiple, tandemly integrated HPV genomes interspersed with host DNA (type III). Analysis of the organization of type II and type III integration sites is complicated by their repetitive nature, as sequences of individual repeats are difficult to distinguish from each other. This article presents a method for directly visualizing HPV integration sites using molecular combing combined with fluorescent in situ hybridization, also known as fiber-FISH. In this technique, genomic DNA is stretched across a glass coverslip and individual integrated HPV sequences are detected and directly visualized by in situ hybridization with a resolution of ∼1 kb. Fiber-FISH allows comprehensive characterization of the genomic organization of HPV integration sites containing type II and type III integration.