Integrated human papillomavirus type 16 (HPV-16) DNA was directly visualized on metaphase chromosomes in the two human cervical carcinoma cell lines SiHa and CaSki by fluorescence in situ hybridization with a biotinylated DNA probe (7.9 kb). The fluorescence intensities of hybridization signals from single copies and dispersed clusters of integrated HPV-16 DNA were quantified using a microscope equipped with a cooled-CCD camera that was interfaced to an image processor and host computer. Hybridization signals were localized on chromosomes using separate, registered images of 4',6-diamidino-2-phenylindole (DAPI) or propidium iodide stained metaphase chromosome spreads. In both SiHa and CaSki spreads, a single fluorescein signal was observed on one or both chromatids of chromosome 13, which was identified by simultaneous hybridization with a biotinylated centromere probe specific for chromosomes 13 and 21. Ratios of the dis- Key terms: Microscopy, fluorescence/image processing, computer-assistedhucleic acid hybridizatiodDNA probes, HPV The technique of in situ hybridization allows one to detect, localize, and quantify unique nucleic acid sequences within single cells and upon individual chromosomes. Radioactive probes and autoradiographic techniques have been required for quantitation of target sequence copy number and are often necessary in order to detect specific nucleic acid sequences present in low abundance (4,12). However, recent advances have been made in the labeling and detection of hybridized nonradioactive probes; thus it is now possible to detect specific nucleic acid sequences only a few kilobases in length on metaphase chromosomes (3,10,16,17,26,30). In physical mapping studies, fluorescence detection of hybridization signals on chromosomes provides better microscopic spatial resolution and greater precision than microautoradiography (6,18,20). In addition, fluorescence detection of nonra-
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