Simultaneous molecular karyotyping and mapping of viral DNA integration sites by 25-color COBRA-FISH

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Abstract
Combined binary ratio labeling (COBRA) fluorescence in situ hybridization (FISH) allows 24‐color FISH karyotyping of human metaphase chromosomes utilizing only four fluorochromes, instead of the five required for combinatorial labeling procedures. Here we show that by introduction of a fifth fluorochrome, COBRA‐FISH permits molecular cytogenetic mapping of viral integration sites in complex karyotypes in the context of a 24‐color hybridization. We were able to detect a single copy of the human papillomavirus 16 in the SiHa cell line and to confirm the site of integration at 13q21–31. We also demonstrate the gene mapping possibility of 25‐color hybridization by detecting a MYC cosmid on normal metaphase chromosomes. The possibility of mapping single‐copy probes in the background of 24‐color hybridization expands the tools for cytogenetic mapping of DNA sequences and will contribute to the understanding of the role of viral integration and chromosome rearrangement in virus‐mediated carcinogenesis. Genes Chromosomes Cancer 28:92–97, 2000.