Simultaneous molecular karyotyping and mapping of viral DNA integration sites by 25-color COBRA-FISH.
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.
Szuhai
K
K
Laboratory for Cytochemistry, Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands.
Bezrookove
V
V
Wiegant
J
J
Vrolijk
J
J
Dirks
R W
RW
Rosenberg
C
C
Raap
A K
AK
Tanke
H J
HJ
D002877:Chromosomes, Human / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics*; D005260:Female; D006801:Humans; D007089:Image Enhancement / Q000379:methods; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D007621:Karyotyping / Q000379:methods; D008856:Microscopy, Fluorescence; D027383:Papillomaviridae / Q000235:genetics; D030361:Papillomavirus Infections; D014407:Tumor Cells, Cultured; D014412:Tumor Virus Infections; D002583:Uterine Cervical Neoplasms; D016662:Virus Integration / Q000235:genetics*
D004279:DNA, Viral