IN-SITU HYBRIDIZATION TECHNIQUES FOR ELECTRON-MICROSCOPY DETECTION OFHUMAN-PAPILLOMAVIRUS GENE-SEQUENCES

At present, in situ hybridization (ISH) study of human papillomavirus (HPV) at the ultrastructural level is limited because of the complexit y of the techniques. We have developed a method for electron microscop y ISH that is easily adapted for routine use. The method permits the u ltrastructural localization of gene sequences of HPV and of any other DNA or RNA sequences, in a technically simple way. CaSki cells contain ing 400-600 copies of HPV type 16 were grown for 3 days, fixed and pro cessed for embedding in LRW resin at 56 degrees C. An ISH technique wa s then carried out using biotinylated DNA probes for HPV types 6/11, 1 6/18, and 31/33/51. Antibiotin antibody conjugated with 15-nm colloida l-gold particles was used to detect the hybrid. All stages of the tech nique were carried out on slides with wells and on microtiter plates. Monkey kidney cells (BGM) with no evidence of any type of integrated H PV in the genome were used as a negative control. The CaSki cells incu bated with the probe specific for HPV types 16/18 yielded a positive i ntranuclear reaction, manifested as either large accumulations of gold particles located next to the membrane in the nuclear periphery or sm all groupings (4-8 gold particles) spread throughout the nucleus. No p ositive reaction was observed in CaSki cells treated with the probe fo r HPV 6/11 and 31/33/51 nor BGM cells with any of the probes used.