Non-Fluorescent Differentiation of Viral and Chromosomal Nucleic Acids in Individual Nuclei

Non-fluorescent multiplein situnucleic acid detection is most efficiently achieved by simultaneous hybridization using probes labelled with different reporter molecules. Any reporter molecules which can be used individually can be combined to allow dual nucleic acid detection, but we have found digoxigenin and biotin the most generally useful as they are safe and sensitive. We find that nick-translated probes give more consistent and sensitive results than those labelled by random priming and the protocols described here therefore utilise nick-translated probes (Herrington et al., 1989a). The maximum sensitivity achieved to date with high signal resolution is one to two copies of human papilloma virus (HPV) (Herrington et al., 1992a) in cultured cells and 2.5-12 copies of HPV in archival biopsies (Herrington et al., 1991), and digoxigenin-labelled HPV probes have been used to investigate the role of HPV infection in cervical neoplasia both in biopsies and cervical smears (Herrington 1994; Herrington, 1995). Minor modification to these techniques allows analysis of numerical chromosome abnormalities in both cytological (Herrington et al., 1995) and histological (Southern and Herrington 1996; Southern and Herrington, 1997) material.