The parameter Tm(t) has been defined by non-isotopic in situ hybridization and describes the tissue melting temperature (Tm(t)) of human papillomavirus (HPV) DNA sequences. In this study, multiple in situ hybridization signals for HPV types 16, 31 and 33 in individual archival biopsies hybridized with genomic probes are shown by polymerase chain reactions to be due to cross-hybridization of probe sequences to a single tissue target. Tm(t) is independent of viral type but depends on the homology between probe and target when using nick-translated whole genomic probes. The difference between Tm and Tm(t) is not due to the presence of viral capsid protein. Multiple HPV signals in archival material should not therefore be interpreted as indicative of multiple HPV infection unless adequate stringency conditions have been employed or they are present in morphologically distinct areas of the biopsy.
Furthermore, extrapolation of calculated DNA homologies to non-isotopic in situ hybridization analysis may not be appropriate. A hybridization signal does not imply probe and target identity: this has implications for HPV typing in clinical material.