Summary
The experimentally derived parameter Tmt (tissue Tm) was defined previously to describe the end-point used for evaluation of the stringency of non-isotopic in situ hybridization and was found to differ from the theoretical melting temperature (Tm) for several HPV types. In this paper, the reasons for this discrepancy were investigated by performing a series of experiments with a variety of probes for both human genomic and integrated viral sequences in isolated and cultured normal and abnormal cells in addition to paraffin-embedded material. Tmt was shown to be dependent on several parameters of probe and target, and on the sensitivity of the detection system used but was not affected by aldehyde fixation or paraffin wax embedding under optimal conditions of nucleic acid unmasking. These data support the hypothesis that differences between Tmt and Tm may be due to the use of a different end-point for in situ hybridization analysis rather than biochemical alteration of DNA-DNA interactions in intact cells. Appropriate stringency conditions should therefore be determined by experiment rather than calculated theoretically for gene evaluation in cells and tissues.
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References
Alexandrov, I. I., Mitkevich, S. P. & Yurov, Y. B. (1988) The phylogeny of human chromosome specific alpha satellites. Chromosoma (Berlin) 96, 443–53.
Baker, C. C., Phelps, W. C., Lindgren, V., Braun, M. J., Gonda, M. A. & Howley, P. M. (1987) Structural and transcriptional analysis of human papillomavirus type 16 sequences in cervical carcinoma cell lines. J. Virol. 61, 962–971.
Britten, R. J., Graham, D. E. & Neufeld, B. R. (1974) Analysis of repeating DNA sequences by reassociation. In Methods in Enzymology (edited by Grossmann, L. & Moldave, K.), Vol. 29, pp. 363–418. London: Academic Press.
Burns, J., Graham, A. K. & Mcgee, J. O'D. (1988) Non-isotopic detection of in situ nucleic acid in cervix: an updated protocol. J. Clin. Pathol. 41, 897–99.
Cooke, H. J., Schmidtke, H. & Gosden, J. R. (1982) Characterisation of a human Y chromosome repeated sequence and related sequences in higher primates. Chromosoma (Berlin) 87, 491–502.
Cox, K. H., Deleon, D. V., Angerer, L. M. & Angerer, R. C. (1984) Detection of mRNA in sea urchin embryos by in situ hybridization using asymmetric RNA probes. Dev. Biol. 101, 485–502.
Hames, B. D. & Higgins, S. J., eds (1985) Nucleic Acid Hybridisation: a Practical Approach. Oxford: Oxford University Press.
Herrington, C. S., Burns, J., Graham, A. K., Evans, M. F. & Mcgee, J. O'D. (1989a) Interphase cytogenetics using biotin and digoxigenin labelled probes I: relative sensitivity of both reporters for detection of HPV16 in CaSki cells. J. Clin. Pathol. 42, 592–600.
Herrington, C. S., Burns, J., Graham, A. K., Bhatt, B. & Mcgee, J. O'D. (1989b) Interphase cytogenetics using biotin and digoxigenin labelled probes II: simultaneous detection of two nucleic acid species in individual nuclei. J. Clin. Pathol. 42, 601–6.
Herrington, C. S., Burns, J., Graham, A. K. & Mcgee, J. O'D. (1990) Discrimination of closely homologous HPV types by in situ hybridisation: definition and derivation of Tmts. Histochem. J. 22, 545–54.
Herrington, C. S., Graham, A. K. & Mcgee, J. O'D. (1991) Interphase cytogenetics using biotin and digoxigenin labelled probes: III. Increased sensitivity and flexibility for detecting HPV in cervical biopsy specimens and cell lines. J. Clin. Pathol. 44, 33–8.
Herrington, C. S., DeAngelis, M., Evans, M. F., Troncone, G. & Mcgee, J. O'D. (1992) High risk HPV detection in routine cervical smears: strategy for screening. J. Clin. Pathol. 45, 385–90.
Herrington, C. S., Anderson, S. M., Graham, A. K. & Mcgee, J. O'D. (1993a) The discrimination of high risk HPV types by in situ hybridization and the polymerase chain reaction. Histochem. J. 25, 191–8.
Herrington, C. S., Cooper, K. & Mcgee, J. O'D. (1994) Interphase cytogenetics: the determination of chromosome ploidy independent of cellular proliferation. J. Pathol. (in press).
Mincheva, A., Gissmann, L. & Zur Hausen, H. (1987) Chromosomal integration sites of human papillomavirus DNA in three cervical cancer cell lines mapped by in situ hybridisation. Med. Microbiol. Immunol. 176, 245–56.
Traganos, F., Darzynkiewicz, Z., Shaprless, T. & Melamed, M. R. (1975) Denaturation of deoxyribonucleic acid in situ: effect of formaldehyde. J. Histochem. Cytochem. 23, 431–8.
Waye, J. S. & Willard, H. F. (1989) Chromosome specificity of satellite DNAs: short- and long-range organization of a diverged dimeric subset of human alpha satellite from chromosome 3. Chromosoma 97, 475–80.
Waye, J. S., Durfy, S., Pinkel, D., et al. (1987a) Chromsome-specific alpha satellite DNA from human chromosome 1: hierarchical structure and genomic organization of a polymorphic domain spanning several hundred kilobase pairs of centromeric DNA. Genomics 1, 43–51.
Waye, J. S., England, S. B. & Willard, H. F. (1987b) Genomic organization of alpha satellite DNA on human chromosome 7: evidence for two distinct alphoid domains on a single chromosome. Mol. Cell Biol. 7, 349–56.
Waye, J. S., Creeper, L. & Willard, H. F. (1987c) Organization and evolution of alpha satellite DNA from human chromosome 11. Chromosoma 95, 182–8.
Willard, H. F. & Waye, J. S. (1987) Hierarchical order in chromosome-specific human alpha satellite DNA. Trends Genetics 3, 193–8.
Willard, H. F., Smith, K. & Sutherland, J. (1983) Isolation and characterization of a major tandem repeat family from the human X chromosome. Nucleic Acids Res. 11, 2017–33.
Willard, H. F., Greig, G., Powers, V. & Waye, J. (1987) Molecular organization and haplotype analysis of centromeric DNA from human chromsome 17: implications for linkage in neurofibromatosis. Genomics 1, 368–73.
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Herrington, C.S., O'D. Mcgee, J. Discrimination of closely homologous human genomic and viral sequences in cells and tissues: further characterization of Tmt . Histochem J 26, 545–552 (1994). https://doi.org/10.1007/BF00158588
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DOI: https://doi.org/10.1007/BF00158588