Summary
A method of in situ hybridization for visualizing individual human chromosomes from pter to qter, both in metaphase spreads and interphase nuclei, is reported. DNA inserts from a single chromosomal library are labeled with biotin and partially preannealed with a titrated amount of total human genomic DNA prior to hybridization with cellular or chromosomal preparations. The cross-hybridization of repetitive sequences to nontargeted chromosomes can be markedly suppressed under appropriate preannealing conditions. The remaining single-stranded DNA is hybridized to specimens of interest and detected with fluorescent or enzymelabeled avidin conjugates following post-hybridization washes. DNA inserts from recombinant libraries for chromosomes 1, 4, 7, 8, 13, 14, 18, 20, 21, 22, and X were assessed for their ability to decorate specifically their cognate chromosome; most libraries proved to be highly specific. Quantitative densitometric analyses indicated that the ratio of specific to nonspecific hybridization signal under optimal preannealing conditions was at least 8:1. Interphase nuclei showed a cohesive territorial organization of chromosomal domains, and laserscanning confocal fluorescence microscopy was used to aid the 3-D visualization of these domains. This method should be useful for both karyotypic studies and for the analysis of chromosome topography in interphase cells.
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References
Agard DA, Sedat JW (1983) Three-dimensional architecture of a polytene nucleus. Nature 302:676–681
Brakenhoff GJ, Blom P, Barends P (1979) Confocal scanning light microscopy with high aperture immersion lenses. J Microsc 117:219–232
Brigati DJ, Myerson D, Leary JJ, Spalholz B, Travis SZ, Fong CKY, Hsiung GD, Ward DC (1983) Detection of viral genomes in cultured cells and paraffin-embedded tissue sections using biotinlabeled hybridization probes. Virology 126:32–50
Britten RJ, Kohne DE (1968) Repeated sequences in DNA. Science 161:529–540
Cooke HJ, Hindley J (1979) Cloning of human satellite III DNA: different components are on different chromosomes. Nucleic Acids Res 6:3177–3197
Cremer C, Cremer T (1978) Considerations on a laser-scanning-microscope with high resolution and depth of field. Microscopica Acta 81:31–44
Cremer T, Cremer C, Baumann H, Luedtke EK, Sperling K, Teuber V, Zorn C (1982) Rabl's model of the interphase chromosome arrangement tested in Chinese hamster cells by premature chromosome condensation and laser-UV-microbeam experiments. Hum Genet 60:46–56
Cremer T, Landegent J, Brückner A, Scholl HP, Schardin M, Hager HD, Devilee P, Pearson P, Ploeg M van der (1986) Detection of chromosome aberrations in the human interphase nucleus by visualization of specific target DNAs with radioactive and non-radioactive in situ hybridization techniques: diagnosis of trisomy 18 with probe L1.84. Hum Genet 74:346–352
Cremer T, Tesin D, Hopman AHN, Manuelidis L (1988a) Rapid interphase and metaphase assessment of specific chromosomal changes in neuroectodermal tumor cells by in situ hybridization with chemically modified DNA probes. Exp Cell Res 176:199–220
Cremer T, Lichter P, Borden J, Ward DC, Manuelidis L (1988b) Detection of chromosome aberrations in metaphase and interphase tumor cells by in situ hybridization using chromosome-specific library probes. Hum Genet 80:235–246
Davis LG, Dibner MD, Battey JF (1986) Basic methods in molecular biology. Elsevier, New York Amsterdam
Emmerich P, Loos P, Jauch A, Hopman AHN, Wiegant J, Higgins M, White BN, Ploeg M van der, Cremer C, Cremer T (1988) Double in situ hybridization in combination with digitized image analysis: a new approach to study interphase chromosome topography. Exp Cell Res (in press)
Finney DJ (1971) Statistical methods in biological assay, 2nd edn. Hafner Press, New York
Gosden JR, Lawrie SS, Cooke HJ (1981) A cloned repeated DNA sequence in human chromosome heteromorphisms. Cytogenet Cell Genet 29:32–39
Hamada H, Petrino MG, Kakunaga T (1982) A novel repeated element with Z-DNA-forming potential is widely found in evolutionary diverse eukaryotic genomes. Proc Natl Acad Sci USA 79:6465–6469
Hens L, Baumann H, Cremer T, Sutter A, Cornelis JJ, Cremer C (1983) Immunocytochemical localization of chromatin regions UV-microirradiated in S-phase or anaphase: evidence for a territorial organization of chromosomes during the cell cycle of Chinese hamster cells. Exp Cell Res 149:257–269
Johnson GD, Davidson RS, McNamee KC, Russell G, Goodwin D, Holborow EJ (1982) Fading of immunofluorescence during microscopy: a study of the phenomenon and its remedy. J Immunol Methods 55:231–242
Landegent JE, Jansen in de Wal N, Dirks RW, Baas F, Ploeg M van der (1987) Use of whole cosmid cloned genomic sequences for chromosomal localization by non-radioactive in situ hybridization. Hum Genet 77:366–370
Langer PR, Waldrop AA, Ward DC (1981) Enzymatic synthesis of biotin-labeled polynucleotides: novel nucleic acid affinity probes. Proc Natl Acad Sci USA 78:6633–6637
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NJ
Manuelidis L (1984) Different central nervous system cell types display sistinct and nonrandom arrangements of satellite DNA sequences. Proc Natl Acad Sci USA 81:3123–3127
Manuelidis L (1985a) Individual interphase chromosome domains revealed by in situ hybridization. Hum Genet 71:288–293
Manuelidis L (1985b) Indications of centromere movement during interphase and differentiation. Ann NY Acad Sci 450:205–221
Manuelidis L, Borden J (1988) Reproducible compartmentalization of individual chromosome domains in human CNS cells revealed by in situ hybridization and three dimensional reconstruction. Chromosoma 96:397–410
Manuelidis L, Ward DC (1984) Chromosomal and nuclear distribution of theHindIII 1.9-kb human DNA repeat segments. Chromosoma 91:28–38
McDermid HE, Duncan AMV, Higgins MJ, Hamerton JL, Rector E, Brasch KR, White BN (1986) Isolation and characterization of an α-satellite repeated sequence from human chromosome 22. Chromosoma 94:228–234
Mendelsohn ML, Mayall BH, Bogart E, Moore DH II, Perry BH (1973) DNA content and DNA-based centromeric index of the 24 human chromosomes. Science 179:1126–1129
Pinkel D, Straume T, Gray JW (1986a) Cytogenetic analysis using quantitative, high sensitivity, fluorescence hybridization. Proc Natl Acad Sci USA 83:2934–2938
Pinkel D, Gray JW, Trask B, Engh G van den, Fuscoe J, Dekken H van (1986b) Cytogenetic analysis by in situ hybridization with fluorescently labeled nucleic acid probes. Cold Spring Harbor Symp Quant Biol 51:151–157
Rappold GA, Cremer T, Hager HD, Davies KE, Müller CR, Yang T (1984) Sex chromosome positions in human interphase nuclei as studied by in situ hybridization with chromosome specific DNA probes. Hum Genet 67:317–325
Schardin M, Cremer T, Hager HD, Lang M (1985) Specific staining of human chromosomes in Chinese hamster x man hybrid cell lines demonstrates interphase chromosome territories. Hum Genet 71: 281–287
Sealey PG, Whittaker PA, Southern EM (1985) Removal of repeated sequences from hybridization probes. Nucleic Acids Res 13: 1905–1922
Trask B, Engh G van den, Pinkel D, Mullikin J, Waldman F, Dekken H van, Gray J (1988) Fluorescence in situ hybridization to interphase cell nuclei in suspension allows flow cytometric analysis of chromosome content and microscopic analysis of nuclear organization. Hum Genet 78:251–259
Van Dilla MA, Deaven LL, Albright KL, Allen NA, Aubuchon MR, Bartholdi MF, Brown NC, Campbell EW, Carrano AV, Clark LM, Cram LS, Crawford BD, Fuscoe JC, Gray JW, Hildebrand CE, Jackson PJ, Jett JH, Longmire JL, Lozes CR, Luedemann ML, Martin JC, McNinch JS, Meincke LJ, Mendelsohn ML, Meyne J, Moyzis RK, Munk AC, Perlman J, Peters DC, Silva AJ, Trask BJ (1986) Human chromosome-specific DNA libraries: construction and availability. Biotechnology 4:537–552
Waye JS, England SB, Willard HF (1987) Genomic organization of alpha satellite DNA on human chromosome 7: evidence for two distinct alphoid domains on a single chromosome. Mol Cell Biol 7:349–356
White JG, Amos WB, Fordman M (1987) An evaluation of confocal versus conventional imaging of biological structures by fluorescence light microscopy. J Cell Biol 105:41–48
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Lichter, P., Cremer, T., Borden, J. et al. Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization using recombinant DNA libraries. Hum Genet 80, 224–234 (1988). https://doi.org/10.1007/BF01790090
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DOI: https://doi.org/10.1007/BF01790090