The Technique of In Situ Hybridization

Principles and Applications
  • Desirée du Sart
  • K. H. Andy Choo
Part of the Springer Protocols Handbooks book series (SPH)


The aim of this chapter is to give a general overview of the technique of in situ hybridization By way of introducing the technique, the history and background of in situ hybridization will first be discussed, followed by the principles and basic steps involved and lastly, the various applications of in situ hybridization Detailed descriptions of the individual procedures and methods are outside the scope of this chapter, but readers are referred to In Situ Hybridization Protocols, edited by K H. Andy Choo, 1994, and Nonradioactive In Situ Hybridization Application Manual, Boehnnger Mannheim, 2nd Edition, 1996.


Comparative Genome Hybridization Structural Chromosome Rearrangement PRINS Technique Centromeric Repeat Sequence Clone Restriction Endonuclease 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. 1.
    Pardue, M L and Gall, J G (1969) Molecular hybridization of radioactive DNA to the DNA of cytological preparations Proc Natl Acad Sci USA 64, 600–604PubMedCrossRefGoogle Scholar
  2. 2.
    John, H, Birnstiel, M, and Jones, K (1969) RNA-DNA hybrids at the cytological level Nature 223, 582–587PubMedCrossRefGoogle Scholar
  3. 3.
    Kessler, C (1990) The digoxygenin system principle and applications of the novel nonradioactive DNA labeling and detection system Bio Technology Int 1990, 183–194Google Scholar
  4. 4.
    Wiegant, J, Ried, T, Nederlof, P M, van der Ploeg, M, Tanke, H J, and Raap, A K (1991) In situ hybridization with fluoresceinated DNA Nucleic Acids Res 19, 3237–3241PubMedCrossRefGoogle Scholar
  5. 5.
    Earle, E and Choo, K (1994) in Methods in Molecular Biology, vol 33 In Situ Hybridization Protocols (Choo, K,ed),vol 33, pp 147–158, Humana, Totowa, NJ, pp 147–158CrossRefGoogle Scholar
  6. 6.
    Wulf, M, Bosse, A, Voss, B, and Muller, K (1995) Improvement of fluorescence in situ hybridization (RNA-FISH) on human paraffin sections by Propidium Iodide counterstaining Biotechniques 19, 168–172Google Scholar
  7. 7.
    Pinkel, D, Gray, J W, Trask, B, van den Engh, G, Fuscoe, J, and van Dekken, H (1986) Cytogenetic analysis by in situ hybridization with fluorescently labeled nucleic acid probes Cold Spring Harb Symp Quant Biol 51(Pt 1), 151–157PubMedGoogle Scholar
  8. 8.
    Nederlof, P M, van der Flier, S, Wiegant, J, Raap, A K, Tanke, H J, Ploem, J S, and van derPloeg, M (1990) Multiple fluorescence in situ hybridization Cytometry 11, 126–131PubMedCrossRefGoogle Scholar
  9. 9.
    Ried, T, Baldim, A, Rand, T C, and Ward, D C (1992) Simultaneous visualization of seven different DNA probes by in situ hybridization using combinatorial fluorescence and digital imaging microscopy Proc Natl Acad Sci USA 89, 1388–1392PubMedCrossRefGoogle Scholar
  10. 10.
    Speicher, M, Ballard, S, and Ward, D (1996) Karyotyping human chromosomes by combinatorial multi-fluor FISH Nature Genet 12, 368–375PubMedCrossRefGoogle Scholar
  11. 11.
    Lichter, P, Cremer, T, Tang, C J, Watkins, P C, Manuehdis, L, and Ward, D C (1988) Rapid detection of human chromosome 21 aberrations by in situ hybridization Proc Natl Acad Sci USA 85, 9664–9668PubMedCrossRefGoogle Scholar
  12. 12.
    Jauch, A, Daumer, C, Lichter, P, Murken, J, Schroeder Kurth, T, and Cremer, T (1990) Chromosomal in situ suppression hybridization of human gonosomes and autosomes and its use in clinical cytogenetics Hum Genet 85,145–150PubMedCrossRefGoogle Scholar
  13. 13.
    Kievits, T, Dauwerse, J G, Wiegant, J, Devilee, P, Breuning, M H, Cornehsse, C J, van Ommen, G J, and Pearson, P L (1990) Rapid subchromosomal localization of cosmids by nonradioactive in situ hybridization Cytogenet Cell Genet 53, 134–136PubMedCrossRefGoogle Scholar
  14. 14.
    Boyle, A, Lichter, P, and Ward, D (1990) Rapid analysis of mouse-hamster hybrid cell lines by in situ hybridization Genomics 7 Google Scholar
  15. 15.
    Lengauer, C, Riethman, H, and Cremer, T (1990) Painting of human chromosomes with probes generated from hybrid cell lines by PCR with Alu and LI primers Hum Genet 86, 1–6PubMedCrossRefGoogle Scholar
  16. 16.
    Lichter, P, Ledbetter, S, Ledbetter, D, and Ward, D (1990) Fluorescence in situ hybridization with Alu and LI polymerase chain reaction probes for rapid characterization of human chromosomes in hybrid cell lines Proc Natl Acad Sci USA 87, 6634–6638PubMedCrossRefGoogle Scholar
  17. 17.
    Baldim, A, Ross, M, Nizetic, D, Vatcheva, R, Lindsay, E, Lehrach, H, and Simscalco, M (1992) Chromosomal assignment of human YAC clones by fluorescence in situ hybridization use of single colony PCR and multiple labeling Genomics 14, 181–184CrossRefGoogle Scholar
  18. 18.
    Liu, P, Sicihano, J, Seong, D, Craig, J, Zhao, Y, Jong, P D, and Siciliano, M (1993) Dual PCR primers and conditions for isolation of human chromosome painting probes from hybrid cell Cancer Genet Cytogenet 65, 93–99PubMedCrossRefGoogle Scholar
  19. 19.
    Muller, S, Koehler, U, Wienberg, J, Marsella, R, Finelh, P, Antonacci, R, Rocchi, M, and Archidiacono, N (1996) Comparative fluorescence in situ hybridization mapping of primate chromosomes with Alu polymerase chain reaction generated probes from human/ rodent somatic cell hybrids Chromosome Res 4, 38–42PubMedCrossRefGoogle Scholar
  20. 20.
    Bohlander, S K, Espinosa, R d, Le Beau, M M, Rowley, J D, and Diaz, M O (1992) A method for the rapid sequence-independent amplification of microdissected chromosomal material Genomics 13, 1322–1324PubMedCrossRefGoogle Scholar
  21. 21.
    Meltzer, P S, Guan, X Y, Burgess, A, and Trent, J M (1992) Rapid generation of region specific probes by chromosome microdissection and their application Nat Genet 1, 24–28PubMedCrossRefGoogle Scholar
  22. 22.
    Guan, X Y, Meltzer, P S, and Trent, J M (1994) Rapid generation of whole chromosome painting probes (WCPs) by chromosome microdissection Genomics 22, 101–107PubMedCrossRefGoogle Scholar
  23. 23.
    Lengauer, C, Eckelt, A, Weith, A, Endhch, N, Ponehes, N, Lichter, P, Greuhch, K O, and Cremer, T (1991) Painting of defined chromosomal regions by in situ suppression hybridization of libraries from laser-microdissected chromosomes Cytogenet Cell Genet 56, 27–30PubMedCrossRefGoogle Scholar
  24. 24.
    Gray, J, Langlois, G, Carrano, A, Burkhart-Schultz, K, and Dilla, M V (1979) High resolution chromosome analysis One and two parameter flow cytometry Chromosoma 73, 9–27CrossRefGoogle Scholar
  25. 25.
    Telenius, H, Pelmear, A H, Tunnacliffe, A, Carter, N P, Behmel, A., Ferguson Smith, M A, Nordenskjold, M, Pfragner, R, and Ponder, B A (1992) Cytogenetic analysis by chromosome painting using DOP-PCR amplified flow-sorted chromosomes Genes Chromosom Cancer 4, 257–263PubMedCrossRefGoogle Scholar
  26. 26.
    Telenius, H, Carter, N P, Bebb, C E, Nordenskjold, M, Ponder, B A, and Tunnacliffe, A (1992) Degenerate oligonucleotide-pnmed PCR general amplification of target DNA by a single degenerate primer Genomics 13, 718–725PubMedCrossRefGoogle Scholar
  27. 27.
    Vooijs, M, Yu, L C, Tkachuk, D, Pinkel, D, Johnson, D, and Gray, J W (1993) Libraries for each human chromosome, constructed from sorter-enriched chromosomes by using linker-adaptor PCR Am J Hum Genet 52, 586–597PubMedGoogle Scholar
  28. 28.
    Saltman, D L, Dolganov, G M, Pearce, B S, Kuo, S S, Callahan, P J, Cleary, M L, and Lovett, M (1992) Isolation of region-specific cosmids from chromosome 5 by hybridization with microdissection clones Nucleic Acids Res 20, 1401–1404PubMedCrossRefGoogle Scholar
  29. 29.
    Pinkel, D, Landegent, J, Collins, C, Fuscoe, J, Segraves, R, Lucas, J, and Gray, J (1988) Fluorescence in situ hybridization with human chromosome-specific libraries detection of tnsomy 21 and translocations of chromosome 4 Proc Natl Acad Sci USA 85,9138–9142PubMedCrossRefGoogle Scholar
  30. 30.
    Suijkerbuijk, R F, Matthopoulos, D, Kearney, L, Monard, S, Uhut, S, Cotter, F E, Herbergs, J, van Kessel, A G, and Young, B D (1992) Fluorescence in situ identification of human marker chromosomes using flow sorting and Alu element-mediated PCR Genomics 13, 355–362PubMedCrossRefGoogle Scholar
  31. 31.
    Langford, C F, Telenius, H, Carter, N P, Miller, N G, and Tucker, E M (1992) Chromosome painting using chromosome-specific probes from flow-sorted pig chromosomes Cytogenet Cell Genet 61,221–223PubMedCrossRefGoogle Scholar
  32. 32.
    Langford, C, Fischer, P, Binns, M, Holmes, N, and Carter, N (1996) Chromosome-specific paints from a high resolution flow karyotype of the dog Chromosome Res 4, 115–123PubMedCrossRefGoogle Scholar
  33. 33.
    Fuchs, J, Houben, A, Brandes, A, and Schubert, I (1996) Chromosome ‘painting’ in plants-a feasible technique? Chromosoma 104, 315–320PubMedGoogle Scholar
  34. 34.
    Kalhoniemi, A, Kalhoniemi, O P, Sudar, D, Rutovitz, D, Gray, J W, Waldman, F, and Pinkel, D (1992) Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors Science 258, 818–821CrossRefGoogle Scholar
  35. 35.
    du Manoir, S, Speicher, M R, Joos, S, Schrock, E, Popp, S, Dohner, H, Kovacs, G, Robert Nicoud, M, Lichter, P, and Cremer, T (1993) Detection of complete and partial chromosome gains and losses by comparative genomic in situ hybridization Hum Genet 90, 590–610PubMedCrossRefGoogle Scholar
  36. 36.
    James, L and Varley, J (1996) Preparation, labeling and detection of DNA from archival tissue sections suitable for comparative genomic hybridization Chromosome Res 4, 163,164Google Scholar
  37. 37.
    Bryndorf, T, Kirchhoff, M, Rose, H, Maahr, J, Gerdes, T, Karhu, R, Kalhoniemi, A, Chnstensen, B, Lundsteen, C, and Philip, J (1995) Comparative genomic hybridization in clinical cytogenetics Am J Hum Genet 57, 1211–1220PubMedGoogle Scholar
  38. 38.
    Manuehdis, L (1990) A view of interphase chromosomes Science 250, 1533–1540CrossRefGoogle Scholar
  39. 39.
    Haaf, T and Schmid, M (1991) Chromosome topology in mammalian interphase nuclei Exp Cell Res 192, 325–332PubMedCrossRefGoogle Scholar
  40. 40.
    Gilson, E, Laroche, T, and Gasser, S (1993) Telomeres and the functional architecture of the nucleus Trends Cell Biol 3,128–134PubMedCrossRefGoogle Scholar
  41. 41.
    Nagele, R, Freeman, T, McMorrow, L, and Lee, H Y (1995) Precise spatial positioning of chromosomes during prometaphase evidence for chromosomal order Science 270, 1831–1835PubMedCrossRefGoogle Scholar
  42. 42.
    Henikoff, S and Dreesen, T D (1989) Trans-inactivation of the Drosophila brown gene evidence for transcnptional repression and somatic pairing dependence Proc Natl Acad Sci USA 86, 6704–6708PubMedCrossRefGoogle Scholar
  43. 43.
    Loidl, J (1990) The initiation of meiotic chromosome pairing the cytological view Genome 33, 759–778PubMedGoogle Scholar
  44. 44.
    Tease, C (1996) Analysis using dual-color fluorescence in situ hybridization of meiotic chromosome segregation in male mice heterozygous for a reciprocal translocation Chromosome Res 4,61–68PubMedCrossRefGoogle Scholar
  45. 45.
    Trask, B J. (1991) DNA sequence localization in metaphase and interphase cells by fluorescence in situ hybridization Methods Cell Biol 35, 3–35PubMedCrossRefGoogle Scholar
  46. 46.
    Haaf, T and Ward, D C (1994) Structural analysis of alpha-satellite DNA and centromere proteins using extended chromatin and chromosomes Hum Mol Genet 3,697–709PubMedCrossRefGoogle Scholar
  47. 47.
    Trask, B, Pinkel, D, and van den Engh, G (1989) The proximity of DNA sequences in interphase cell nuclei correlated to genomic distance and permits ordering of cosmids spanning 250 kilobase pairs Genomics 5, 710–717PubMedCrossRefGoogle Scholar
  48. 48.
    Lawrence, J B, Carter, K C, and Gerdes, M J (1992) Extending the capabilities of interphase chromatin mapping [news] Nat Genet 2, 171,172CrossRefGoogle Scholar
  49. 49.
    Wiegant, J, Kalle, W, Mullenders, L, Brookes, S, Hoovers, J M, Dauwerse, J G, van Ommen, G J, and Raap, A K (1992) High-resolution in situ hybridization using DNA halo preparations Hum Mol Genet 1,587–591PubMedCrossRefGoogle Scholar
  50. 50.
    Lawrence, J B, Singer, R H, and McNeil, J A (1990) Interphase and metaphase resolution of different distances within the human dystrophin gene Science 249,928–932PubMedCrossRefGoogle Scholar
  51. 51.
    Heng, H H, Squire, J, and Tsui, L C (1992) High-resolution mapping of mammalian genes by in situ hybridization to free chromatin Proc Natl Acad Sci USA 89,9509–9513PubMedCrossRefGoogle Scholar
  52. 52.
    Parra, I and Windle, B (1993) High resolution visual mapping of stretched DNA by fluorescent hybridization [see comments] Nat Genet 5, 1721CrossRefGoogle Scholar
  53. 53.
    Haaf, T and Ward, D C (1994) High resolution ordering ofYACcontigs using extended chromatin and chromosomes Hum Mol Genet 3, 629–633PubMedCrossRefGoogle Scholar
  54. 54.
    Fidlerova, H, Senger, G, Kost, M, Sanseau, P, and Sheer, D (1994) Two simple procedures for releasing chromatin from routinely fixed cells for fluorescence in situ hybridization Cytogenet Cell Genet 65, 203–205PubMedCrossRefGoogle Scholar
  55. 55.
    Heiskanen, M, Karhu, R, Hellsten, E, Peltonen, L, Kalhoniemi, O P, and Palotie, A (1994) High resolution mapping using fluorescence in situ hybridization to extended DNA fibers prepared from agarose-embedded cells Biotechniques 17, 928,929, 932,923Google Scholar
  56. 56.
    Heiskanen, M, Hellsten, E, Kalhoniemi, O P, Makela, T P, Ahtalo, K, Peltonen, L, and Palotie, A (1995) Visual mapping by fiber-FISH Genomics 30, 31–36PubMedCrossRefGoogle Scholar
  57. 57.
    Senger, G, Jones, T A, Fidlerova, H, Sanseau, P, Trowsdale, J, Duff, M, and Sheer, D (1994) Released chromatin linearized DNA for high resolution fluorescence in situ hybridization Hum Mol Genet 3, 1275–1280PubMedCrossRefGoogle Scholar
  58. 58.
    Hudson, P, Penschow, J, Shine, J, Ryan, G, Niall, H, and Coghlan, J (1981) Hybridization histochemistry Use of recombinant DNA as a ‘homing probe’ for tissue localization of specific mRNA populations Endocrinology 108, 353–356PubMedCrossRefGoogle Scholar
  59. 59.
    Gerfen, C (1989) Quantification of in situ hybridization histochemistry for analysis of brain function Methods Neurosci 1, 79–97Google Scholar
  60. 60.
    Pringle, J H, Ruprai, A K, Primrose, L, Keyte, J, Potter, L, Close, P, and Lauder, I (1990) In situ hybridization of immunoglobuhn light chain mRNA in paraffin sections using biotmylated or hapten-labeled ohgonucleotide probes J Pathol 162, 197–207PubMedCrossRefGoogle Scholar
  61. 61.
    McNeil, J A, Johnson, C V, Carter, K C, Singer, R H, and Lawrence, J B (1991) Localizing DNA and RNA within nuclei and chromosomes by fluorescence in situ hybridization Genet Anal Tech Appl 8,41–58PubMedGoogle Scholar
  62. 62.
    Birk, P and Grimm, P (1994) Rapid nonradioactive in situ hybridization for mterleuken-2 mRNA with riboprobes generated using the polymerase chain reaction J Immunol Methods 167, 83PubMedCrossRefGoogle Scholar
  63. 63.
    Dirks, R, Van de Rijke, F, Fujishita, S, Van der Ploeg, M, and Raap, A (1993) Methodology for specific intron and exon RNA detection in cultured cells by haptenized and fluorochromized probes J Cell Sci 104, 1187–1197PubMedGoogle Scholar
  64. 64.
    Wilkinson, D and Green, J (1990) In Situ Hybridization and the Three Dimensional Reconstruction of Serial Sections IRL, Oxford, UKGoogle Scholar
  65. 65.
    DeChiara, T M, Robertson, E J, and Efstratadis, A (1991) Parental imprinting of the mouse insulin-like growth factor II gene Cell 64, 849–859CrossRefGoogle Scholar
  66. 66.
    Koch, J, Kolvraa, S, Petersen, K, Gregersen, N, and Bolund, L (1989) Ohgonucleotide-priming methods for the chromosome specific labeling of alpha satellite DNA in situ Chromosoma 98, 259–265PubMedCrossRefGoogle Scholar
  67. 67.
    Hindkjaer, J, Koch, J, Mogensen, J, Kolvraa, S, and Bolund, L (1994) Primed in situ (PRINS) labeling of DNA Methods Mol Biol 33,95–107PubMedGoogle Scholar
  68. 68.
    Hindkjaer, J, Koch, J, and Mogensen, J (1991) In situ labeling of nucleic acids for gene mapping, diagnostics and functional cytogenetics Adv Mol Genet 4,45–59Google Scholar
  69. 69.
    Volpi, E V and Baldim, A (1993) MULTIPRINS a method for multicolor primed in situ labeling Chromosome Res 1, 257–260PubMedCrossRefGoogle Scholar
  70. 70.
    Speel, E J, Lawson, D, Hopman, A H, and Gosden, J (1995) Multi-PRINS multiple sequential ohgonucleotide primed in situ DNA synthesis reactions label specific chromosomes and produce bands Hum Genet 95, 29–33PubMedCrossRefGoogle Scholar
  71. 71.
    Speel, E J, Jansen, M P, Ramaekers, F C, and Hopman, A H (1994) A novel triplecolor detection procedure for brightfield microscopy, combining in situ hybridization with lmmunocytochemistry J Histochem Cytochem 42, 1299–1307PubMedGoogle Scholar
  72. 72.
    Pellestor, F, Girardet, A, Lefort, G, Andreo, B, and Charheu, J P (1995) Use of the primed in situ labeling (PRINS) technique for a rapid detection of chromosomes 13, 16, 18, 21, X and Y Hum Genet 95, 12–17PubMedCrossRefGoogle Scholar
  73. 73.
    Macas, J, Dolezel, J, Pich, U, Schubert, I, and Lucretti, S (1995) Primer-induced labeling of pea and field bean chromosomes in situ and in suspension Biotechmques 19, 402–408Google Scholar
  74. 74.
    Mogensen, J, Kolvraa, S, Hindkjaer, J, Petersen, S, Koch, J, Nygard, M, Jensen, T, Gregersen, N, Junker, S, and Bolund, L(1991) Nonradioactive, sequence-specific detection of RNA in situ by primed in situ labeling (PRINS) Exp Cell Res 196,92–98PubMedCrossRefGoogle Scholar
  75. 75.
    Chen, R H and Fuggle, S V (1993) In situ cDNA polymerase chain reaction A novel technique for detecting mRNA expression Am J Pathol 143, 1527–1534PubMedGoogle Scholar
  76. 76.
    Haaf, T and Ward, D (1996) Inhibition of RNA polymerase transcription causes chromatin decondensation, loss of nucleolar structure, and dispersion of chromosomal domains Exp Cell Res 224, 163–173PubMedCrossRefGoogle Scholar
  77. 77.
    Weber-Matthiesen, K, Deerberg, J, Muller-Hermehnk, A, Schlegelberger, B, and Grote, W (1993) Rapid immunophenotypic characterization of chromosomally aberrant cells by the new fiction methodCytogenet Cell Genet 63, 123–125PubMedCrossRefGoogle Scholar
  78. 78.
    Speel, E J, Herbergs, J, Ramaekers, F C, and Hopman, A H (1994) Combined lmmunocytochemistry and fluorescence m situ hybridization for simultaneous tricolor detection of cell cycle, genomic, and phenotypic parameters of tumor cells J Histochem Cytochem 42,961–966PubMedGoogle Scholar
  79. 79.
    Fair, C J, Bayne, R A L, Kipling, D, Mills, W, Cntcher, R, and Cooke, H (1995) Generation of a human X-denved minichromosome using telomere-associated chromosome fragmentation EMBO J 14,5444–5454Google Scholar
  80. 80.
    Haase, A, Brahic, M, Stowrmg, L, and Blum, H (1984) Detection of viral nucleic acids by in situ hybridization Meth Virol 7,189–226Google Scholar
  81. 81.
    Gowans, E, Jilbert, A, and Burrell, C (1989) in Nucleic Acid Probes (Symons, R, ed), CRC, Boca Raton, FL, pp 130–158Google Scholar
  82. 82.
    Brigati, D J, Myerson, D, Leary, J. J, Spalholz, B N, Travis, S Z, Fong, C K Y, Hsiung, D G, and Ward, D C (1983) Detection of viral genomes in cultured cells and paraffin embedded tissue sections using biotin labeled hybridization probes Virology 126Google Scholar
  83. 83.
    McDougall, J K, Myerson, D, and Beckmann, A M (1986) Detection of viral DNA and RNA by insitu hybridization J Histochem Cytochem 34, 33–38PubMedGoogle Scholar
  84. 84.
    Brahic, M, Haase, A, and Cash, E (1984) Simultaneous in situ detection of viral RNA and antigens Proc Natl Acad Sci USA 81, 5445PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc , Totowa, NJ. 1998

Authors and Affiliations

  • Desirée du Sart
    • 1
  • K. H. Andy Choo
    • 1
  1. 1.Murdoch Institute for Birth DefectsRoyal Chtldrens HospttalAustralia

Personalised recommendations