Overview of Fluorescence In Situ Hybridization Techniques for Molecular Cytogenetics

A.K. Raap1

1 Leiden University, Leiden
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 8.1
DOI:  10.1002/0471142956.cy0801s00
Online Posting Date:  May, 2001
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This unit presents an overview of the FISH methodology. It covers such topics as direct versus indirect methods, sensitivity, multiplicity, resolution, and applications.

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Table of Contents

  • History
  • Direct Versus Indirect Methods
  • Sensitivity
  • Multiplicity
  • Resolution
  • Applications
  • Literature Cited
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Literature Cited

   Arnoldus, E.P.J., Wiegant, J., Noordermeer, I.A., Wessels, J.W., Beverstock, G.C., Grosveld, G.C., van der Ploeg, M., and Raap, A.K. 1990. Detection of the Philadelphia chromosome in interphase nuclei. Cytogenet. Cell Genet. 54:108‐111.
   Arnoldus, E.P.J., Noordermeer, I.A., Peters, A.B.C., Voormolen, J.H.C., Bots, G.T.A.M., Raap, A.K., and van der Ploeg, M. 1991. Interphase cytogenetics of brain tumors. Genes Chrom. Cancer 3:101‐107.
   Bentz, M., Cabot, G., Moos, M., Speicher, M., Ganser, M., Lichter, P., and Dohner, H. 1994. Detection of chimeric BCR‐ABL genes on bone marrow samples and blood smears in chronic myeloid and acute lymphoblastic leukemia by in situ hybridization. Blood 83:1922‐1928.
   Carter, N.P., Ferguson‐Smith, M.A., Perryman, M.T., Telenius, H., Pelmear, A.H., Leversha, M.A., Glancy, M.T., Wood, S.L., Cook, K., Dyson, H.M., Ferguson‐Smith, M.E., and Willat, L.R. 1992. Reverse chromosome painting: A method for the rapid analysis of aberrant chromosomes in clinical cytogenetics. J. Med. Genet. 29:299‐307.
   Cremer, T., Landegent, J.E., Brueckner, A., Scholl, H.P., Schardin, M., Hager, H.D., Devilee, P., Pearson, P., and van der Ploeg, M. 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., Kurz, A., Zirbel, S., Dietzel, S., Rinke, E., Schröck, E., Speicher, M., Mathieu, R.U., Jauch, A., Emmerlich, P., Scherthan, H., Ried, T., Cremer, P., and Lichter, P. 1993. The role of chromosome territories in the functional compartmentalization of the cell nucleus. Cold Spring Harbor Symp. Quant. Biol. 58:777‐792.
   Dauwerse, J.G., Wiegant, J., Raap, A.K., Breuning, M.H., and van Ommen, G.J.B. 1992. Multiple colors by fluorescence in situ hybridization using ratio‐labelled DNA probes create a molecular karyotype. Hum. Mol. Genet. 1:593‐598.
   Dirks, R.W., van de Rijke, F.M., Fujishita, S., van der Ploeg, M., and Raap, A.K. 1993. Methodologies for specific intron and exon localization in cultured cells by haptenized and fluorochromized probes. J. Cell Sci. 104:1187‐1197.
   du Manoir, S., Speicher, M.R., Joos, S., Schröck, E., Popp, S., Doehner, 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. 90:590‐610.
   Emanuel, B.S. 1993. The use of fluorescence in situ hybridization to identify human chromosomal anomalies. Growth Genet. Hormones 9:6‐12.
   Florijn, R.J., Blonden, L.A.J., Vrolijk, H., Wiegant, J., Vaandrager, J.W., Baas, F., den Dunnen, J.T., Tanke, H.J., van Ommen, G.J.B., and Raap, A.K. 1995. High‐resolution DNA Fiber‐FISH for genomic DNA mapping and colour‐bar coding large genes. Hum. Mol. Genet. 4:831‐836.
   Florijn, R.J., van de Rijke, F.M., Vrolijk, H., Blonden, L.A.J., Hofker, M.H., den Dunnen, J.T., Tanke, H.J., van Ommen, G.J.B., and Raap, A.K. 1996. Exon mapping by Fiber‐FISH or LR‐PCR. Genomics. In press.
   Haaf, T. and Ward, D.C. 1994. High resolution ordering of YAC contigs using extended chromatin and chromosomes. Hum. Mol. Genet. 3:629‐633.
   Heiskanen, M., Karhu, R., Hellsten, E., Peltonen, L., Kallioniemi, O.P., and Palotie, A. 1994. High resolution mapping by FISH to extended DNA fibers prepared from agarose embedded cells. Biotechniques. 17:928‐933.
   Hopman, A.H.N., Ramaekers, F.C.S., Raap, A.K., Beck, J.L.M., Devilee, P., van der Ploeg, M., and Vooijs, G.P. 1988. In situ hybridization as a tool to study numerical chromosome aberrations in solid bladder tumors. Histochemistry 89:307‐316.
   Kallioniemi, A., Kallioniemi, O.P., Sudar, D., Rutovitz, D., Gray, J.W., Waldman, F.M., and Pinkel, D. 1992a. Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors. Science 258:818‐821.
   Kallioniemi, O.P., Kallioniemi, A., Kurisu, W., Thor, A., Chen, L.C., Smith, H.S., Waldman, F.M., Pinkel, D., and Gray, J.W. 1992b. ERBB2 amplification in breast cancer analyzed by fluorescence in situ hybridization. Proc. Natl. Acad. Sci. U.S.A. 89:5321‐5325.
   Kerstens, H.M.J., Poddighe, P.J., and Hanselaar, A.G.J.M. 1995. CARD‐ISH signal amplification. J. Histochem. Cytochem. 43:347‐350.
   Kievits, T., Devilee, P., Wiegant, J., Wapenaar, M.C., Cornelisse, C.J., van Ommen, G.J.B., and Pearson, P.L. 1990. Direct non‐radioactive in situ hybridization of somatic cell hybrid DNA to human lymphocyte chromosomes. Cytometry 1:105‐109.
   Kitsberg, D., Selig, S., Brandeis, M., Simon, I., Keshet, I., Driscoll, D.J., Nicholls, R.D., and Cedar, H. 1993. Allele‐specific replication timing of imprinted gene regions. Nature 364:459‐463.
   Landegent, J.E., Jansen in de Wal, N., van Ommen, G.J.B., Baas, F., de Vijlder, G., van Duijn, P., and van der Ploeg, M. 1985. Chromosomal localization of a unique gene by non‐autoradiographic in situ hybridization. Nature 317:175‐177.
   Landegent, J.E., Jansen in de Wal, N., Dirks, R.W., Baas, F., and van der Ploeg, M. 1987. Use of whole cosmid cloned genomic sequences for chromosomal localization by non‐radioactive in situ hybridization. Hum. Genet. 77:366‐370.
   Langer, P.R., Waldrop, A.A., and Ward, D.C. 1981. Enzymatic synthesis of biotin‐labeled polynucleotides: Novel nucleic acid affinity probes. Proc. Natl. Acad. Sci. U.S.A. 78:6633‐6637.
   Lawrence, J.B., Singer, R.H., and Marselle, L.M. 1989. Highly localized tracks of specific transcripts within interphase nuclei visualized by in situ hybridization. Cell 57:493‐502.
   Lawrence, J.B., Singer, R.H., and McNeil, J.A. 1990. Interphase and metaphase resolution of different distances within the human dystrophin gene. Science 249:928‐932.
   Lengauer, C., Speicher, M.R., Popp, S., Jauch, A., Taniwaki, M., Nagaraja, R., Riethman, H.C., Donis‐Keller, H., D'Urso, M., Schlessinger, D., and Cremer, T. 1993. Chromosomal bar codes produced by multicolor fluorescence in situ hybridization with multiple YAC clones and whole chromosome painting probes. Hum. Mol. Genet. 2:505‐512.
   Lichter, P., Cremer, T., Borden, J., Manuelidis, L., and Ward, D.C. 1988. Delineation of individual human chromosomes in metaphase and interphase cells by in situ hybridization using recombinant DNA libraries. Hum. Genet. 80:224‐234.
   Lichter, P., Tang, C.C., Call, K., Hermanson, G., Evans, G., Housman, D., and Ward, D.C. 1990. High resolution mapping of human chromosome 11 by in situ hybridization with cosmid probes. Science 247:64‐69.
   Melzer, P.S., Guan, X.U., Burgers, A., and Trent, J.M. 1993. Rapid generation of region specific probes by chromosome microdissection and their application. Nature Genet. 1:24‐28.
   Nederlof, P.M., Robinson, D., Abuknesha, R., Wiegant, J., Hopman, A.H.N., Tanke, H.J., and Raap, A.K. 1989. Three‐color fluorescence in situ hybridization for the simultaneous detection of multiple nucleic acid sequences. Cytometry 10:20‐27.
   Nederlof, P.M., Flier, S., Wiegant, J., Raap, A.K., Tanke, H.J., Ploem, J.S., and van der Ploeg, M. 1990. Multiple fluorescence in situ hybridization. Cytometry 11:126‐131.
   Nederlof, P.M., Flier, S., Vrolijk, J., Tanke, H.J., and Raap, A.K. 1992. Fluorescence ratio measurements of double‐labeled probes for multiple in situ hybridization by digital imaging microscopy. Cytometry 13:839‐845.
   Parra, I. and Windle, B. 1993. High resolution visual mapping of stretched DNA by fluorescent hybridization. Nature Genet. 5:17‐21.
   Pinkel, D., Landegent, J.E., Collins, C., Fuscoe, J., Segraves, R., Lucas, J., and Gray, J. 1988. Fluorescence in situ hybridization with human chromosome‐specific libraries: Detection of trisomy 21 and translocations of chromosome 4. Proc. Natl. Acad. Sci. U.S.A. 85:9138‐9142.
   Raap, A.K., Nederlof, P.M., Dirks, R.W., Wiegant, J.C.A.G., and van der Ploeg, M. 1990. Use of haptenised nucleic acid probes in fluorescent in situ hybridisation. In In Situ Hybridization: Application to Developmental Biology and Medicine (N. Harris, and D.G. Williams, eds.) pp. 33‐41. Cambridge University Press, Cambridge.
   Raap, A.K., van de Corput, M.P.C., Vervenne, R.A.W., van Gijlswijk, R.P.M., Tanke, H.J., and Wiegant, J. 1995. Ultrasensitive FISH using peroxidase‐mediated deposition of biotin‐ or fluorochrome‐tyramides. Hum. Mol. Genet. 4:529‐534.
   Ried, T., Baldini, A., Rand, T.C., and Ward, D.C. 1992a. Simultaneous visualization of seven different DNA probes by in situ hybridization using combinatorial fluorescence and digital imaging microscopy. Proc. Natl. Acad. Sci. U.S.A. 89:1388‐1392.
   Ried, T., Landes, G., Dackowski, W., Klinger, K., and Ward, D.C. 1992b. Multicolor fluorescence in situ hybridization for the simultaneous detection of probe sets for chromosomes 13, 18, 21, X and Y in uncultured amniotic fluid cells. Hum. Mol. Genet. 1:307‐313.
   Ried, T., Lengauer, C., Cremer, T., Wiegant, J., Raap, A.K., van der Ploeg, M., Groitl, P., and Lipp, M. 1992c. Specific metaphase and interphase detection of the breakpoint region in 8q24 of Burkitt lymphoma cells by triple colour fluorescence in situ hybridization. Genes Chrom. Cancer 4:69‐74.
   Schröck, E., du Manoir, S., Veldman, T., Schoell, B., Wienberg, J.Y., Ferguson‐Smith, M.A., Ning, Y., Ledbetter, D.H., Bar‐Am, I., Soenksen, D., Garini, Y., and Ried, T. 1996. Multicolor spectral karyotyping of human chromosomes. Science 273:494‐497.
   Selig, S., Okumura, K., Ward, D.C., and Cedar, H. 1993. Delineation of DNA replication time zones by fluorescence in situ hybridization. EMBO J. 11:1217‐1225.
   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. 3:1275‐1280.
   Smit, V.T., Wessels, J.W., Mollevanger, P., Dauwerse, J.G., van Vliet, M., Beverstock, G.C., Breuning, M.H., Devilee, P., Raap, A.K., and Cornelisse, C.J. 1991. Improved interpretation of complex chromosomal rearragements by combined GTG banding and in situ suppression hybridization using chromosome‐specific libraries and cosmid probes. Genes Chrom. Cancer 3:239‐248.
   Speicher, M.R., Gwyn Ballard, S., and Ward, D.C. 1996. Karyotyping human chromosomes by combinatorial multi‐fluor FISH. Nature Genet. 12:368‐375.
   Stilgenbauer, S., Dohner, H., Bulgay‐Morschel, M., Weitz, S., Bentz, M., and Lichter, P. 1993. High frequency of monoallelic retinoblastoma gene deletion in B‐cell chronic lymphocytic leukemia shown by interphase cytogenetics. Blood 81:2118‐2222.
   Tkachuk, D.C., Westbrook, C.A., Andreeff, M., Donlon, T.A., Cleary, M.L., Suryanarayan, K., Homge, M., Redner, A., Gray, J., and Binkel, D. 1990. Detection of bcr‐abl fusion in chronic myelogeneous leukemia by in situ hybridization. Science 250:559‐561.
   Trask, B.J., Massa, H., Kenwrick, S., and Gitschier, J. 1991. Mapping of human chromosome Xq28 by two‐color fluorescence in situ hybridization of DNA sequences in interphase cell nuclei. Am. J. Hum. Genet. 48:1‐15.
   van den Engh, G., Sachs, R., and Trask, B.J. 1992. Estimating genomic distance from DNA sequence location in cell nuclei by a random walk model. Science 257:1410‐1412.
   van Gijlswijk, R.P.M., Wiegant, J., Raap, A.K., and Tanke, H.J. 1996. Improved localization of fluorescent tyramides for fluorescence in situ hybridization using dextran sulfate and polyvinyl alcohol. J. Histochem. Cytochem. 44:389‐392.
   van Ommen, G.J.B., Breuning, M.H., and Raap, A.K. 1995. FISH in genome research and molecular diagnostics. Curr. Opin. Genet. Devel. 5:304‐308.
   Wiegant, J., Kalle, W., Mullenders, L., Brookes, S., Hoovers, J.M.N., Dauwerse, J.G., van Ommen, G.J.B., and Raap, A.K. 1992. High‐resolution in situ hybridization using DNA halo preparations. Hum. Mol. Genet. 1:587‐591.
   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. Nucl. Acids Res. 19:3237‐3241.
   Wiegant, J., Wiesmeijer, C.C., Hoovers, J.M.N., Schuuring, E., d'Azzo, A., Vrolijk, J., Tanke, H.J., and Raap, A.K. 1993. Multiple and sensitive fluorescence in situ hybridization with rhodamine‐, fluorescein‐, and coumarin‐labeled DNAs. Cytogenet. Cell Genet. 63:73‐76.
   Wiegant, J., Verwoerd, N.P., Mascheretti, S., Bolk, M., Tanke, H.J., and Raap, A.K. 1996. An evaluation of a new series of fluorescent dUTPs for fluorescence in situ hybridization. J. Histochem. Cytochem. 44:525‐529.
   Zachar, Z., Kramer, J., Mims, I.P., and Bingham, P.M. 1993. Evidence for channeled diffusion of pre‐mRNA during nuclear RNA transport in metazoans. J. Cell Biol. 12:729‐742.
   Zirbel, R.M., Mathieu, U.R., Kurz, A., Cremer, T., and Lichter, P. 1993. Evidence for a nuclear compartment of transcription and splicing located at chromosome boundaries. Chromosome Res. 1:93‐106.
Key References
   Pardue, M.L. and Gall, J.G. 1969. Molecular hybridization of radioactive DNA to the DNA of cytological preparations. Proc. Natl. Acad. Sci. U.S.A. 64:600‐604.
  Very first report of in situ hybridization using autoradiography for detection.
   Rudkin, G.T. and Stollar, B.D. 1977. High resolution detection of DNA⋅RNA hybrids in situ by indirect immunofluorescence. Nature 265:472‐473.
  First description of indirect FISH using anti‐DNA⋅RNA antibodies.
   Bauman, J.G.J., Wiegant, J., Borst, P., and Van Duijn, P. 1980. A new method for fluorescence microscopical localization of specific DNA sequences by in situ hybridization of fluorochrome labeled RNA. Exp. Cell Res. 138:485‐490.
  First report of direct FISH using fluorochrome‐labeled RNA.
   Langer et al., 1981. See above.
  First description of the synthesis of allylamine dUTP, its conjugation to biotin, and its use in DNA polymerase reactions.
   Landegent et al., 1985. See above.
  First report of single‐copy gene detection by nonradioactive in situ hybridization using reflection microscopy.
   Cremer et al., 1986. See above.
  Introduction of “interphase cytogenetics.”
   Landegent et al., 1987; Lichter et al., 1988; and Pinkel et al., 1988. See above.
  First reports of the repeat suppression hybridization principle, also called chromosomal in situ suppression hybridization.
   Wiegant et al., 1992; Parra and Windle, 1993. See above.
  First Fiber‐FISH reports.
   Hopman et al., 1986. See above.
  First bicolor FISH paper.
   Kallioniemi et al., 1992a. See above.
  First CGH report.
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