FISH for Mapping Single Copy Genes

  • Terje Raudsepp
  • Bhanu P. Chowdhary
Part of the Methods in Molecular Biology™ book series (MIMB, volume 422)


During the past two decades fluorescent in-situ hybridization (FISH) has become a standard technique to directly localize, orient, and order genes in the genomes of a wide range of species. Despite the availability of a variety of probes, probe labeling and signal-detection systems, and advanced image analysis software, the core procedures used to carry out FISH remain the same. A detailed overview of these procedures, including target preparation (metaphase/interphase chromosomes and DNA fibers), probe labeling, in-situ hybridization, signal detection, and imaging, is here provided in a stepwise manner.

Key Words

FISH gene mapping metaphase chromosomes interphase chromosomes DNA fibers DNA labeling 


  1. 1.
    Nath, J. and Johnson, K. L. (2000) A review of fluorescence in situ hybridization (FISH): current status and future prospects. Biotech. Histochem. 75, 54–78.CrossRefPubMedGoogle Scholar
  2. 2.
    van der Ploeg, M. (2000) Cytochemical nucleic acid research during the twentieth century. Eur. J. Histochem. 44, 7–42.PubMedGoogle Scholar
  3. 3.
    Liehr, T. and Claussen, U. (2002) Current developments in human molecular cytogenetic techniques. Curr. Mol. Med. 2, 283–297.CrossRefPubMedGoogle Scholar
  4. 4.
    Trask, B. J. (2002) Human cytogenetics: 46 chromosomes, 46 years and counting. Nat. Rev. Genet. 3, 769–778.CrossRefPubMedGoogle Scholar
  5. 5.
    Levsky, J. M. and Singer, R. H. (2003) Fluorescence in situ hybridization: past, present and future. J. Cell Sci. 116, 2833–2838.CrossRefPubMedGoogle Scholar
  6. 6.
    Chowdhary, B. P. and Raudsepp, T. (2005) Mapping genomes at the chromosome level. In Mammalian Genomics (Ruvinsky, A. and Graves, J. A. M. eds), pp. 23–66, CABI, Wallingford, UK.CrossRefGoogle Scholar
  7. 7.
    Speicher, M. R. and Carter, N. P. (2005) The new cytogenetics: blurring the boundaries with molecular biology. Nat. Rev. Genet. 6, 782–792.CrossRefPubMedGoogle Scholar
  8. 8.
    Nederlof, P. M., Robinson, D., Abuknesha, R., et al. (1989) Three-color fluorescence in situ hybridization for the simultaneous detection of multiple nucleic acid sequences. Cytometry 10, 20–27.CrossRefPubMedGoogle Scholar
  9. 9.
    Nederlof, P. M., van der Flier, S., Wiegant, J., et al. (1990) Multiple fluorescence in situ hybridization. Cytometry 11, 126–131.CrossRefPubMedGoogle Scholar
  10. 10.
    Birren, B., Green, E. D., Klapholtz, S., Myers, R. M., and Rskams, J. (eds) (1997) Genome Analysis. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.Google Scholar
  11. 11.
    Daniel, S. G., Westling, M. E., Moss, M. S., and Kanagy, B. D. (1998) FastTag nucleic acid labeling system: a versatile method for incorporating haptens, fluorochromes and affinity ligands into DNA, RNA and oligonucleotides. Biotechniques 24, 484–489.PubMedGoogle Scholar
  12. 12.
    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.CrossRefPubMedGoogle Scholar
  13. 13.
    Trask, B., Pinkel, D., and van den Engh, G. (1989) The proximity of DNA sequences in interphase cell nuclei is correlated to genomic distance and permits ordering of cosmids spanning 250 kilobase pairs. Genomics 5, 710–717.CrossRefPubMedGoogle Scholar
  14. 14.
    Trask, B. J., Allen, S., Massa, H., et al. (1993) Studies of metaphase and interphase chromosomes using fluorescence in situ hybridization. Cold Spring Harb. Symp. Quant. Biol. 58, 767–775.PubMedGoogle Scholar
  15. 15.
    Heiskanen, M., Karhu, R., Hellsten, E., et al. (1994) High resolution mapping using fluorescence in situ hybridization to extended DNA fibers prepared from agarose-embedded cells. Biotechniques 17, 928–929, 932–933.PubMedGoogle Scholar
  16. 16.
    Heiskanen, M., Hellsten, E., Kallioniemi, O. P., et al. (1995) Visual mapping by fiber-FISH. Genomics 30, 31–36.CrossRefPubMedGoogle Scholar
  17. 17.
    Heiskanen, M., Kallioniemi, O., and Palotie, A. (1996) Fiber-FISH: experiences and a refined protocol. Genet Anal. 12, 179–184.PubMedGoogle Scholar
  18. 18.
    Watson, J. D. and Crick, F. H. (1953) Molecular structure of nucleic acids; a structure for deoxyribose nucleic acid. Nature, 171, 737–738.CrossRefPubMedGoogle Scholar
  19. 19.
    Sjoberg, A., Peelman, L. J., and Chowdhary, B. P. (1997) Application of three different methods to analyse fibre-FISH results obtained using four lambda clones from the porcine MHC III region. Chromosome Res. 5, 247–253.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2008

Authors and Affiliations

  • Terje Raudsepp
    • 1
  • Bhanu P. Chowdhary
    • 1
  1. 1.Department of Veterinary Integrative BiosciencesTexas A&M UniversityCollege Station

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