Skip to main content

Labeling Fluorescence In Situ Hybridization Probes for Genomic Targets

  • Protocol

Part of the Methods in Molecular Biology™ book series (MIMB,volume 204)

Abstract

Fluorescence in situ hybridization (FISH) requires nucleic acid probes, including deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or nucleic acid analogs, labeled directly with fluorophores, or capable of indirect association with fluorophores. The nucleic acid provides the FISH assay with its specificity through complementary pairing of the probe nucleotides with nucleotides of the target nucleic acid. The appended fluorophores provide the ability to visually detect the homologous regions within the cellular structure using a fluorescence microscope. Photographic or electronic cameras can also be used to provide permanent images of the fluorescence staining patterns, and the latter can be used to provide quantitative measurements of the probe fluorescence.

Keywords

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.

This is a preview of subscription content, log in via an institution.

Buying options

Protocol
EUR   44.95
Price includes VAT (France)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
EUR   117.69
Price includes VAT (France)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
EUR   158.24
Price includes VAT (France)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
EUR   158.24
Price includes VAT (France)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Bueno D., Skinner J., Abud H., and Heath J. K. (1996) Double in situ hybridization on mouse embryos for detection of overlapping regions of gene expression. Trends Genet. 12, 385–387.

    CrossRef  CAS  PubMed  Google Scholar 

  2. Diwu Z., Klaubert D. H., and Haugland R. P. (1999) Spectral properties and biological applications of ELF enzyme substrates that generate bright fluorescent precipitates at the enzymatic activity sites. Proc. SPIE-Intl. Soc. Opt. Eng. 3602, 265.

    CAS  Google Scholar 

  3. Jowett T. and Yan Y. L. (1996) Double fluorescent in situ hybridization to zebrafish embryos. Trends Genet. 12, 387–389.

    CrossRef  CAS  PubMed  Google Scholar 

  4. Bobrow M. N., Harris T. D., Shaughnessy K. J., and Litt G. J. (1989) Catalyzed reporter deposition, a novel method of signal amplification. Application to immunoassays. J. Immunol. Methods 125, 279–285.

    CrossRef  CAS  PubMed  Google Scholar 

  5. Raap A. K., Van De Corput M. P., Vervenne R. A., Van Gijlswijk R. P., Tanke H. J., and Wiegant J. (1995) Ultra-sensitive FISH using peroxidase-mediated deposition of biotin-or fluorochrome tyramides. Hum. Mol. Genet. 4, 529–534.

    CrossRef  CAS  PubMed  Google Scholar 

  6. Speel E. J., Ramaekers F. C., and Hopman A. H. (1997) Sensitive multicolor fluorescence in situ hybridization using catalyzed reporter deposition (CARD) amplification. J. Histochem. Cytochem. 45, 1439–1446.

    CAS  PubMed  Google Scholar 

  7. Bauman J. G., and Van Duijn P. (1981) Hybrido-cytochemical localization of specific DNA sequences by fluorescence microscopy. Histochem. J. 13, 723–733.

    CrossRef  CAS  PubMed  Google Scholar 

  8. Cremer T., Landegent J., Bruckner A., et al. (1986) Detection of chromosome aberrations in the human interphase nucleus by visualization of specific target DNAs with radioactive and nonradioactive in situ hybridization techniques: Diagnosis of trisomy 18 with probe L1.84. Hum. Genet. 74, 346–352.

    CrossRef  CAS  PubMed  Google Scholar 

  9. Cremer T., Tesin D., Hopman A. H., and Manuelidis L. (1988) 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.

    CrossRef  CAS  PubMed  Google Scholar 

  10. Landegent J. E., Jasen in De Wal N., Baan R. A., Hoeijmakers J. H., and Van Der Ploeg M. (1984) 2-Acetylaminofluorene-modified probes for the indirect hybridocytochemical detection of specific nucleic acid sequences. Exp. Cell Res. 153, 61–72.

    CrossRef  CAS  PubMed  Google Scholar 

  11. 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.

    CrossRef  CAS  PubMed  Google Scholar 

  12. Nederlof P. M., Van Der Flier S., Wiegant J., et al. (1990) Multiple fluorescence in situ hybridization. Cytometry 11, 126–131.

    CrossRef  CAS  PubMed  Google Scholar 

  13. Dale R. M. and Ward D. C. (1975) Mercurated polynucleotides: New probes for hybridization and selective polymer fractionation. Biochemistry 14, 2458–2469.

    CrossRef  CAS  PubMed  Google Scholar 

  14. Hopman A. H., Wiegant J., Tesser G. I., and Van Duijn P. (1986) A nonradioactive in situ hybridization method based on mercurated nucleic acid probes and sulfhydryl-hapten ligands. Nucl. Acids Res. 14, 6471–6488.

    CrossRef  CAS  PubMed  Google Scholar 

  15. Hopman A. H., Wiegant J., and Van Duijn P. (1987) Mercurated nucleic acid probes, a new principle for nonradioactive in situ hybridization. Exp. Cell Res. 169, 357–368.

    CrossRef  CAS  PubMed  Google Scholar 

  16. Van Belkum A., Linkels E., Jelsma T., Van Den Berg F. M., and Quint W. (1994) Nonisotopic labeling of DNA by newly developed hapten-containing platinum compounds. Biotechniques 16, 148–153.

    PubMed  Google Scholar 

  17. Draper D. E. (1984) Attachment of reporter groups to specific, selected cytidine residues in RNA using a bisulfite-catalyzed transamination reaction. Nucl. Acids Res. 12, 989–1002.

    CrossRef  CAS  PubMed  Google Scholar 

  18. Reisfeld A., Rothenberg J. M., Bayer E. A., and Wilchek M. (1987) Nonradioactive hybridization probes prepared by the reaction of biotin hydrazide with DNA. Biochem. Biophys. Res. Commun. 142, 519–526.

    CrossRef  CAS  PubMed  Google Scholar 

  19. Keller G. H., Huang D. P., and Manak M. M. (1989) Labeling of DNA probes with a photoactivatable hapten. Anal. Biochem. 177, 392–395.

    CrossRef  CAS  PubMed  Google Scholar 

  20. Keller G. H., Cumming C. U., Huang D. P., Manak M. M., and Ting R. (1988) A chemical method for introducing haptens onto DNA probes. Anal. Biochem. 170, 441–450.

    CrossRef  CAS  PubMed  Google Scholar 

  21. Chu B. C., Wahl G. M., and Orgel L. E. (1983) Derivatization of unprotected polynucleotides. Nucl. Acids Res. 11, 6513–6529.

    CrossRef  CAS  PubMed  Google Scholar 

  22. Morrison L. E., Halder T. C., and Stols L. M. (1989) Solution-phase detection of polynucleotides using interacting fluorescent labels and competitive hybridization. Anal. Biochem. 183, 231–244.

    CrossRef  CAS  PubMed  Google Scholar 

  23. Langer P. R., Waldrop A. A., and Ward D. C. (1981) Enzymatic synthesis of biotinlabeled polynucleotides: novel nucleic acid affinity probes. Proc. Natl. Acad. Sci. USA 78, 6633–6637.

    CrossRef  CAS  PubMed  Google Scholar 

  24. Langer-Safer P. R., Levine M., and Ward D. C. (1982) Immunological method for mapping genes on Drosophila polytene chromosomes. Proc. Natl. Acad. Sci. USA 79, 4381–4385.

    CrossRef  CAS  PubMed  Google Scholar 

  25. Manuelidis L., Langer-Safer P. R., and Ward D. C. (1982) High-resolution mapping of satellite DNA using biotin-labeled DNA probes. J. Cell. Biol. 95, 619–625.

    CrossRef  CAS  PubMed  Google Scholar 

  26. Ried T., Landes G., Dackowski W., Klinger K., and Ward D. C. (1992) 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.

    CrossRef  CAS  PubMed  Google Scholar 

  27. Arnoldus E. P., Wiegant J., Noordermeer I. A., et al. (1990) Detection of the Philadelphia chromosome in interphase nuclei. Cytogenet. Cell. Genet. 54, 108–111.

    CrossRef  CAS  PubMed  Google Scholar 

  28. 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.

    CrossRef  CAS  PubMed  Google Scholar 

  29. Ried T., Baldini 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, 1388–1392.

    CrossRef  CAS  PubMed  Google Scholar 

  30. Telenius H., Pelmear A. H., Tunnacliffe A., et al. (1992) Cytogenetic analysis by chromosome painting using DOP-PCR amplified flow-sorted chromosomes. Genes Chromosomes Cancer 4, 257–263.

    CrossRef  CAS  PubMed  Google Scholar 

  31. Morrison L. E. and Legator M. S. (1999) Multi-color fluorescence in situ hybridizations techniques, in An Introduction to Fluorescence in situ Hybridization: Principles and Clinical Applications (eiAndreeff M. and Pinkel D., eds.), Wiley-Liss, New York, pp. 77–118.

    Google Scholar 

  32. Sambrook J., Fritsch E. F., and Maniatis T. (1989) Molecular Cloning, A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor.

    Google Scholar 

  33. Collins C., Kuo W. L., Segraves R., Fuscoe J., Pinkel D., and Gray J. W. (1991) Construction and characterization of plasmid libraries enriched in sequences from single human chromosomes. Genomics 11, 997–1006.

    CrossRef  CAS  PubMed  Google Scholar 

  34. Deaven L. L., Van Dilla M. A., Bartholdi M. F., et al. (1986) Construction of human chromosome-specific DNA libraries from flow-sorted chromosomes. Cold Spring Harb. Symp. Quant. Biol. 51, 159–167.

    CAS  PubMed  Google Scholar 

  35. Carter N. P., Ferguson-Smith M. A., Perryman M. T., et al. (1992) Reverse chromosome painting: A method for the rapid analysis of aberrant chromosomes in clinical cytogenetics. J. Med. Genet. 29, 299–307.

    CrossRef  CAS  PubMed  Google Scholar 

  36. Telenius H., De Vos D., Blennow E., Willat L. R., Ponder B. A., and Carter N. P. (1993) Chromatid contamination can impair the purity of flow sorted metaphase chromosomes. Cytometry 14, 97–101.

    CrossRef  CAS  PubMed  Google Scholar 

  37. Guan X. Y., Meltzer P. S., and Trent J. M. (1994) Rapid generation of whole chromosome painting probes (WCPs) by chromosome microdissection. Genomics 22, 101–107.

    CrossRef  CAS  PubMed  Google Scholar 

  38. Telenius H., Carter N. P., Bebb C. E., Nordenskjold M., Ponder B. A., and Tunnacliffe A. (1992) Degenerate oligonucleotide-primed PCR: General amplification of target DNA by a single degenerate primer. Genomics 13, 718–725.

    CrossRef  CAS  PubMed  Google Scholar 

  39. Cherif D., Bernard O., and Berger R. (1989) Detection of single-copy genes by nonisotopic in situ hybridization on human chromosomes. Hum. Genet. 81, 358–362.

    CrossRef  CAS  PubMed  Google Scholar 

  40. Kelly R. B., Cozzarelli N. R., Deutscher M. P., Lehman I. R., and Kornberg A. (1970) Enzymatic synthesis of deoxyribonucleic acid. XXXII. Replication of duplex deoxyribonucleic acid by polymerase at a single strand break. J. Biol. Chem. 245, 39–45.

    CAS  PubMed  Google Scholar 

  41. Feinberg A. P. and Vogelstein B. (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132, 6–13.

    CrossRef  CAS  PubMed  Google Scholar 

  42. Feinberg A. P. and Vogelstein B. (1984) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Addendum. Anal. Biochem. 137, 266–267.

    CrossRef  CAS  Google Scholar 

  43. Speicher M. R., Gwyn Ballard S., and Ward D. C. (1996) Karyotyping human chromosomes by combinatorial multi-fluor FISH. Nat. Genet. 12, 368–375.

    CrossRef  CAS  PubMed  Google Scholar 

  44. Schrock E., Du Manoir S., Veldman T., et al. (1996) Multicolor spectral karyotyping of human chromosomes. Science 273, 494–497.

    CrossRef  CAS  PubMed  Google Scholar 

  45. Morrison L., Legator M., Ramakrishnan R., Zimmerman D., Volling-Kwasneski M., and Koeppen P. (1998) FISH karyotyping with combinations of whole chromosome probes directly labeled with five visible fluorescent labels. Cytometry Supplement 9, 150.

    Google Scholar 

  46. Morrison L. E. (1995) Detection of energy transfer and fluorescence quenching, in Nonisotopic probing, blotting, and sequencing (Kricka L. J., ed.), Academic Press, San Diego, pp. 429–471.

    CrossRef  Google Scholar 

  47. Van Belkum A., Linkels E., Jelsma T., Houthoff H. J., Van Den Berg F., and Quint W. (1993) Application of a new, universal DNA labeling system in the PCR mediated diagnoses of Chlamydia trachomatis and human papillomavirus type 16 infection in cervical smears. J. Virol. Methods 45, 189–200.

    CrossRef  PubMed  Google Scholar 

  48. Garvey J. S., Cremer N. E., and Sussdorf D. H. (1977) Methods in Immunology: A Laboratory Text for Instruction and Research. 3rd ed. W. A. Benjamin, Inc., Reading, PA.

    Google Scholar 

  49. Morrison L. E. (1988) Time-resolved detection of energy transfer: Theory and application to immunoassays. Anal. Biochem. 174, 101–120.

    CrossRef  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2002 Humana Press Inc.

About this protocol

Cite this protocol

Morrison, L.E., Ramakrishnan, R., Ruffalo, T.M., Wilber, K.A. (2002). Labeling Fluorescence In Situ Hybridization Probes for Genomic Targets. In: Fan, YS. (eds) Molecular Cytogenetics. Methods in Molecular Biology™, vol 204. Humana Press. https://doi.org/10.1385/1-59259-300-3:21

Download citation

  • DOI: https://doi.org/10.1385/1-59259-300-3:21

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-006-9

  • Online ISBN: 978-1-59259-300-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics