Advertisement

Fluorescence In Situ Hybridization on Electrophoresed Cells to Detect Sequence Specific DNA Damage

  • Julian Laubenthal
  • Diana Anderson
Protocol
  • 4k Downloads
Part of the Methods in Molecular Biology book series (MIMB, volume 1054)

Abstract

Fluorescence in situ hybridization (FISH) to label fragments of DNA with probes which can specifically locate a genomic region of interest, combined with the single cell electrophoresis (Comet) assay, also termed Comet-FISH, allows the quantification of DNA damage and repair at a specific genomic locus. While the Comet assay alone quantifies only the overall DNA damage of an individual cell, subsequent FISH on the electrophoresed single cell genome enables the coincidental localization of fluorescently labelled sequences (i.e., probes) to the respective damaged or undamaged genes or specific genomic regions of interest. In that way sequence specific DNA damage, global genomic and transcription coupled repair or the three dimensional ultrastructure of cells from any tissue can be comparatively investigated. This protocol provides a detailed description of the principles and basic methodology of a standard Comet-FISH experiment to study interphase cells of any tissue. Also important variations of the protocol (e.g., neutral conditions to detect double strand breaks) as well as the production of fluorochrome-labelled DNA probes via random priming are described.

Key words

Fluorescence in situ hybridization (FISH) Single cell electrophoresis (Comet) assay Sequence specific DNA damage DNA repair 

References

  1. 1.
    Pardue ML, Gall JG (1970) Chromosomal localization of mouse satellite DNA. Science 168:1356–1358PubMedCrossRefGoogle Scholar
  2. 2.
    Pinkel D, Straume T, Gray JW (1986) Cytogenetic analysis using quantitative, high-sensitivity, fluorescence hybridization. Proc Natl Acad Sci U S A 83:2934–2938PubMedCrossRefGoogle Scholar
  3. 3.
    Baumgartner A, Schmid TE, Cemeli E, Anderson D (2004) Parallel evaluation of doxorubicin-induced genetic damage in human lymphocytes and sperm using the comet assay and spectral karyotyping. Mutagenesis 19:313–318PubMedCrossRefGoogle Scholar
  4. 4.
    Swiger RR, Tucker JD (1996) Fluorescence in situ hybridization: a brief review. Environ Mol Mutagen 27:245–254PubMedCrossRefGoogle Scholar
  5. 5.
    Anderson D, Dhawan A (2009) The comet assay. Royal Society of Chemistry, CambridgeGoogle Scholar
  6. 6.
    Cook PR, Brazell IA (1976) Conformational constraints in nuclear DNA. J Cell Sci 22:287–302PubMedGoogle Scholar
  7. 7.
    Ostling O, Johanson KJ (1984) Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochem Biophys Res Commun 123:291–298PubMedCrossRefGoogle Scholar
  8. 8.
    Collins AR, Oscoz AA, Brunborg G, Gaivao I, Giovannelli L, Kruszewski M, Smith CC, Stetina R (2008) The comet assay: topical issues. Mutagenesis 23:143–151PubMedCrossRefGoogle Scholar
  9. 9.
    Klaude M, Eriksson S, Nygren J, Ahnstrom G (1996) The comet assay: mechanisms and technical considerations. Mutat Res 363:89–96PubMedCrossRefGoogle Scholar
  10. 10.
    Sipinen V, Laubenthal J, Baumgartner A, Cemeli E, Linschooten JO, Godschalk RW, Van Schooten FJ, Anderson D, Brunborg G (2010) In vitro evaluation of baseline and induced DNA damage in human sperm exposed to benzo[a]pyrene or its metabolite benzo[a]pyrene-7,8-diol-9,10-epoxide, using the comet assay. Mutagenesis 25:417–425PubMedCrossRefGoogle Scholar
  11. 11.
    Albertini RJ, Anderson D, Douglas GR, Hagmar L, Hemminki K, Merlo F, Natarajan AT, Norppa H, Shuker DE, Tice R et al (2000) IPCS guidelines for the monitoring of genotoxic effects of carcinogens in humans. International Programme on Chemical Safety. Mutat Res 463:111–172PubMedCrossRefGoogle Scholar
  12. 12.
    Santos SJ, Singh NP, Natarajan AT (1997) Fluorescence in situ hybridization with comets. Exp Cell Res 232:407–411PubMedCrossRefGoogle Scholar
  13. 13.
    Rapp A, Bock C, Dittmar H, Greulich KO (2000) UV-A breakage sensitivity of human chromosomes as measured by Comet-FISH depends on gene density and not on the chromosome size. J Photochem Photobiol B 56:109–117PubMedCrossRefGoogle Scholar
  14. 14.
    Kumaravel TS, Bristow RG (2005) Detection of genetic instability at HER-2/neu and p53 loci in breast cancer cells sing Comet-FISH. Breast Cancer Res Treat 91:89–93PubMedCrossRefGoogle Scholar
  15. 15.
    McKelvey-Martin VJ, Ho ET, McKeown SR, Johnston SR, McCarthy PJ, Rajab NF, Downes CS (1998) Emerging applications of the single cell gel electrophoresis (comet) assay. I. Management of invasive transitional cell human bladder carcinoma. II. Fluorescent in situ hybridization comets for the identification of damaged and repaired DNA sequences in individual cells. Mutagenesis 13:1–8PubMedCrossRefGoogle Scholar
  16. 16.
    Mosesso P, Palitti F, Pepe G, Pinero J, Bellacima R, Ahnstrom G, Natarajan AT (2010) Relationship between chromatin structure, DNA damage and repair following X-irradiation of human lymphocytes. Mutat Res 701:86–91PubMedCrossRefGoogle Scholar
  17. 17.
    Amendola R, Basso E, Pacifici PG, Piras E, Giovanetti A, Volpato C, Romeo G (2006) Ret, Abl1 (cAbl) and Trp53 gene fragmentations in Comet-FISH assay act as in vivo biomarkers of radiation exposure in C57BL/6 and CBA/J mice. Radiat Res 165:553–561PubMedCrossRefGoogle Scholar
  18. 18.
    Glei M, Schaeferhenrich A, Claussen U, Kuechler A, Liehr T, Weise A, Marian B, Sendt W, Pool-Zobel BL (2007) Comet fluorescence in situ hybridization analysis for oxidative stress-induced DNA damage in colon cancer relevant genes. Toxicol Sci 96:279–284PubMedCrossRefGoogle Scholar
  19. 19.
    Park E, Glei M, Knobel Y, Pool-Zobel BL (2007) Blood mononucleocytes are sensitive to the DNA damaging effects of iron overload – in vitro and ex vivo results with human and rat cells. Mutat Res 619:59–67PubMedCrossRefGoogle Scholar
  20. 20.
    Arutyunyan R, Rapp A, Greulich KO, Hovhannisyan G, Haroutiunian S, Gebhart E (2005) Fragility of telomeres after bleomycin and cisplatin combined treatment measured in human leukocytes with the Comet-FISH technique. Exp Oncol 27:38–42PubMedGoogle Scholar
  21. 21.
    Escobar PA, Smith MT, Vasishta A, Hubbard AE, Zhang L (2007) Leukaemia-specific chromosome damage detected by comet with fluorescence in situ hybridization (Comet-FISH). Mutagenesis 22:321–327PubMedCrossRefGoogle Scholar
  22. 22.
    Fernandez JL, Vazquez-Gundin F, Rivero MT, Genesca A, Gosalvez J, Goyanes V (2001) DBD-fish on neutral comets: simultaneous analysis of DNA single- and double-strand breaks in individual cells. Exp Cell Res 270:102–109PubMedCrossRefGoogle Scholar
  23. 23.
    Menke M, Angelis KJ, Schubert I (2000) Detection of specific DNA lesions by a combination of comet assay and FISH in plants. Environ Mol Mutagen 35:132–138PubMedCrossRefGoogle Scholar
  24. 24.
    Glei M, Hovhannisyan G, Pool-Zobel BL (2009) Use of Comet-FISH in the study of DNA damage and repair: review. Mutat Res 681:33–43PubMedCrossRefGoogle Scholar
  25. 25.
    Spivak G, Cox RA, Hanawalt PC (2009) New applications of the comet assay: Comet-FISH and transcription-coupled DNA repair. Mutat Res 681:44–50PubMedCrossRefGoogle Scholar
  26. 26.
    Baumgartner A, Cemeli E, Laubenthal J, Anderson A (2009) The comet assay in sperm-assessing genotoxins in male germ cells. Elsevier. RSC Publishing in London, UK. DOI:  10.1039/9781847559746-00331
  27. 27.
    Singh NP, McCoy MT, Tice RR, Schneider EL (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 175:184–191PubMedCrossRefGoogle Scholar
  28. 28.
    Tice RR, Agurell E, Anderson D, Burlinson B, Hartmann A, Kobayashi H, Miyamae Y, Rojas E, Ryu JC, Sasaki YF (2000) Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen 35:206–221PubMedCrossRefGoogle Scholar
  29. 29.
    Wienberg J, Stanyon R (1997) Comparative painting of mammalian chromosomes. Curr Opin Genet Dev 7:784–791PubMedCrossRefGoogle Scholar
  30. 30.
    Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning : a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NYGoogle Scholar

Copyright information

© Springer Science+Business Media, New York 2013

Authors and Affiliations

  • Julian Laubenthal
    • 1
  • Diana Anderson
    • 1
  1. 1.School of Life SciencesUniversity of BradfordBradfordUK

Personalised recommendations