Skip to main content

In Situ DNA Damaging Foci Analysis on Metaphase Chromosomes

  • Protocol
  • First Online:
Radiation Cytogenetics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1984))

Abstract

DNA damage foci such as ionizing radiation-induced foci (IRIF) can visually distinguish the location and number of specific types of DNA damages. This method is widely used to detect DNA damage in interphase cells. These DNA damage foci can be also visualized on metaphase chromosomes. The technique has an advantage as it provides an easy method of quantifying chromosomal DNA damage. Radiation-induced DNA double strand breaks can be assessed for gamma-H2AX foci formation on metaphase chromosomes.

Gamma-H2AX foci can be observed at the break point of chromosomes and can persist in newly repair chromosomes. Foci observation may be advantageous compared to classical cytogenetic analysis due to less time required for analysis. Metaphase DNA damage analysis can be also used for the estimation of DNA damage persistence in daughter cells and capacity of DNA repair. Not only DNA double strand breaks can be visualized, but also other types of DNA damage and modification such as oxidative damage, crosslinking, and methylation of DNA can be visualized with appropriate antibodies. This IRIF immunostaining technique can be combined with FISH analysis for the immunoFISH method.

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

Access this chapter

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 149.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 199.99
Price excludes VAT (USA)
  • 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

Institutional subscriptions

Similar content being viewed by others

References

  1. Bonner WM, Boon EP, Rogakou EP (1998) Antibody to histone H2AX phosphorylated on serine 139 detects structures formed immediately after introduction of DNA double-stranded breaks. Mol Biol Cell 9:320A–320A

    Google Scholar 

  2. van Veelen LR, Essers J, van de Rakt MW, Odijk H, Pastink A, Zdzienicka MZ, Paulusma CC, Kanaar R (2005) Ionizing radiation-induced foci formation of mammalian Rad51 and Rad54 depends on the Rad51 paralogs, but not on Rad52. Mutat Res 574:34–49

    Article  Google Scholar 

  3. Kobayashi J (2004) Molecular mechanism of the recruitment of NBS1/hMRE11/hRAD50 complex to DNA double-strand breaks: NBS1 binds to gamma-H2AX through FHA/BRCT domain. J Radiat Res 45:473–478

    Article  CAS  Google Scholar 

  4. Limoli CL, Giedzinski E, Bonner WM, Cleaver JE (2002) UV-induced replication arrest in the xeroderma pigmentosum variant leads to DNA double-strand breaks, gamma-H2AX formation, and Mre11 relocalization. Proc Natl Acad Sci U S A 99:233–238

    Article  CAS  Google Scholar 

  5. Kato TA, Okayasu R, Bedford JS (2008) Comparison of the induction and disappearance of DNA double strand breaks and gamma-H2AX foci after irradiation of chromosomes in G1-phase or in condensed metaphase cells. Mutat Res 639:108–112

    Article  CAS  Google Scholar 

  6. Kato TA, Wilson PF, Nagasawa H, Fitzek MM, Weil MM, Little JB, Bedford JS (2007) A defect in DNA double strand break processing in cells from unaffected parents of retinoblastoma patients and other apparently normal humans. DNA Repair (Amst) 6:818–829

    Article  CAS  Google Scholar 

  7. Kato TA, Okayasu R, Bedford JS (2009) Signatures of DNA double strand breaks produced in irradiated G1 and G2 cells persist into mitosis. J Cell Physiol 219:760–765

    Article  CAS  Google Scholar 

  8. Jeppesen P (2000) Immunofluorescence in cytogenetic analysis: method and applications. Genet Mol Biol 23:1107–1114

    Article  Google Scholar 

  9. Zhang X, Rosenstein BS, Wang Y, Lebwohl M, Mitchell DM, Wei H (1997) Induction of 8-oxo-7,8-dihydro-2′-deoxyguanosine by ultraviolet radiation in calf thymus DNA and HeLa cells. Photochem Photobiol 65:119–124

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Takamitsu A. Kato .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2019 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Omata, C., Kato, T.A. (2019). In Situ DNA Damaging Foci Analysis on Metaphase Chromosomes. In: Kato, T., Wilson, P. (eds) Radiation Cytogenetics. Methods in Molecular Biology, vol 1984. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9432-8_10

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9432-8_10

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9430-4

  • Online ISBN: 978-1-4939-9432-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics