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A Historical Overview of Bromo-Substituted DNA and Sister Chromatid Differentiation

  • Roberto Mezzanotte
  • Mariella Nieddu
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1094)

Abstract

The thymidine analogue 5-bromo-2′-deoxyuridine (BrdU) has been widely used to make sister chromatid differentiation (SCD) evident in metaphase chromosomes of cells grown for two cycles in BrdU and, thus, containing varying amounts of the thymidine analogue. A direct consequence was the possibility of making sister chromatid exchange (SCE) evident without using autoradiographic procedures. The latter phenomenon was first discovered in 1953, and its frequency is considered a reliable marker of pathological cell situations, as well as an indicator of mutagenic compounds. Several experimental procedures were found which produced SCD, such as the use of fluorochromes like 33258 Hoechst or acridine orange, whose observation under fluorescence microscopy was directly recorded by photos or stained with Giemsa to make chromosome preparations permanent. Other treatments followed by Giemsa staining required the use of saline hot solutions, acid solutions, nuclease attack and specific monoclonal antibodies. Basically two molecular mechanisms were invoked to explain the different affinity of Giemsa stain for differential BrdU-substituted chromatid DNA. The first implied debromination of chromatid DNA, whose occurrence would be greater in chromatids containing an amount of BrdU greater than that present in sister chromatids. The second mechanism, although not denying the importance of DNA debromination, postulated that chromatin structural organization, in terms of DNA–protein and/or protein–protein DNA interaction, is responsible for SCD production.

Key words

DNA replication BrdU Sister chromatid differentiation Sister chromatid exchange 

Notes

Acknowledgements

The authors wish to thank Ms. Mary Ann Groeneweg for checking and correcting the English manuscript.

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Copyright information

© Springer Science+Business Media, New York 2014

Authors and Affiliations

  • Roberto Mezzanotte
    • 1
  • Mariella Nieddu
    • 2
  1. 1.Dipartimento di Scienze BiomedicheUniversity of CagliariCagliariItaly
  2. 2.Dipartimento di Scienze BiomedicheCittadella Universitaria Monserrato, Università di CagliariCagliariItaly

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