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DNA Isolation and Sample Preparation for Quantification of Adduct Levels by Accelerator Mass Spectrometry

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Molecular Toxicology Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1105))

Abstract

Accelerator mass spectrometry (AMS) is a highly sensitive technique used for the quantification of adducts following exposure to carbon-14- or tritium-labeled chemicals, with detection limits in the range of one adduct per 1011–1012 nucleotides. The protocol described in this chapter provides an optimal method for isolating and preparing DNA samples to measure isotope-labeled DNA adducts by AMS. When preparing samples, special precautions must be taken to avoid cross-contamination of isotope among samples and produce a sample that is compatible with AMS. The DNA isolation method described is based upon digestion of tissue with proteinase K, followed by extraction of DNA using Qiagen isolation columns. The extracted DNA is precipitated with isopropanol, washed repeatedly with 70 % ethanol to remove salt, and then dissolved in water. DNA samples are then converted to graphite or titanium hydride and the isotope content measured by AMS to quantify adduct levels. This method has been used to reliably generate good yields of uncontaminated, pure DNA from animal and human tissues for analysis of adduct levels.

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Acknowledgements

The authors thank Kristin Stoker for help with preparation of the manuscript. This work performed under the auspices of the US DOE (DE-AC52-07NA27344) with support from the NIH/National Center for Research Resources (RR13461).

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Authors and Affiliations

  1. Biology and Biotechnology Research Program, Center for Accelerator Mass Spectroscopy, Lawrence Livermore National Laboratory, Livermore, CA, 94551, USA

    Karen H. Dingley

  2. Biology and Biotechnology Research Program, Center for Accelerator Mass Spectroscopy, Lawrence Livermore National Laboratory, Livermore, CA, USA

    Esther A. Ubick, John S. Vogel, Ted J. Ognibene, Michael A. Malfatti, Kristen Kulp & Kurt W. Haack

Authors
  1. Karen H. Dingley
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  2. Esther A. Ubick
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  3. John S. Vogel
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  4. Ted J. Ognibene
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  5. Michael A. Malfatti
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  6. Kristen Kulp
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  7. Kurt W. Haack
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Editor information

Editors and Affiliations

  1. Occupational Health, University of Pittsburgh Dept. Environmental &, Pittsburgh, Pennsylvania, USA

    Phouthone Keohavong

  2. Dept. of Pharmaceutical Sciences, Nova Southeastern University College of Pharmacy, Fort Lauderdale, Florida, USA

    Stephen G. Grant

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© 2014 Humana Press

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Dingley, K.H. et al. (2014). DNA Isolation and Sample Preparation for Quantification of Adduct Levels by Accelerator Mass Spectrometry. In: Keohavong, P., Grant, S. (eds) Molecular Toxicology Protocols. Methods in Molecular Biology, vol 1105. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-739-6_12

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  • DOI: https://doi.org/10.1007/978-1-62703-739-6_12

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-738-9

  • Online ISBN: 978-1-62703-739-6

  • eBook Packages: Springer Protocols

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