DNA isolation and sample preparation for quantification of adduct levels by accelerator mass spectrometry.

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

Research output: Contribution to journalArticle

3 Citations (Scopus)

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 10(11)-10(12) 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.

Original languageEnglish (US)
Pages (from-to)147-157
Number of pages11
JournalMethods in molecular biology (Clifton, N.J.)
Volume1105
DOIs
StatePublished - 2014
Externally publishedYes

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Mass Spectrometry
DNA
Isotopes
Endopeptidase K
Graphite
2-Propanol
Tritium
DNA Adducts
Titanium
Limit of Detection
Digestion
Ethanol
Carbon
Nucleotides
Salts
Water

ASJC Scopus subject areas

  • Medicine(all)

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DNA isolation and sample preparation for quantification of adduct levels by accelerator mass spectrometry. / Dingley, Karen H.; Ubick, Esther A.; Vogel, John S.; Ognibene, Ted J.; Malfatti, Michael A.; Kulp, Kristen; Haack, Kurt W.

In: Methods in molecular biology (Clifton, N.J.), Vol. 1105, 2014, p. 147-157.

Research output: Contribution to journalArticle

Dingley, Karen H. ; Ubick, Esther A. ; Vogel, John S. ; Ognibene, Ted J. ; Malfatti, Michael A. ; Kulp, Kristen ; Haack, Kurt W. / DNA isolation and sample preparation for quantification of adduct levels by accelerator mass spectrometry. In: Methods in molecular biology (Clifton, N.J.). 2014 ; Vol. 1105. pp. 147-157.
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AU - Haack, Kurt W.

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