A sample preparation protocol for quantification of radiolabeled nucleoside incorporation into DNA by accelerator mass spectrometry

Sang Soo Hah, Janna M. Mundt, Esther A. Ubick, Ken W Turteltaub, Jeffrey Gregg, Paul Henderson

Research output: Contribution to journalArticle

8 Scopus citations

Abstract

A general protocol is described for measuring the incorporation of radiocarbon-labeled 2′-deoxynucleosides into DNA using accelerator mass spectrometry (AMS). This technology provides attomole (10-18 mol) sensitivity, with detection limits for DNA analysis in the range of one 14C atom per 1011-1012 total carbons. In practice this corresponds to approximately 1 labeled nucleoside per 1011 normal bases. A key aspect of the method is the use of precautions aimed at prevention of artifactual DNA oxidation during the sample preparation by the use of antioxidants and chaotropic salts during the DNA isolation. In principle, any type of appropriately labeled nucleoside derivative can be studied using the described protocol, provided that there is incorporation of the deoxynucleoside into DNA. We demonstrated this protocol using MCF-7 human breast cancer cells and a mouse model for mammary carcinoma, which we dosed with 14C-labeled 2′-deoxyguanosine (dG) and 14C-labeled 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxodG). The nucleoside 8-oxodG is a ubiquitous compound that forms in cells by the reaction of dG with reactive oxygen species which has been associated with numerous disease, carcinogenesis and aging. DNA from cells treated with 14C-labeled nucleosides was isolated and prepared for analysis by AMS in order to measure the DNA-bound radioactivity. The method allows the generation of reliable and sufficient yields of pure DNA from human cells and animal tissues for analysis of radiocarbon levels. Ultimately, this protocol will be applied to understanding the role of modified nucleoside incorporation into DNA in cancer initiation and progression, but could also be used to study any DNA metabolism process where 14C-labeled nucleosides are used.

Original languageEnglish (US)
Pages (from-to)763-766
Number of pages4
JournalNuclear Instruments and Methods in Physics Research, Section B: Beam Interactions with Materials and Atoms
Volume259
Issue number1
DOIs
StatePublished - Jun 2007

Keywords

  • Accelerator mass spectrometry (AMS)
  • Carbon-14
  • DNA damage
  • DNA digestion
  • DNA isolation
  • Nucleoside incorporation

ASJC Scopus subject areas

  • Surfaces, Coatings and Films
  • Instrumentation
  • Surfaces and Interfaces

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