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Quantitative gas chromatography mass spectrometric analysis of 2′-deoxyinosine in tissue DNA

Nature Protocols volume 1, pages 1995–2002 (2006)Cite this article

Abstract

Several studies examining DNA deamination have published levels of 2′-deoxyinosine that illustrated a large variation between studies. Most of them are the result of artifactual DNA deamination that occurs during the process of sample preparation, particularly acid hydrolysis. This protocol for measurement of 2′-deoxyinosine describes the use of nuclease P1 and alkaline phosphatase to achieve release of nucleosides from DNA, followed by HPLC prepurification with subsequent gas chromatography–mass spectrometry analysis of the nucleosides. It has been used in the measurement of the levels of 2′-deoxyinosine in DNA of commercial sources and DNA from cells and animal tissues, and gives values ranging from 3 to 7 2′-deoxyinosine per 106 2-deoxyadenosine. This protocol should take approximately 7 days to complete.

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Figure 1: Deamination of 2′-deoxyadenosine forms 2′-deoxyinosine.
Figure 2: Overview of the three approaches in the study of DNA deamination products.
Figure 3: Reversed-phase HPLC separation of nucleosides using (a) authentic standards and (b) digested salmon testes DNA.
Figure 4: Mass spectra of (a) tri-TMS-2′-deoxyinosine, (b) tri-TMS-2′-deoxyadenosine and (c) tetra-TMS-2′-deoxyguanosine.
Figure 5
Figure 6: Gas chromatogram of purified 2′-deoxyguanosine.
Figure 7: Total ion chromatogram of the derivatized 2′-deoxyinosine (dIno, 6.89 min), 2′-deoxyadenosine (dAdo, 7.17 min) and 2′-amino-2′-deoxyadenosine (2′-aminoAdo, 8.05 min).

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Acknowledgements

We thank the Academic Research Fund of the National University of Singapore for support.

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  1. Kok Seong Lim

    Present address: Department of Neurosciences, School of Medicine, University of California San Diego, La Jolla, California, USA

Authors and Affiliations

  1. Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore

    Kok Seong Lim, Andrew Jenner & Barry Halliwell

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Correspondence to Barry Halliwell.

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Lim, K., Jenner, A. & Halliwell, B. Quantitative gas chromatography mass spectrometric analysis of 2′-deoxyinosine in tissue DNA. Nat Protoc 1, 1995–2002 (2006). https://doi.org/10.1038/nprot.2006.301

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