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Isotope-Dilution Liquid Chromatography–Tandem Mass Spectrometry for Detection of 5-Hydroxymethyluracil and 5-Formyluracil in DNA

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DNA Modification Detection Methods

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Abstract

5-Hydroxymethyluracil (5hmU) and 5-formyluracil (5fU) are mutagenic and major oxidized nucleobases in DNA that are derived from thymine (T) or 5-methylcytosine (5mC). 5hmU, and possibly 5fU as well, in DNA may also assume an epigenetic role. The analysis of 5hmU and 5fU in DNA has suffered from multiple drawbacks such as a lack of specificity, artifacts generated in sample preparation, as well as poor applicability to analysis of nucleoside forms of DNA base damage. To overcome these problems, we developed a capillary liquid chromatography–electrospray ionization–tandem mass spectrometry (LC-ESI-MS3) method that is combined with the stable isotope-dilution technique, which allows quantification of 5hmU, 5fU, and several other oxidation products in DNA with high sensitivity, specificity, and accuracy. In this chapter, we describe in detail the protocols from DNA extraction, through DNA digestion and off-line HPLC enrichment, to LC-MS3 analysis. The method is readily applicable to other modified nucleosides in DNA.

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Acknowledgments

The development of the methodology used on which this procedure was prepared was supported by the National Institutes of Health (Grants R01 CA101864, R01 DK082779, R01 DK071111, and P30-DK41296). The preparation of this procedure was supported by Inner Mongolia University with a grant from its “Outstanding Young Talents” Program (to YL) and a grant from its “Steed Plan” High-Level Talents Program (to JW).

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Authors and Affiliations

  1. State Key Laboratory of Reproductive Regulation and Breeding of Grassland Livestock, School of Life Sciences, Inner Mongolia University, Hohhot, People’s Republic of China

    Yu Liu & ** Wang

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  1. Yu Liu
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  2. ** Wang
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Correspondence to ** Wang .

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Editors and Affiliations

  1. Department of Chemistry, Wuhan University, Wuhan, China

    Bi-Feng Yuan

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Liu, Y., Wang, J. (2022). Isotope-Dilution Liquid Chromatography–Tandem Mass Spectrometry for Detection of 5-Hydroxymethyluracil and 5-Formyluracil in DNA. In: Yuan, BF. (eds) DNA Modification Detection Methods . Springer Protocols Handbooks. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1229-3_12

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  • DOI: https://doi.org/10.1007/978-1-0716-1229-3_12

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1228-6

  • Online ISBN: 978-1-0716-1229-3

  • eBook Packages: Springer Protocols

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