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Global DNA Methylation Analysis Using Methylcytosine Dioxygenase

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DNA Modification Detection Methods

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Abstract

Because the alteration of global DNA methylation levels is detected in various cancer cells, the global DNA methylation level is expected as a biomarker for cancer diagnostics. Bisulfite-based assays have been widely used as a traditional method for the quantification of global DNA methylation levels. High-performance liquid chromatography (HPLC)-based assay and enzyme-linked immunosorbent assay (ELISA) have also been used; however, these methods require time-consuming DNA treatments, such as bisulfite treatment, digestion, or immobilization of genomic DNA before analysis. To analyze global DNA methylation levels in simple steps, an enzymatic assay using Ten-eleven translocation (TET), which oxidizes 5-methylcytosine (5mC) on genomic DNA, was developed. Succinate is produced during 5mC oxidation; thus, the amount of succinate produced by TET-mediated oxidation is correlated with the global DNA methylation level of genomic DNA. In this chapter, we describe the details of the expression of TET in Escherichia coli and the measurement of the amount of succinate produced from the TET-mediated reaction. The TET-based assay detects global DNA methylation levels in at least 100 ng genomic DNA.

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Acknowledgments

This work was supported by the Precise Measurement Technology Promotion Foundation.

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Authors and Affiliations

  1. Graduate School of Bionics, Tokyo University of Technology, Hachioji, Tokyo, Japan

    Natsumi Taka & Wataru Yoshida

  2. School of Bioscience and Biotechnology, Tokyo University of Technology, Hachioji, Tokyo, Japan

    Wataru Yoshida

Authors
  1. Natsumi Taka
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  2. Wataru Yoshida
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Corresponding author

Correspondence to Wataru Yoshida .

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Editors and Affiliations

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

    Bi-Feng Yuan

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Taka, N., Yoshida, W. (2022). Global DNA Methylation Analysis Using Methylcytosine Dioxygenase. 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_9

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  • DOI: https://doi.org/10.1007/978-1-0716-1229-3_9

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1228-6

  • Online ISBN: 978-1-0716-1229-3

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