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DNA Methylation Analysis of Free-Circulating DNA in Body Fluids

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DNA Methylation Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1708))

Abstract

Circulating cell-free DNA in body fluids is an analyte of great interest in basic and clinical research. The analyses of DNA methylation and hydroxymethylation patterns in body fluids might allow one to determine the certain state of a disease, in particular of cancer. DNA methylation biomarkers in liquid biopsies, i.e. blood plasma samples, may help optimizing personalized therapy for individual patients. DNA methylation analyses of specific loci usually require a bisulfite conversion of the DNA, which requires a sufficiently high amount of DNA at the appropriate concentration. However, free-circulating DNA is generally low concentrated. Therefore, high volumes of body fluids need to be analyzed. This high volume needs to be reduced in order to facilitate the bisulfite conversion. In addition, disease-related free-circulating DNA is even less abundant than normal DNA in the total amount of free-circulating DNA. Accordingly, analytical and pre-analytical methods are needed, which permit an accurate and sensitive quantification of single methylated DNA copies in the presence of unmethylated DNA in abundance.

This protocol describes two methods for DNA enrichment from body fluids: DNA extraction by means of magnetic beads and polymer-mediated enrichment of DNA. Subsequent bisulfite conversion is achieved by means of a high-speed conversion protocol. Adaptions of the workflow required for the analysis of hydroxymethylation via oxidation 5-hydroxymethylcytosines to 5-formylcytosines prior to the bisulfite conversion are introduced. A quantitative real-time PCR based on the methylation-specific and HeavyMethyl PCR methodologies is introduced. This qPCR assay allows for an accurate and sensitive quantification of single copies of the DNA methylation biomarkers SHOX2 and SEPT9 in blood plasma. Specific issues regarding the analysis of body fluids and respective trouble shooting approaches are discussed.

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Competing Interests

Dimo Dietrich has been an employee and is a stockholder of Epigenomics AG, a company that aims to commercialize the DNA methylation biomarkers SEPT9 and SHOX2. Dimo Dietrich is coinventor and owns patents on methylation biomarkers and related technologies. These patents are commercially exploited by Epigenomics AG. Dimo Dietrich receives inventor’s compensation from Epigenomics AG. Dimo Dietrich is a consultant for AJ Innuscreen GmbH (Berlin, Germany), a 100% daughter company of Analytik Jena AG (Jena, Germany), and receives royalties from the sale of innuCONVERT Bisulfite Kits.

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Authors and Affiliations

  1. Institute of Pathology, University Hospital Bonn (UKB), Sigmund-Freud-Str. 25, 53127, Bonn, Germany

    Maria Jung, Glen Kristiansen & Dimo Dietrich

Authors
  1. Maria Jung
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  2. Glen Kristiansen
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  3. Dimo Dietrich
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Correspondence to Dimo Dietrich .

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Editors and Affiliations

  1. Laboratory for Epigenetics and Environment, Centre National de Recherche en Génomique Humaine, CEA—Institut de Biologie Francois Jacob, Evry, France

    Jörg Tost

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Jung, M., Kristiansen, G., Dietrich, D. (2018). DNA Methylation Analysis of Free-Circulating DNA in Body Fluids. In: Tost, J. (eds) DNA Methylation Protocols. Methods in Molecular Biology, vol 1708. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7481-8_32

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  • DOI: https://doi.org/10.1007/978-1-4939-7481-8_32

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7479-5

  • Online ISBN: 978-1-4939-7481-8

  • eBook Packages: Springer Protocols

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