Methodology for Accurate Detection of Mitochondrial DNA Methylation

Mie Mechta, Lars Roed Ingerslev, Romain Barrès

    Research output: Contribution to journalJournal articleResearchpeer-review

    13 Citations (Scopus)

    Abstract

    Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert unmethylated cytosine into uracil, in a single-stranded DNA context. Bisulfite sequencing can be targeted (using PCR) or performed on the whole genome and provides absolute quantification of cytosine methylation at the single base-resolution. Given the distinct nature of nuclear- and mitochondrial DNA, notably in the secondary structure, adaptions of bisulfite sequencing methods for investigating cytosine methylation in mtDNA should be made. Secondary and tertiary structure of mtDNA can indeed lead to bisulfite sequencing artifacts leading to false-positives due to incomplete denaturation poor access of bisulfite to single-stranded DNA. Here, we describe a protocol using an enzymatic digestion of DNA with BamHI coupled with bioinformatic analysis pipeline to allow accurate quantification of cytosine methylation levels in mtDNA. In addition, we provide guidelines for designing the bisulfite sequencing primers specific to mtDNA, in order to avoid targeting undesirable NUclear MiTochondrial segments (NUMTs) inserted into the nuclear genome.

    Original languageEnglish
    Article numbere57772
    JournalJournal of Visualized Experiments
    Volume135
    ISSN1940-087X
    DOIs
    Publication statusPublished - 2018

    Keywords

    • Bisulfite sequencing
    • DNA methylation
    • Epigenetics
    • Genetics
    • Issue 135
    • Mitochondria
    • Mitochondrial DNA
    • Mitochondrial DNA methylation
    • Mitoepigenetics

    Cite this