Dataset Information

Non-CpG methylation biases bisulphite PCR towards low or unmethylated mitochondrial DNA: recommendations for the field.

ABSTRACT: Mitochondrial DNA (mtDNA) is a circular genome of 16?kb that is present in multiple copies in mitochondria. mtDNA codes for genes that contribute to mitochondrial structure and function. A long-standing question has asked whether mtDNA is epigenetically regulated similarly to the nuclear genome. Recently published data suggest that unlike the nuclear genome where CpG methylation is the norm, mtDNA is methylated predominantly at non-CpG cytosines. This raises important methodological considerations for future investigations. In particular, existing bisulphite PCR techniques may be unsuitable due to primers being biased towards amplification from unmethylated mtDNA. Here, we describe how this may have led to previous studies underestimating the level of mtDNA methylation and reiterate methodological strategies for its accurate assessment.

SUBMITTER: Morris MJ

PROVIDER: S-EPMC7055202 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Non-CpG methylation biases bisulphite PCR towards low or unmethylated mitochondrial DNA: recommendations for the field.

Morris Margaret J MJ Hesson Luke B LB Youngson Neil A NA

Environmental epigenetics 20200101 1

Mitochondrial DNA (mtDNA) is a circular genome of 16 kb that is present in multiple copies in mitochondria. mtDNA codes for genes that contribute to mitochondrial structure and function. A long-standing question has asked whether mtDNA is epigenetically regulated similarly to the nuclear genome. Recently published data suggest that unlike the nuclear genome where CpG methylation is the norm, mtDNA is methylated predominantly at non-CpG cytosines. This raises important methodological consideratio ...[more]

PMID: 32154030

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Project description:BackgroundDNA methylation is a well-studied epigenetic mark that is frequently altered in diseases such as cancer, where specific changes are known to reflect the type and severity of the disease. Therefore, there is a growing interest in assessing the clinical utility of DNA methylation as a biomarker for diagnosing disease and guiding treatment. The development of an accurate loci-specific methylation assay, suitable for use on low-input clinical material, is crucial for advancing DNA methylation biomarkers into a clinical setting. A targeted multiplex bisulphite PCR sequencing approach meets these needs by allowing multiple DNA methylated regions to be interrogated simultaneously in one experiment on limited clinical material.ResultsHere, we provide an updated protocol and recommendations for multiplex bisulphite PCR sequencing (MBPS) assays for target DNA methylation analysis. We describe additional steps to improve performance and reliability: (1) pre-sequencing PCR optimisation which includes assessing the optimal PCR cycling temperature and primer concentration and (2) post-sequencing PCR optimisation to achieve uniform coverage of each amplicon. We use a gradient of methylated controls to demonstrate how PCR bias can be assessed and corrected. Methylated controls also allow assessment of the sensitivity of methylation detection for each amplicon. Here, we show that the MBPS assay can amplify as little as 0.625 ng starting DNA and can detect methylation differences of 1% with a sequencing coverage of 1000 reads. Furthermore, the multiplex bisulphite PCR assay can comprehensively interrogate multiple regions on 1-5 ng of formalin-fixed paraffin-embedded DNA or circulating cell-free DNA.ConclusionsThe MBPS assay is a valuable approach for assessing methylated DNA regions in clinical samples with limited material. The optimisation and additional quality control steps described here improve the performance and reliability of this method, advancing it towards potential clinical applications in biomarker studies.

Dataset Information

Non-CpG methylation biases bisulphite PCR towards low or unmethylated mitochondrial DNA: recommendations for the field.

Publications

Non-CpG methylation biases bisulphite PCR towards low or unmethylated mitochondrial DNA: recommendations for the field.

Similar Datasets

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