Abstract
Pyrosequencing is a technique that uses a sequencing-by-synthesis system which is designed to quantify single-nucleotide polymorphisms (SNPs). Artificial C/T SNP creation via bisulfite modification permits measurement of DNA methylation locally and globally in real time. Alteration in DNA methylation has been implicated in aging, as well as aging-related conditions such as cancer, as well as cardiovascular, neurodegenerative, and autoimmune diseases. Considering its ubiquitous presence in divergent clinical pathologies, quantitative analysis of DNA CpG methylation both globally and at individual genes helps to elucidate the regulation of genes involved in pathophysiological conditions. The ability to detect and quantify the methylation pattern of DNA has the potential to serve as an early detection marker and potential drug target for several diseases. Here, we provide a detailed technical protocol for pyrosequencing supplemented by critical information about assay design and nuances of the system that provides a strong foundation for beginners in the field.
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Acknowledgement
This work was supported by National Institutes of Health National Institute on Aging (AG020628, AG028268), National Institute of Environmental Health Science (P30 ES017885), University of Michigan (Claude D. Pepper Older American Independence Center, Nathan Shock Center for the Basic Biology of Aging, Rheumatic Disease Clinical Center, Caner Center Microarray Core, Michigan Diabetes and Research Training Center Animal Phenotyping Core), Geriatrics Research, Education and Clinical Care Center (GRECC), and the VA Ann Arbor Healthcare System. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
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Delaney, C., Garg, S.K., Yung, R. (2015). Analysis of DNA Methylation by Pyrosequencing. In: Shaw, A. (eds) Immunosenescence. Methods in Molecular Biology, vol 1343. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2963-4_19
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DOI: https://doi.org/10.1007/978-1-4939-2963-4_19
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2962-7
Online ISBN: 978-1-4939-2963-4
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