De novo quantitative bisulfite sequencing using the pyrosequencing technology
Abstract
Current protocols for DNA methylation analysis are either labor intensive or limited to the measurement of only one or two CpG positions. Pyrosequencing is a real-time sequencing technology that can overcome these limitations and be used as an epigenotype-mapping tool. Initial experiments demonstrated reliable quantification of the degree of DNA methylation when 2-6 CpGs were analyzed. We sought to improve the sequencing protocol so as to analyze as many CpGs as possible in a single sequencing run. By using an improved enzyme mix and adding single-stranded DNA-binding protein to the reaction, we obtained reproducible results for as many as 10 successive CpGs in a single sequencing reaction spanning up to 75 nucleotides. A minimum amount of 10 ng of bisulfite-treated DNA is necessary to obtain good reproducibility and avoid preferential amplification. We applied the assay to the analysis of DNA methylation patterns in four CpG islands in the vicinity of IGF2 and H19 genes. This allowed accurate and quantitative de novo sequencing of the methylation state of each CpG, showing reproducible variations of methylation state in contiguous CpGs, and proved to be a useful adjunct to current technologies.
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Two technical replicates of bisulfite pyrosequencing provided estimates of the percentage methylation of each CpG island.
“Two technical replicates of bisulfite pyrosequencing provided estimates of the percentage methylation of each CpG island (Dupont, Tost, Jammes, & Gut, 2004).”
From Associations Between Early Life Stress and Gene Methylation in Children (2015) · cites this paper as Dupont et al., 2004
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- Authors
- Dupont, J.-M., Tost, J., Jammes, H., & Gut, I. G.
- Affiliations
- Hôpital Cochin (FR)
- Inserm (FR)
- Corresponding author
- Dupont, Jean-Michel
- Venue
- Analytical Biochemistry
- Publisher
- Elsevier BV
- Published
- 2004-10
- Volume
- 333
- Issue
- 1
- Pages
- 119-127
- ISSN
- 0003-2697 (ISSN-L)
- OpenAlex
- W2008284862
- Cited by
- 253
- References
- 31
- Citations by year
- 2026: 1 · 2025: 1 · 2024: 1 · 2023: 5 · 2022: 5 · 2021: 5 · 2020: 2 · 2019: 7 · 2018: 7 · 2017: 4 · 2016: 17 · 2015: 20 · 2014: 14 · 2013: 9 · 2012: 17
- FWCI
- 6.27
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Analytical Biochemistry (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Epigenetics and DNA Methylation
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genetic Syndromes and Imprinting
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA and Nucleic Acid Chemistry
- Keywords
- DNA methylation, bisulfite sequencing, pyrosequencing, quantitative methylation analysis, CpG islands, insulin-like growth factor II, H19, single-stranded DNA binding protein
- MeSH terms
- DNA-Binding Proteins, Genotype, Humans, Sulfites, Polymerase Chain Reaction, DNA Fingerprinting, Sequence Analysis, DNA, CpG Islands, DNA Methylation
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