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Cited by 3,238Epigenetics and DNA MethylationGenetic Syndromes and ImprintingDNA and Nucleic Acid Chemistry

A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands

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Frommer, M, McDonald, L E, Millar, D S, Collis, C M, Watt, F, Grigg, G W, Molloy, P L, Paul, C L·1992·Proceedings of the National Academy of Sciences
DOI 10.1073/pnas.89.5.1827PMC PMC48546
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Abstract

The modulation of DNA-protein interactions by methylation of protein-binding sites in DNA and the occurrence in genomic imprinting, X chromosome inactivation, and fragile X syndrome of different methylation patterns in DNA of different chromosomal origin have underlined the need to establish methylation patterns in individual strands of particular genomic sequences. We report a genomic sequencing method that provides positive identification of 5-methylcytosine residues and yields strand-specific sequences of individual molecules in genomic DNA. The method utilizes bisulfite-induced modification of genomic DNA, under conditions whereby cytosine is converted to uracil, but 5-methylcytosine remains nonreactive. The sequence under investigation is then amplified by PCR with two sets of strand-specific primers to yield a pair of fragments, one from each strand, in which all uracil and thymine residues have been amplified as thymine and only 5-methylcytosine residues have been amplified as cytosine. The PCR products can be sequenced directly to provide a strand-specific average sequence for the population of molecules or can be cloned and sequenced to provide methylation maps of single DNA molecules. We tested the method by defining the methylation status within single DNA strands of two closely spaced CpG dinucleotides in the promoter of the human kininogen gene. During the analysis, we encountered in sperm DNA an unusual methylation pattern, which suggests that the high methylation level of single-copy sequences in sperm may be locally modulated by binding of protein factors in germ-line cells.

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  • The c.C127 is located in a CpG dinucleotide and can be methylated at the 5 carbon, yielding 5-methylcytosine, and deaminated to produce thymine.

    “The c.C127 is located in the CpG dinucleotide and can be methylated at the 5 carbon, yielding 5-methylcytosine, and deaminated to produce thymine [25].”

    From Identification of a founder haplotype of <i>APOE Kyoto</i> variant in lipoprotein glomerulopathy (2025) · cites this paper as [25]

    The paper’s method description (p. 1827) discusses 5-methylcytosine and tests methylation at CpG dinucleotides. It does not identify c.C127 or state that 5-methylcytosine deaminates to thymine; its thymine result instead concerns PCR amplification after bisulfite converts unmethylated cytosine to uracil. (LLM, conf=0.85)

    Experimental — accuracy not yet measured

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Research works printed but not linked: 24

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Cited by

Sorted by citations

Authors
Frommer, M., McDonald, L. E., Millar, D. S., Collis, C. M., Watt, F., Grigg, G. W., Molloy, P. L., & Paul, C. L.
Affiliations
Royal Prince Alfred Hospital (AU)
ORCID
Venue
Proceedings of the National Academy of Sciences
Publisher
National Academy of Sciences
Published
1992-03
Volume
89
Issue
5
Pages
1827-1831
ISSN
  • 0027-8424 (ISSN-L)
  • 1091-6490
DOI
10.1073/pnas.89.5.1827
PMC
PMC48546
OpenAlex
W1979273476
Cited by
3,238
References
3
Citations by year
2026: 78 · 2025: 91 · 2024: 128 · 2023: 98 · 2022: 112 · 2021: 136 · 2020: 92 · 2019: 103 · 2018: 109 · 2017: 111 · 2016: 129 · 2015: 141 · 2014: 109 · 2013: 128 · 2012: 137
FWCI
5.69
Type
journal article
Language
en
Open access
Locations
  • Proceedings of the National Academy of Sciences (publishedVersion)
  • PubMed Central (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, 5-methylcytosine, strand-specific sequencing, bisulfite modification, CpG, sperm DNA methylation, protein-DNA interactions, genomic imprinting, X chromosome inactivation, fragile X syndrome
MeSH terms
Base Sequence, Cytosine, DNA, Humans, Kininogens, Methylation, Molecular Sequence Data, Oligodeoxyribonucleotides, Promoter Regions, Genetic, Sulfites, Polymerase Chain Reaction, 5-Methylcytosine

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