A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands - PubMed (original) (raw)
A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands
M Frommer et al. Proc Natl Acad Sci U S A. 1992.
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.
Similar articles
- High sensitivity mapping of methylated cytosines.
Clark SJ, Harrison J, Paul CL, Frommer M. Clark SJ, et al. Nucleic Acids Res. 1994 Aug 11;22(15):2990-7. doi: 10.1093/nar/22.15.2990. Nucleic Acids Res. 1994. PMID: 8065911 Free PMC article. - High-speed conversion of cytosine to uracil in bisulfite genomic sequencing analysis of DNA methylation.
Shiraishi M, Hayatsu H. Shiraishi M, et al. DNA Res. 2004 Dec 31;11(6):409-15. doi: 10.1093/dnares/11.6.409. DNA Res. 2004. PMID: 15871463 - DNA methylation analysis by bisulfite conversion, cloning, and sequencing of individual clones.
Zhang Y, Rohde C, Tierling S, Stamerjohanns H, Reinhardt R, Walter J, Jeltsch A. Zhang Y, et al. Methods Mol Biol. 2009;507:177-87. doi: 10.1007/978-1-59745-522-0_14. Methods Mol Biol. 2009. PMID: 18987815 - DNA methylation: a profile of methods and applications.
Fraga MF, Esteller M. Fraga MF, et al. Biotechniques. 2002 Sep;33(3):632, 634, 636-49. doi: 10.2144/02333rv01. Biotechniques. 2002. PMID: 12238773 Review. - Sequencing 5-methylcytosine residues in genomic DNA.
Grigg G, Clark S. Grigg G, et al. Bioessays. 1994 Jun;16(6):431-6. doi: 10.1002/bies.950160612. Bioessays. 1994. PMID: 8080433 Review.
Cited by
- DNA methylation controls stemness of astrocytes in health and ischaemia.
Kremer LPM, Cerrizuela S, El-Sammak H, Al Shukairi ME, Ellinger T, Straub J, Korkmaz A, Volk K, Brunken J, Kleber S, Anders S, Martin-Villalba A. Kremer LPM, et al. Nature. 2024 Sep 4. doi: 10.1038/s41586-024-07898-9. Online ahead of print. Nature. 2024. PMID: 39232166 - Analyzing single-cell bisulfite sequencing data with MethSCAn.
Kremer LPM, Braun MM, Ovchinnikova S, Küchenhoff L, Cerrizuela S, Martin-Villalba A, Anders S. Kremer LPM, et al. Nat Methods. 2024 Sep;21(9):1616-1623. doi: 10.1038/s41592-024-02347-x. Epub 2024 Jul 31. Nat Methods. 2024. PMID: 39085432 Free PMC article. - MethSCAn: accurate exploratory analysis of single-cell methylomes.
[No authors listed] [No authors listed] Nat Methods. 2024 Sep;21(9):1595-1596. doi: 10.1038/s41592-024-02348-w. Nat Methods. 2024. PMID: 39085431 No abstract available. - Comparing methylation levels assayed in GC-rich regions with current and emerging methods.
Guanzon D, Ross JP, Ma C, Berry O, Liew YJ. Guanzon D, et al. BMC Genomics. 2024 Jul 30;25(1):741. doi: 10.1186/s12864-024-10605-7. BMC Genomics. 2024. PMID: 39080541 Free PMC article. - Single-cell omics: experimental workflow, data analyses and applications.
Sun F, Li H, Sun D, Fu S, Gu L, Shao X, Wang Q, Dong X, Duan B, Xing F, Wu J, Xiao M, Zhao F, Han JJ, Liu Q, Fan X, Li C, Wang C, Shi T. Sun F, et al. Sci China Life Sci. 2024 Jul 23. doi: 10.1007/s11427-023-2561-0. Online ahead of print. Sci China Life Sci. 2024. PMID: 39060615 Review.
References
- Biochemistry. 1970 Jul 7;9(14):2858-65 - PubMed
- Cell. 1991 Jul 12;66(1):77-83 - PubMed
- Nucleic Acids Res. 1980 Oct 24;8(20):4777-90 - PubMed
- Proc Natl Acad Sci U S A. 1983 May;80(9):2422-6 - PubMed
- Proc Natl Acad Sci U S A. 1984 Apr;81(7):1991-5 - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Molecular Biology Databases