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

Frommer M., McDonald L.E., Millar D.S., Collis C.M., Watt F., Grigg G.W., Molloy P.L. and Paul C.L. (1992) A genomic sequencing protocol that yields a positive display of 5- methylcytosine residues in individual DNA strands. Proceedings of the National Academy of Sciences of the United States of America, 89 5: 1827-1831. doi:10.1073/pnas.89.5.1827


Author Frommer M.
McDonald L.E.
Millar D.S.
Collis C.M.
Watt F.
Grigg G.W.
Molloy P.L.
Paul C.L.
Title A genomic sequencing protocol that yields a positive display of 5- methylcytosine residues in individual DNA strands
Journal name Proceedings of the National Academy of Sciences of the United States of America   Check publisher's open access policy
ISSN 0027-8424
Publication date 1992-01-01
Sub-type Article (original research)
DOI 10.1073/pnas.89.5.1827
Open Access Status Not yet assessed
Volume 89
Issue 5
Start page 1827
End page 1831
Total pages 5
Language eng
Subject 1000 General
1311 Genetics
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.
Keyword bisulfite modification
DNA methylation
genomic sequencing
kininogen gene
PCR
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status Unknown

Document type: Journal Article
Sub-type: Article (original research)
Collection: Scopus Import - Archived
 
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