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A genomic sequencingprotocol that yieldsa positive display of5-methylcytosineresidues inindividual DNAstrands. Marianne FrommerLouise E. McDonaldDavid Millar1992 год

A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands.
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Аннотация: 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.
Год издания: 1992
Авторы: Marianne Frommer, Louise E. McDonald, David Millar, Christina M. Collis, Fujiko Watt, G. W. GRIGG, Peter L. Molloy, Cheryl Paul
Издательство: National Academy of Sciences
Источник: Proceedings of the National Academy of Sciences
Ключевые слова: Genetic Syndromes and Imprinting, Epigenetics and DNA Methylation, Cancer-related gene regulation
Другие ссылки: Proceedings of the National Academy of Sciences (HTML)
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Europe PMC (PubMed Central) (HTML)
PubMed Central (HTML)
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