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

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 bisulfiteinduced 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 derming 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.Cytosine methylation has long been recognized as an important factor in the silencing of genes in mammalian cells. Recent studies have shown that cytosine methylation at single CpG dinucleotides within the recognition sites of a number of transcription factors is sufficient to block binding of the factors to DNA (1-6) and to inhibit transcription (3-5). Therefore, to determine the role of cytosine methylation in specific regulatory mechanisms in vivo, it has become important to know the methylation status of individual CpG dinucleotides in genomic DNA. Genomic sequencing protocols, which have been developed to ascertain the methylation status of selected regions within genes, utilize the Maxam and Gilbert chemical cleavage reactions carried out on genomic DNA (7) with various additional procedures to enhance the signal from the sequence under investigation (8, 9). These protocols are versatile in that they can be adapted for identification of protein-binding sites on genomic DNA in vivo (8, 10) but have two major drawbacks with respect to the identification of 5-methylcytosine residues. First, 5-methylcytosine is identified by the lack of a band in all tracks of a sequencing gel; any background cleavage ladder or close spacing of bands can result in difficulties of interpretation. Second, the sequence obtained represents a population average for many DNA molecules, so that the protocols cannot be adapted for sequencing s...

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