What question did this study set out to answer?

To develop a strand-specific genomic sequencing protocol that provides positive identification and single-molecule resolution of 5-methylcytosine residues in genomic DNA.

March 1, 1992Proceedings of the National Academy of SciencesOpen Access

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

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Authors

MFMarianne FrommerUNSW Sydney LMLouise E. McDonaldThe University of Queensland DMDavid MillarRoyal Jubilee Maternity Services

Discussion

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Implication

Methodological study demonstrates positive identification of single-strand 5-methylcytosine residues in human genomic DNA, highlighting strand-specific methylation mapping capabilities.

Key Points

To develop a strand-specific genomic sequencing protocol that provides positive identification and single-molecule resolution of 5-methylcytosine residues in genomic DNA.
Treated genomic DNA with bisulfite to selectively deaminate unmethylated cytosine to uracil while leaving 5-methylcytosine unreactive.
Amplified targets using strand-specific PCR primers so that uracil and thymine amplify as thymine, whereas 5-methylcytosine amplifies as cytosine.
Evaluated the protocol by direct sequencing and single-molecule clone sequencing of two CpG dinucleotides within the human kininogen gene promoter, including sperm DNA samples.
Generated positive, strand-specific displays of 5-methylcytosine residues yielding both population-wide average sequences and individual single-molecule methylation maps.
Detected an atypical methylation profile at two promoter CpG dinucleotides in human sperm DNA, indicating localized modulation driven by germ-line protein binding.

Cite This Study

Frommer et al. (1992) studied this question.

synapsesocial.com/papers/69d8efd52c39562886ae345e https://doi.org/10.1073/pnas.89.5.1827

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March 1, 1992Proceedings of the National Academy of SciencesOpen Access

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

View Full Paper

Ask AI

Bookmark

Authors

MFMarianne FrommerUNSW Sydney LMLouise E. McDonaldThe University of Queensland DMDavid MillarRoyal Jubilee Maternity Services

Discussion

Member takes

Implication

Methodological study demonstrates positive identification of single-strand 5-methylcytosine residues in human genomic DNA, highlighting strand-specific methylation mapping capabilities.

Key Points

To develop a strand-specific genomic sequencing protocol that provides positive identification and single-molecule resolution of 5-methylcytosine residues in genomic DNA.
Treated genomic DNA with bisulfite to selectively deaminate unmethylated cytosine to uracil while leaving 5-methylcytosine unreactive.
Amplified targets using strand-specific PCR primers so that uracil and thymine amplify as thymine, whereas 5-methylcytosine amplifies as cytosine.
Evaluated the protocol by direct sequencing and single-molecule clone sequencing of two CpG dinucleotides within the human kininogen gene promoter, including sperm DNA samples.
Generated positive, strand-specific displays of 5-methylcytosine residues yielding both population-wide average sequences and individual single-molecule methylation maps.
Detected an atypical methylation profile at two promoter CpG dinucleotides in human sperm DNA, indicating localized modulation driven by germ-line protein binding.

Cite This Study

Frommer et al. (1992) studied this question.

synapsesocial.com/papers/69d8efd52c39562886ae345e https://doi.org/10.1073/pnas.89.5.1827

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