Cytosine methylation : quantitation by automated genomic sequencing and GENESCAN analysis
Bisulfite treatment and PCR amplification of genomic DNA permits the methylation analysis of any cytosine residue in a target sequence. By cloning and sequencing the PCR product, the methylation of individual molecules can be determined, whereas direct sequencing of the PCR product can provide an av...
| Veröffentlicht in: | BioTechniques. - 1991. - 21(1996), 1 vom: 17. Juli, Seite 126-33 |
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| Format: | Aufsatz |
| Sprache: | English |
| Veröffentlicht: |
1996
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| Zugriff auf das übergeordnete Werk: | BioTechniques |
| Schlagworte: | Comparative Study Journal Article Research Support, Non-U.S. Gov't Coloring Agents DNA Primers Sulfites Cytosine 8J337D1HZY Taq Polymerase EC 2.7.7.- mehr... |
| Zusammenfassung: | Bisulfite treatment and PCR amplification of genomic DNA permits the methylation analysis of any cytosine residue in a target sequence. By cloning and sequencing the PCR product, the methylation of individual molecules can be determined, whereas direct sequencing of the PCR product can provide an average of the methylation status in the population of molecules. Reliable quantitation of cytosine methylation by direct sequencing, however, has not been possible with current methods. In this paper we describe an accurate and innovative protocol to directly quantitate the methylation of any cytosine residue in the target sequence by fluorescence-based automated genomic sequencing. Only the cytosine and thymine residues of bisulfite-treated and amplified genomic DNA are sequenced. The degree of methylation is obtained by direct comparison of the cytosine and thymine signals, which have been labeled with the same fluorescent dyes. GENESCAN analysis is employed to achieve a fast and accurate estimate of methylation at every cytosine in the target sequence. Combining direct bisulfite genomic sequencing and GENESCAN analysis permits the rapid survey of detailed DNA methylation profiles. Using this approach we have found the unexpected result that multicopy plasmid DNA grown in a Dcm host is not always fully methylated as suggested by restriction enzyme data |
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| Beschreibung: | Date Completed 19.02.1997 Date Revised 28.09.2018 published: Print GENBANK: M12379 Citation Status MEDLINE |
| ISSN: | 1940-9818 |