Rapid quantification of DNA methylation through dNMP analysis following bisulfite-PCR
| Autor: | In Young Park, Lian Hua Shi, Hyung-Keun Ku, Sung-Moon Jang, Inchul Yang, Sang-Ryoul Park |
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| Jazyk: | angličtina |
| Rok vydání: | 2006 |
| Předmět: |
Oligonucleotide
Hydrolysis Deoxyribonucleotides Electrophoresis Capillary Methylation Biology DNA Methylation Reference Standards Polymerase Chain Reaction Thymine Bisulfite genomic DNA chemistry.chemical_compound Cytosine Capillary electrophoresis chemistry Biochemistry DNA methylation Genetics Methods Online Sulfites |
| Zdroj: | Nucleic Acids Research |
| ISSN: | 1362-4962 0305-1048 |
| Popis: | We report a novel method for rapid quantification of the degree of DNA methylation of a specific gene. Our method combined bisulfite-mediated PCR and quantification of deoxyribonucleoside monophosphate (dNMP) contents in the PCR product through capillary electrophoresis. A specific bisulfite-PCR product was enzymatically hydrolyzed to dNMP monomers which were quantitatively analyzed through subsequent capillary electrophoresis. PCR following bisulfite treatment converts unmethylated cytosines to thymines while leaving methyl-cytosines unchanged. Then the ratio of cytosine to thymine determined by capillary electrophoresis represents the ratio of methyl-cytosine to cytosine in genomic locus of interest. Pure oligonucleotides with known sequences were processed in parallel as standards for normalization of dNMP peaks in capillary electrophoresis. Sources of quantification uncertainty such as carryovers of dNTPs or primers and incomplete hydrolysis were examined and ruled out. When the method was applied to samples with known methylation levels (by bisulfite-mediated sequencing) as a validation, deviations were within +/-5%. After bisulfite-PCR, the analytical procedure can be completed within 1.5 h. |
| Databáze: | OpenAIRE |
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