NDLI: Rapid quantification of DNA methylation through dNMP analysis following bisulfite-PCR

Content Provider	PubMed Central
Author	Yang, Inchul Park, In Young Jang, Sung-moon Shi, Lian Hua Ku, Hyung-keun Park, Sang-ryoul
Copyright Year	2006
Abstract	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.
Related Links	http://dx.doi.org/10.1093/nar/gkl257
Starting Page	61
File Format	PDF
ISSN	03051048
e-ISSN	13624962
Journal	Nucleic Acids Research
Issue Number	8
Volume Number	34
Language	English
Publisher	Oxford University Press
Publisher Date	2006-04-28
Access Restriction	Open
Rights Holder	Oxford University Press
Subject Keyword	Genetics Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Genetics

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