Quantitation of DNA methylation by melt curve analysis

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dc.contributor.authorSmith, E.
dc.contributor.authorJones, M.
dc.contributor.authorDrew, P.
dc.date.issued2009
dc.description.abstractBackground: Methylation of DNA is a common mechanism for silencing genes, and aberrant methylation is increasingly being implicated in many diseases such as cancer. There is a need for robust, inexpensive methods to quantitate methylation across a region containing a number of CpGs. We describe and validate a rapid, in-tube method to quantitate DNA methylation using the melt data obtained following amplification of bisulfite modified DNA in a real-time thermocycler. Methods: We first describe a mathematical method to normalise the raw fluorescence data generated by heating the amplified bisulfite modified DNA. From this normalised data the temperatures at which melting begins and finishes can be calculated, which reflect the less and more methylated template molecules present respectively. Also the T50, the temperature at which half the amplicons are melted, which represents the summative methylation of all the CpGs in the template mixture, can be calculated. These parameters describe the methylation characteristics of the region amplified in the original sample. Results: For validation we used synthesized oligonucleotides and DNA from fresh cells and formalin fixed paraffin embedded tissue, each with known methylation. Using our quantitation we could distinguish between unmethylated, partially methylated and fully methylated oligonucleotides mixed in varying ratios. There was a linear relationship between T50 and the dilution of methylated into unmethylated DNA. We could quantitate the change in methylation over time in cell lines treated with the demethylating drug 5-aza-2'-deoxycytidine, and the differences in methylation associated with complete, clonal or no loss of MGMT expression in formalin fixed paraffin embedded tissues. Conclusion: We have validated a rapid, simple in-tube method to quantify methylation which is robust and reproducible, utilizes easily designed primers and does not need proprietary algorithms or software. The technique does not depend on any operator manipulation or interpretation of the melt curves, and is suitable for use in any laboratory with a real-time thermocycler. The parameters derived provide an objective description and quantitation of the methylation in a specimen, and can be used to for statistical comparisons of methylation between specimens.
dc.description.statementofresponsibilityEric Smith, Michael E Jones and Paul A Drew
dc.identifier.citationBMC Cancer, 2009; 9(1):123-1-123-12
dc.identifier.doi10.1186/1471-2407-9-123
dc.identifier.issn1471-2407
dc.identifier.issn1471-2407
dc.identifier.orcidSmith, E. [0000-0003-2958-3492]
dc.identifier.orcidDrew, P. [0000-0001-5661-4771]
dc.identifier.urihttp://hdl.handle.net/2440/51348
dc.language.isoen
dc.publisherBioMed Central
dc.relation.granthttp://purl.org/au-research/grants/nhmrc/275504
dc.rights© 2009 Smith et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
dc.source.urihttps://doi.org/10.1186/1471-2407-9-123
dc.subjectCell Line, Tumor
dc.subjectHumans
dc.subjectEsophageal Neoplasms
dc.subjectColorectal Neoplasms
dc.subjectAzacitidine
dc.subjectDNA Repair Enzymes
dc.subjectDNA Modification Methylases
dc.subjectTumor Suppressor Proteins
dc.subjectTissue Inhibitor of Metalloproteinase-3
dc.subjectDNA, Neoplasm
dc.subjectOligonucleotides
dc.subjectAntimetabolites, Antineoplastic
dc.subjectImmunohistochemistry
dc.subjectPolymerase Chain Reaction
dc.subjectDNA Methylation
dc.subjectNucleic Acid Denaturation
dc.subjectAlgorithms
dc.subjectCyclin-Dependent Kinase Inhibitor p16
dc.subjectHot Temperature
dc.subjectPromoter Regions, Genetic
dc.subjectDecitabine
dc.titleQuantitation of DNA methylation by melt curve analysis
dc.typeJournal article
pubs.publication-statusPublished

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