Quantitation of DNA methylation by melt curve analysis.
Background: 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...
| Publicado en: | BMC Cancer Vol. 9; pp. 123 - 124 |
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| Autores principales: | , , , , , |
| Formato: | research Journal Article |
| Publicado: |
BioMed Central
2009
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| Acceso en línea: | Ver este registro en EBSCOhost |
| fields | @attributes: recordID: 1 pdfLink: plink: https://search.ebscohost.com/login.aspx?direct=true&db=ccm&AN=105524773&site=ehost-live header: @attributes: shortDbName: ccm uiTerm: 105524773 longDbName: CINAHL Complete uiTag: AN controlInfo: bkinfo: dissinfo: jinfo: jid: 14712407 1CH9 jtl: BMC Cancer issn: 14712407 maglogo: N pubinfo: dt: 2009 vid: 9 pid: 24147 pub: BioMed Central artinfo: ui: 105524773 NLM19393074 2010275434 10.1186/1471-2407-9-123 NLM19393074 105524773 ppf: 123 ppct: 1 formats: tig: atl: Quantitation of DNA methylation by melt curve analysis. aug: au: Smith E Jones ME Drew PA Smith, Eric Jones, Michael E Drew, Paul A affil: School of Nursing and Midwifery, Flinders University, Bedford Park, South Australia 5042, Australia sug: subj: DNA Heat Algorithms Antimetabolites, Antineoplastic Pharmacodynamics Biochemical Phenomena Drug Effects Cells Colorectal Neoplasms Metabolism Colorectal Neoplasms Pathology Colorectal Neoplasms Enzyme Inhibitors Pharmacodynamics Enzyme Inhibitors Enzymes Metabolism Enzymes Esophageal Neoplasms Metabolism Esophageal Neoplasms Pathology Esophageal Neoplasms Genes Immunohistochemistry Nucleotides Polymerase Chain Reaction Proteins Metabolism Proteins Human ab: Background: 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. pubtype: Academic Journal doctype: research Journal Article ougenre: Article language: English refInfo: holdings: @attributes: islocal: N |
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