title: Quantitation of DNA methylation by melt curve analysis.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: School of Nursing and Midwifery, Flinders University, Bedford Park, South Australia 5042, Australia. eric.smith@adelaide.edu.au
authors.forename: Eric
authors.initials: E
authors.lastname: Smith
authors.forename: Michael E
authors.initials: ME
authors.lastname: Jones
authors.forename: Paul A
authors.initials: PA
authors.lastname: Drew
mesh_terms: D000465:Algorithms; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics; D019175:DNA Methylation; D015254:DNA Modification Methylases / Q000235:genetics / Q000378:metabolism; D045643:DNA Repair Enzymes / Q000235:genetics / Q000378:metabolism; D004273:DNA, Neoplasm / Q000737:chemistry / Q000235:genetics*; D000077209:Decitabine; D004938:Esophageal Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D006358:Hot Temperature; D006801:Humans; D007150:Immunohistochemistry; D009691:Nucleic Acid Denaturation / Q000187:drug effects; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D019717:Tissue Inhibitor of Metalloproteinase-3 / Q000235:genetics; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000964:Antimetabolites, Antineoplastic; D019941:Cyclin-Dependent Kinase Inhibitor p16; D004273:DNA, Neoplasm; D009841:Oligonucleotides; C088390:TIMP3 protein, human; D019717:Tissue Inhibitor of Metalloproteinase-3; D025521:Tumor Suppressor Proteins; D000077209:Decitabine; D015254:DNA Modification Methylases; C515491:MGMT protein, human; D045643:DNA Repair Enzymes; D001374:Azacitidine