Chapter17MeltingCurveAssaysforDNAMethylationAnalysisTomaszK.WojdaczandAlexanderDobrovicAbstractTheabilityofsodiumbisulfitetomodifycytosinesinamethylation-dependentmannerallowstheconservationofDNAmethylationinformationduringPCRamplification.PCRproductsamplifiedfrombisulfite-modifiedDNAhavesignificantlydifferentbasecompositionsaccordingtowhethertheyorigi-natefrommethylatedorunmethylatedvariantsofthetargettemplate.DifferentbasecompositionsgiverisetodifferentthermalpropertiesofthePCRproducts.Hence,meltinganalysisofamplificationprod-uctsinmethylationstudiesallowsthedeterminationofwhetherthePCRproductsoriginatefrommethy-latedorunmethylatedtemplates.Here,webrieflyreviewrecentadvancesinmethodologiesbasedonmeltinganalysesofPCRproductsderivedfrombisulfite-modifiedtemplatesandprovideamethodologyformethylation-sensitivehigh-resolutionmelting.Keywords:Methylation,meltingcurve,sodiumbisulfite,high-resolutionmelting,PCRbias,Methylation-sensitivehigh-resolutionmelting(MS-HRM).1.IntroductionTheintroductionofbisulfitemodificationofgenomicDNAenabledthegeneraluseofPCRamplificationinmethylationstudies(1).Sodiumbisulfitedeaminatesunmethylatedcytosinestouracilsleaving5-methylcytosinesintact.Asaconsequence,methylatedcytosinesareamplifiedduringthesubsequentpoly-merasechainreaction(PCR)ascytosineswhereasunmethylatedcytosinesareamplifiedasthymines.Hence,thebasecompositionofthePCRproductdependsonthe5-methylcytosinecontentofthetemplate.ThetwocomplementarystrandsofDNAareheldtogetherbyhydrogenbondsandstackinginteractions.Dissociationofdouble-strandedDNAisknownasDNAmeltingordenaturationJörgTost(ed.),DNAMethylation:MethodsandProtocols,SecondEdition,vol.507C⃝2009HumanaPress,apartofSpringerScience+BusinessMediaDOI10.1007/978-1-59745-522-017Springerprotocols.com229230WojdaczandDobrovicandcanbeinducedeitherbyincreasedtemperatureordena-turingchemicals.ThedissociationofthetriplehydrogenbondbetweenCandGrequiresmoreenergythanthedissociationofthedoublehydrogenbondbetweenTandA,thereforeGC-richsequencesm...
