TheEpiQuik™MeDIPUltraKitisacompletesetofoptimizedreagentstoenrichandcapturemethylatedDNAfragmentsinaconvenientmicroplate-basedformat,andisafurtherrefinementofthepredecessorMethylamp™MeDIPKit bysignificantlyimprovingsensitivityandspecificitywhilereducingbackgroundsignals.Themethod,methylatedDNAimmunoprecipitation(MeDIP),usesamonoclonalantibodyspecificto5-methylcytosinetoimmunoprecipitatemethylatedgenomicDNA.Theenrichedmethylatedfractionscanthenbeusedforgene-specificDNAmethylationanalysisonagenomewidescale.ThehighlysensitiveandspecificformatofthekitcanuseDNAisolatedfromvariousspecies.ThemethylatedDNAthatisenrichedwiththiskitcanbeusedforvariousdownstreamapplicationsincludingqualitativeandquantitativePCR(MeDIP-PCR),microarray(MeDIP-chip)andespeciallysequencing(MeDIP-seq).Thekithasthefollowingadvantagesandfeatures: - Extremelyfastandconvenientprotocolwithatotalproceduretime(frominputsampletoready-to-usemethylatedDNA)oflessthan3hours,whichincludesaminimalhandlingtimeoflessthan20minutes.
- Optimizedbuffersandprotocolallowminimalbackgroundbyovercomingtheweaknessesthatcausenon-specificenrichment.
- Ahighlyspecific5-mCmonoclonalantibodyincludedinthekitcanstronglybindbothsingleanddoublestrandedDNAfragmentscontaining2ormore5-mCswhichenableshighlysensitiveenrichmentofmethylatedDNAwith>99%specificity.
- FlexIBLe96strip-wellmicroplateformatmakestheassayveryeasytohandle:manualmethodwithonereactionatatimeorhighthroughputmethodwith24-48reactionsatatime.
- SpincolumnsandcollectiontubesconvenientlyincludedforaDNApurificationstep.
- LowDNAinputrequirementaslowas50ng(10,000cells)perreaction.
- Highreproducibilityusingpre-optimizedMeDIPconditions.
- CompatiblewithvariousdownstreamanalysisworkflowsincludingMeDIP-PCRandMeDIP-chip,andspecificallyforMeDIP-seq.
BackgroundInformation
CoremechanismsforepigeneticalterationofgenomicDNAarehypermethylationofCpGislandsinspecificgenesandglobalDNAhypomethylation.Region-specificDNAmethylationplaysanimportantroleintherepressionofgenetranscriptionandismainlyfoundin5’-CpG-3’dinucleotideswithinpromotersorinthefirstexonofgenes.GlobalDNAhypomethylationislikelycausedbymethyl-deficiencyduetoavarietyofenvironmentalinfluences.IthasbeendemonstratedthatalterationsinDNAmethylationareassociatedwithmanydiseases,especiallycancer. HighlyspecificisolationofmethylatedDNAcombinedwithnextgenerationsequencingforgenome-widemethylationanalysisshouldprovideanadvantageforconvenientandcomprehensiveidentificationofmethylationstatusofnormalanddiseasedcells,suchascancercells[1].SuchanalysisrequirestheisolatedmethylatedDNAtocontainminimalbackgroundinordertoachievehighspecificity(>98%)forreliablyidentifyingtruemethylatedregions.ThemajormethodforenrichingmethylatedDNAusedforgenome-widemethylationprofilingismethylatedDNAimmunoprecipitation(MeDIP)[2].However,currentlyusedMeDIPmethods,representedbymostcommerciallyavailablekits,havesignificantweaknessesincludinghighlynon-specificenrichment(amountofenrichedDNAis>75%oftheamountofinputDNA)[3],timeconsuming,laborintensive,andhaslowthroughput.Thus,foreffectivelyandspecificallycapturingmethylatedDNAusedfornextgenerationsequencinganalysis,anidealMeDIPmethodrequiresmaximumsensitivitywithminimalbackgroundlevels.Epigentek’sEpiQuik™MeDIPUltraKitisdesignedtoachievethesegoalsbymaximizingsensitivityandminimizingnon-specificbackgroundsignals,andisasignificantimprovementoverpreviousMeDIPkits. 1.RuikeYetal:BMCGenomics,11:137,2010.
2.WeberMetal:NatureGenetics,37:853-862,2005.
3.Brebi-MievillePetal: Epigenetics,7:106-112,2012. Principle&Procedure
ThiskitincludesamethylatedDNAcontrolandanunmethylatedDNAcontrol,anegativecontrolnon-immuneIgG,andcontrolprimersthatcanbeusedwiththecontrolDNAtodemonstratetheenrichmentefficacyandspecificityformethylatedDNA. The5-methylcytosinemonoclonalantibodyprovidedinthiskitishighlyspecificagainstmethylatedDNAfragments,bothsingleanddoublestranded,andisnotcross-reactivetohydroxymethylatedandunmethylatedDNAfragments. Thisantibodycancapture>50%ofDNAfragmentscontainingasfewastwo5-mCsandenrichesallDNAfragmentscontainingfourormore5-mCs. ThepositivecontrolDNAcontaining5-mCcanbeimmunoprecipitatedbythe5-mCantibodybutnotbythenon-immuneIgG.InthisMeDIP,immunoprecipitationof5-mC-enrichedDNAfragmentsisprocessedinamicroplateunderoptimizedreactionconditions,whichenablesMeDIPtobecompletedwithin3hourswithhighefficiency.ImmunoprecipitatedmethylatedDNAisthencleaned,released,andeluted.ElutedDNAcanbeusedforvariousdownstreamapplicationsincludingPCR(MeDIP-PCR)andmicroarray(MeDIP-chip),andisespeciallysuitableforMeDIP-seq. StartingMaterials,InputAmount,&ExpectedYield
ThestartingmaterialshouldbegoodqualitypurifiedDNA.TheamountofDNAforeachreactioncanbe50ng(approximately10,000cells)to500ng.Foranoptimalreaction,theinputDNAamountshouldbe100-200ngperwell.TheyieldedmethylatedDNAisabout4ngfor100nginputDNA(4%),whichisconsistentwiththeexpectedpercentage(4-5%)atwhichthehighestsensitivityandspecificityforenrichedmethylatedDNAhasbeendemonstratedbybisulfitesequencing. | 
 Fig.1. SchematicprocedureoftheEpiQuik™MeDIPUltraKit.
Fig.2. SelectiveenrichmentofmethylatedDNAwiththeEpiQuik™MeDIPUltraKit|50pgofunmethylatedormethylatedDNAcontrolwereeachspikedintofragmentedhumangenomicDNA(100ng).MeDIPwasprocessedwiththeincluded5-mCmonoclonalantibodyandnon-immuneIgGincludedinthekit.ElutedDNAwasanalyzedbyrealtimePCRwiththecontrolprimersincludedinthekittodetectthepresenceofspikedcontrolDNA.Fold-enrichmentrepresentstheamountofrecoveredcontrolDNAandwascalculatedbasedontherealtimePCRCtvalue.
Fig.3. Sensitivedetectionofgene-specificmethylationbyMeDIP-qPCR|FullymethylatedHeLaDNA(500ng)wasfragmentedto100-500bpsusinganEpiSonic™1100.ThefragmentedDNAwasusedformethylatedDNAenrichmentwiththeEpiQuik™MeDIPKit.ElutedDNAwasanalyzedbyrealtimePCRwithprimersspecificforMLH1sequencesinthepromoterregions.Resultsshowthespecificityofthe5-mCantibodyandlowbackgroundfornon-immuneIgG.Fold-enrichmentrepresentstheamountofrecoveredDNAandwascalculatedbasedontherealtimePCRCtvalue.
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