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Epigentek/Methylamp DNA Modification Kit/P-1001-1/40 reactions

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P-1001-1

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InputType:DNAResearchArea:DNAMethylationTargetApplication:SampleModificationVesselFormat:Columns/Tubes100%Guarantee:6monthsProductOverviewTheMethylamp™DNAModificationKitisacompletesetofoptimizedreagentsdesignedtoconvertDNAsothat5-methylcytosinescanbedetectedindownstreamapplications,forthepurposeofgene-specificDNAmethylationanalysis.Thekithasthefollowingadvantagesandfeatures:Totalprotocoltimeislessthan2hours.99.9%conversionrateofunmethylatedcytosineintouracil/thymineExtremelylowdegradationofDNAinthemodificationprocess,preventingover90%ofDNAloss.Requiresaslittleasonly50pgofinputDNA,equivalenttoabout20cells.Extremelysimple,robust,andreliablesodiumbisulfiteconversionprotocol.Suitableforbisulfitesequencing(directandcloning),next-generationsequencing,pyrosequencing,PCR(real-time,end-point,methylation-specific),COBRA(combinedbisulfiterestrictionanalysis),andmethylation-basedmicroarrays.HighlycompatIBLewithIllumina-basedworkflows.BackgroundInformationDNAmethylationoccursbythecovalentadditionofamethylgroupatthe5-carbonofthecytosinering,resultingin5-methylcytosine.DNAmethylationisessentialforthedevelopmentofmanyspeciesandisoftenassociatedwithmanykeyBIOLOGicalprocessesaswellasgeneregulation.TherearevariousmethodsusedtoassessDNAmethylationstates.However,onlybisulfiteconversionofgenomicDNA,followedbyPCRamplification,cloning,andsequencingofindividualPCRamplimersyieldsreliableinformationonthemethylationstatesofindividualcytosinesonindividualDNAmolecules.BytreatingDNAwithsodiumbisulfite,cytosineresiduesaredeaminatedtouracilwhileleaving5-methylcytosineintact,providingresearcherswithdistinguishabemethylatedpositionsintheDNAsequenceduringthedownstreamapplication. Principle&ProcedureDNAischemicallydenaturedtoallowthesodiumbisulfitereagenttoreactspecificallywithsingle-strandedDNA,therebydeaminatingcytosineandcreatingauracilresidue.TheuniqueDNAprotectionreagentscontainedinthemodificationbuffercanpreventchemicalandThermophilicdegradationofDNAduringthesodiumbisulfitetreatmentprocess.Theincludednon-toxic,modifiedDNAcapturebufferenablestheDNAtobindtightlytothecolumnfilter,permittingDNAcleaningtobecarriedoutonthecolumntoeffectivelyremoveresidualsodiumbisulfiteandsalts.ModifiedDNAcanthenbeelutedforuseinvariousmethylationanalysisapplicationsorotherwisestoredat-20°Cforupto2months. Fig.3. Twobisulfite-convertedDNAsfromsixkits,includingtheMethylampDNAModificationKit(Cat#P-1001),weresubjectedtoPCRwithtwoamplicons(655and1109bp)andthreedifferentextensiontemperatures(65°C,68°Cand72°C)toassesscapacityforlongampliconamplification.Bisulfite-convertedDNAfromEpigentek"sMethylampkitwithaPCRextensiontemperatureof65°Chadthemostrobustamplificationofthelonger1109bpamplicon. YangY,etal. BMCGenomics.2015;16(1):350. Fig.1. SchematicprocedureoftheMethylamp™DNAModificationKit. Fig.2. DifferentamountsofDNAisolatedfromaserumsamplewerechemicallymodifiedusingtheMethylamp™DNAModificationKit.RealtimePCRwasthenperformedbyusingapairofprimersandaprobedesignedtoamplifybothmethylatedandunmethylatedallelesofβ-actin.ProductComponentsR1(DNADenatureSolution)R2(DNAModificationPowder)R3(DNAModificationSolution)R4(ModifiedDNACaptureSolution)R5(ModifiedDNACleaningSolution)R6(ModifiedDNAElutionSolution)F-SpinColumnF-CollectionTubeUserGuide

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