TheMethylamp™DNAModificationKitisacompletesetofoptimizedreagentsdesignedtoconvertDNAsothat5-methylcytosinescanbedetectedindownstreamapplications,forthepurposeofgene-specificDNAmethylationanalysis.Thekithasthefollowingadvantagesandfeatures: - Totalprotocoltimeislessthan2hours.
- 99.9%conversionrateofunmethylatedcytosineintouracil/thymine
- ExtremelylowdegradationofDNAinthemodificationprocess,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.
BackgroundInformation
DNAmethylationoccursbythecovalentadditionofamethylgroupatthe5-carbonofthecytosinering,resultingin5-methylcytosine.DNAmethylationisessentialforthedevelopmentofmanyspeciesandisoftenassociatedwithmanykeyBIOLOGicalprocessesaswellasgeneregulation.TherearevariousmethodsusedtoassessDNAmethylationstates.However,onlybisulfiteconversionofgenomicDNA,followedbyPCRamplification,cloning,andsequencingofindividualPCRamplimersyieldsreliableinformationonthemethylationstatesofindividualcytosinesonindividualDNAmolecules.BytreatingDNAwithsodiumbisulfite,cytosineresiduesaredeaminatedtouracilwhileleaving5-methylcytosineintact,providingresearcherswithdistinguishabemethylatedpositionsintheDNAsequenceduringthedownstreamapplication. Principle&Procedure
DNAischemicallydenaturedtoallowthesodiumbisulfitereagenttoreactspecificallywithsingle-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.
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Fig.1. SchematicprocedureoftheMethylamp™DNAModificationKit.
Fig.2. DifferentamountsofDNAisolatedfromaserumsamplewerechemicallymodifiedusingtheMethylamp™DNAModificationKit.RealtimePCRwasthenperformedbyusingapairofprimersandaprobedesignedtoamplifybothmethylatedandunmethylatedallelesofβ-actin.
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