TheBisulFlash™DNABisulfiteConversionMag-96Kitisacompletesetofoptimizedreagentsforfast,highthroughputDNAbisulfiteconversion.Thekitusesauniqueliquidconversionsolutionandmagneticbindingbeadclean-upprocesstoquicklygeneratebisulfite-convertedDNAforhigheryieldsviaa96-wellmicroplate.Amagneticbeadbasedapproachviamicroplatesalsoallowsforautomationworkflows.Thekithasthefollowingadvantages:

• Fast-Completetheentireprocedureinaslittleas1hourand20minforasmanyas96samplessimultaneously,averagingjust50secondspersample.
• Convenient-Ready-to-useliquidconversionmixissimplyaddedtotheDNAsamplesdirectly,withoutneedforpre-preparationoftheconversionreagent.
• Streamlined-ConcurrentlyprocessestheDNAdenaturationandCtoTconversionstepswithouttheneedforaseparateDNAdenaturationstep.
• CompleteConversion-Completelyconvertsunmethylatedcytosineintouracil(>99.9%)withnegligIBLeinappropriateorerrorconversionofmethylcytosinetothymine(<0.1%).
• Flexible-Choiceofeither(a)manualwithonesinglereactioneachtime;or(b)highthroughputwith96reactionseachtimecanbeused,makingtheassayflexible.
• Robust-Simple,reliable,andconsistentreactionconditionswithaneasy-to-followprotocolandhighyield.

BackgroundInformation
DNAmethylationisessentialinregulatinggeneexpressioninnearlyallBIOLOGicalprocessesincludingdevelopment,growth,anddifferentiation.Gene/region-specificorgenome-wideanalysisofDNAmethylationor5-methylcytosine(5-mC)couldprovidevaluableinformationfordiscoveringepigeneticMarkersusedfordiseasediagnosis,andpotentialtargetsusedfortherapeutics.BytreatingDNAwithbisulfite,cytosineresiduesaredeaminatedtouracilwhileleaving5-methylcytosineintact,sothatmethylatedcytosinecanbeproperlyidentifiedviaPCR,array,bisulfitesequencing,ornextgenerationsequencing.

Principle&Procedure
Thiskitcontainsallreagentsrequiredforafastbisulfiteconversioninahighthroughputformat.Withtheuniqueconversionmixsolution,DNAdenaturationstatusissustainedthroughouttheentirebisulfiteconversionprocess,therebyenablingfullconversionofunmethylatedcytosinetouracil.Desulphonationandclean-upoftheconvertedDNAisperformedusingmagneticbead-basedbindingandseparationina96-wellmicroplate.WerecommendusingtheEpiMagHTMagneticSeparator.Highyield,convertedDNAcanbeobtainedandusedforvariousdownstreamapplicationsincludingPCRandnextgenerationsequencing.

StartingMaterials
Startingmaterialsmayincludevarioustissueorcellsamplessuchas:culturedcellsfromaflaskormicroplate,microdissectionsample,paraffin-embeddedtissue,plasma/serumsample,andbodyfluidsample,etc.TheamountofDNAforeachreactioncanbe0.1to1ug.Foranoptimalreaction,theinputDNAamountshouldbe100ng.Ifsmallamounts(e.g.,<10ng)ofstartingDNAareused,thenumberofPCRcyclesshouldbegreaterthan45.

Fig.1. SchematicprocedureoftheBisulFlashDNABisulfiteConversionMag-96Kit.

Fig.2. DifferentamountsofhumangenomicDNAwereconvertedusingtheBisulFlashDNABisulfiteConversionMag-96Kit.ConvertedDNAwasamplifiedusingMethylamp™MS-qPCRFastKit(Cat.#P-1028).

Fig.3. HighaccuracyofDNAconversionisachievedbytheBisulFlashDNABisulfiteConversionMag-96Kit.100ngofgenomicDNAmethylatedinallCpGsitesbyDNAmethylaseswastreatedwiththekitfollowedbyrealtimeqPCRamplificationusingprimersformultiplepromoterscontainingnumerousCpGsitesandthendirectlysequenced.100%Catnon-CpGsitesareconvertedtoTand100%CatCpGsitesremainasC.