EZ-DNA-Methylation-Gold-Kit-说明书

ZYMORESEARCHCORP.TollFree:1-888-882-9682•Fax:[phone]•Web:•E-mail:[e-mail]-Gold™KitCatalogNos.D5005&D5006Highlights•CompletebisulfiteconversionofGC-richDNAinlessthan3hours.•Acoupledheatdenaturation/conversionreactionstepstreamlinestheconversionofunmethylatedcytosinesintouracil.•DNAprecipitationsareomitted.Instead,DNAiscleanedanddesulphonatedinasinglestepusingstate-of-the-artspincolumns.•Eluted,ultra-pureDNAisidealforuseinsubsequentmolecular-basedanalyses.ContentsProductContents..................................................1IntroductiontoDNAMethylation...........................2ProductDescription..............................................3ProductSpecifications..........................................4ReagentPreparation............................................4Protocol................................................................5Appendix..............................................................6FrequentlyAskedQuestions................................7OrderingInformation............................................8ListofRelatedProducts.......................................9ForResearchUseOnlyVer.2.1.0INSTRUCTIONMANUALZYMORESEARCHCORP.TollFree:1-888-882-9682•Fax:[phone]•Web:•E-mail:[e-mail]1ProductContents:EZDNAMethylation-Gold™KitD500550rxns.D5006200rxns.StorageTemperatureCTConversionReagent*5Tubes20TubesRoomTemp.M-DilutionBuffer1.5ml7mlRoomTemp.M-DissolvingBuffer500µl1.2mlRoomTemp.M-BindingBuffer30ml125mlRoomTemp.M-WashBuffer**6ml24mlRoomTemp.M-DesulphonationBuffer10ml40mlRoomTemp.M-ElutionBuffer1ml4mlRoomTemp.Zymo-Spin™ICColumns50ct.200ct.RoomTemp.CollectionTubes50ct.200ct.RoomTemp.InstructionManual11-Note-Integrityofkitcomponentsisguaranteedforoneyearfromdateofpurchase.Reagentsareroutinelytestedonalot-to-lotbasistoensuretheyprovidemaximalperformanceandreliability.*900µlwater,300µlM-DilutionBuffer,and50µlM-DissolvingBuffermustbeaddedpertubeofCTConversionReagentpriortouse.**Add24mlof100%ethanoltothe6mlM-WashBufferconcentrate(D5005)or96mlof100%ethanoltothe24mlM-WashBufferconcentrate(D5006)beforeuse.TheEZDNAMethylation-Gold™Kitsarepatentpending.ThePolymeraseChainReaction(PCR)processiscoveredbyU.S.Pat.Nos.4,683,195and4,683,202assignedtoHoffmann-LaRoche.Patentspendinginothercountries.NolicenseunderthesepatentstousethePCRprocessisconveyedexpresslyorbyimplicationtothepurchaserbythepurchaseofZymoResearch’sEZDNAMethylationkits.FurtherinformationonpurchasinglicensestopracticethePCRprocesscanbeobtainedfromthedirectorofLicensingatAppliedBiosystems,850LincolnCentreDrive,FosterCity,California94404oratRocheMolecularSystems,Inc.,1145AtlanticAvenue,Alameda,California94501.UseofMethylationSpecificPCR(MSP)isprotectedbyUSPatents5,786,146&6,017,704&6,200,756&6,265,171andInternationalPatentWO97/46705.NolicenseunderthesepatentstousetheMSPprocessisconveyedexpresslyorbyimplicationtothepurchaserbythepurchaseofthisproduct.Note-™TrademarksofZymoResearchCorporation.Thisproductisforresearchuseonlyandshouldonlybeusedbytrainedprofessionals.Somereagentsincludedwiththiskitareirritants.Wearprotectiveglovesandeyeprotection.Followthesafetyguidelinesandrulesenactedbyyourresearchinstitutionorfacility.Note:SatisfactionofallZymoResearchproductsisguaranteed.Ifyoushouldbedissatisfiedwiththisproductpleasecall1-888-882-9682.ZYMORESEARCHCORP.TollFree:1-888-882-9682•Fax:[phone]•Web:•E-mail:[e-mail]2IntroductiontoDNAMethylation:DNAmethylationisanaturallyoccurringeventinbothprokaryoticandeukaryoticorganisms.InprokaryotesDNAmethylationprovidesawaytoprotecthostDNAfromdigestionbyrestrictionendonucleasesthataredesignedtoeliminateforeignDNA,andinhighereukaryotesDNAmethylationfunctionsintheregulation/controlofgeneexpression(1).IthasbeendemonstratedthataberrantDNAmethylationisawidespreadphenomenonincancerandmaybeamongtheearliestchangestooccurduringoncogenesis(2).DNAmethylationhasalsobeenshowntoplayacentralroleingeneimprinting,embryonicdevelopment,X-chromosomegenesilencing,andcellcycleregulation.Inmanyplantsandanimals,DNAmethylationconsistsoftheadditionofamethylgrouptothefifthcarbonpositionofthecytosinepyrimidineringviaamethyltransferaseenzyme(3).ThemajorityofDNAmethylationinmammalsoccursin5’-CpG-3’dinucleotides,butothermethylationpatternsdoexist.Infact,about80percentofall5’-CpG-3’dinucleotidesinmammaliangenomesarefoundtobemethylated,whereasthemajorityofthetwentypercentthatremainunmethylatedarewithinpromotersorinthefirstexonsofgenes.TheabilitytodetectandquantifyDNAmethylationefficientlyandaccuratelyhasbecomeessentialforthestudyofcancer,geneexpression,geneticdiseases,aswellasmanyotherimportantaspectsofbiology.Todate,anumberofmethodshavebeendevelopedtodetect/quantifyDNAmethylationincluding:high-performancecapillaryelectrophoresis(4)andmethylation-sensitivearbitrarilyprimedPCR(5).However,themostcommontechniqueusedtodayremainsthebisulfiteconversionmethod(6).ThistechniqueinvolvestreatingmethylatedDNAwithbisulf