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Millipore/S7810 | CpG WIZ® RB1 - Methylation specific PCR assay/S7810/25 assays
  • Millipore/S7810 | CpG WIZ® RB1 - Methylation specific PCR assay/S7810/25 assays

Millipore/S7810 | CpG WIZ® RB1 - Methylation specific PCR assay/S7810/25 assays

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货号: S7810
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    • Description
      CatalogueNumberS7810
      BrandFamilyChemicon®
      TradeName
      • CpGWiz
      • Chemicon
      DescriptionCpGWIZ®RB1-MethylationspecificPCRassay
      OverviewMSP,performedusingtheCpGenome™DNAModificationKitandtheCpGWIZ®RB1AmplificationKit,permitssensitivedetectionofalteredDNA.BecausethisisaPCR-basedassay,itisextremelysensitive,facilitatingthedetectionoflownumbersofmethylatedallelesandthestudyofsamplescontainingsmallamountsofDNA.MSPalsoallowsexaminationofallCpGsites,notjustthosewithinsequencesrecognizedbymethylationsensitiverestrictionenzymes.Increasingthenumberofsuchsiteswhichcanbeassessedallowsrapid,finemappingofmethylationpatternsthroughoutCpGregions.Inaddition,thebisulfitemodificationisideallysuitedforanalysisofCpGislandssinceitconvertsthemajorityofcytosinestouracils,makingaregionofthegenomewhichisCG-richlessdifficulttoamplifybyPCR.



      Methylation-specificPCR(MSP)employsaninitialbisulfitereactiontomodifytheDNA,followedbya"hotstart"PCRamplificationwithspecificprimersdesignedtodistinguishmethylatedDNAfromunmethylatedDNA.AsshowninFigure1,inthebisulfitereaction,allunmethylatedcytosinesareconvertedtouracilswhile5-methylcytosinesremainunaltered.Thus,thesequenceofthetreatedDNAwilldifferiftheDNAisoriginallymethylatedvs.unmethylated.PrimerscontainedintheCpGWIZ®RB1AmplificationKitaredesignedtospecificallyamplifyeachofthesequencesbaseduponthesechemically-induceddifferences.IfthesampleDNAwasoriginallyunmethylated,aproductwillbegeneratedafterPCRusingtheUprimerset.Conversely,aproductwillbegeneratedusingtheMprimersetifthesamplewasoriginallymethylated.
      BackgroundInformationMethylationofcytosineslocated5"toguanosineisknowntohaveaprofoundeffectontheexpressionofseveraleukaryoticgenes(1).Innormalcells,methylationoccurspredominantlyinCG-poorregions,whileCG-richareas,calledCpGislands,remainunmethylated.AberrantmethylationofnormallyunmethylatedCpGislandshasbeendocumentedasarelativelyfrequenteventinimmortalizedandtransformedcells(2)andhasbeenassociatedwithtranscriptionalinactivationofdefinedtumorsuppressorgenesinhumancancers(3,4).Theretinoblastoma1(RB1)geneexhibitscharacteristichypermethylationimmanymaligninanttumors.

      PreviouslydevelopedmethodstodeterminethemethylationstatusofcytosineincludedigestionwithmethylationsensitiverestrictionenzymesandgenomicDNAsequencing.Bothtechniqueshavelimitations:restrictionenzymescanonlydetectmethylationsiteswithintheirrecognitionsequenceandsequencingistimeconsuming.IncreasingthedetectionsensitivityofCpGislandmethylationhasthepotentialtodefinetumorsuppressorgenefunctionandprovidesanewstrategyforearlytumordetection.

      Methylation-specificPCR(MSP)isanewtechnologyforsensitivedetectionofabnormalgenemethylationutilizingsmallamountsofDNA(5).ThisprocessemploysaninitialbisulfitereactiontomodifytheDNA,followedbyPCRamplificationwithspecificprimersdesignedtodistinguishmethylatedfromunmethylatedDNA.TheCpGenome™DNAModificationKit(S7820)containsthereagentsnecessarytoperformtheinitialbisulfitereactions,whiletheCpGWIZ®RB1AmplificationKitcontainsthereagentsrequiredforthePCRamplificationreactions.
      MaterialsRequiredbutNotDeliveredEquipmentandSupplies

      a.MicrocentrifugetubesforPCRamplification

      b.Aerosol-resistantPipettetips

      c.Thermocycler

      d.Gelelectrophoresisapparatus(verticalorhorizontal)

      e.PowerSupply

      f.302nmUVtransIlluminator,cameraandfilm

      Reagents

      a2.5mMdNTPmix(2.5mMofeachnucleotide)

      b."Hotstart"Taqpolymerase

      c."Hotstart"PCRreagents(seeSec.II.Protocols).

      d.Reagentsforgelelectrophoresis(1XTBEand2%agarose,10%acrylamide,orsuitablehighresolutionagarose)

      e.DNAMarkers(sizerange100-300bp)

      f.Ethidiumbromide(10mg/mL)

      g.Gel-loADIngsolution/LoadingDye

      h.BisulfiteModifiedDNA(CpGenome™DNAModificationKit,S7820)
      ProductInformation
      Components
      • ThecomponentsoftheCpGWIZ®RB1AmplificationKitincludethoserequiredforPCRamplificationafterbisulfitemodificationofDNAsamples.Sufficientreagentsareprovidedtoanalyze25sampleswithappropriatecontrols.
      • UPrimerSet7.5μMeachprimer(25X)35μL(neutralcap)90552-15°Cto-25°C
      • MPrimerSet7.5μMeachprimer(25X)35μL(redcap)90553-15°Cto-25°C
      • WPrimerSet7.5μMeachprimer(25X)35μL(greencap)90554-15°Cto-25°C
      • UcontrolDNA0.1μg/μL50μL(whitecap)90393-15°Cto-25°C
      • McontrolDNA0.1μg/μL50μL(redcap)90394-15°Cto-25°C
      • WcontrolDNA0.05μg/μL50μL(greencap)90395-15°Cto-25°C
      • Universal10XPCRBuffer265μL(bluecap)90396-15°Cto-25°C
      StorageandShippingInformation
      Applications
      ApplicationMSP,performedusingtheCpGenomeDNAModificationKit&theCpGWIZRB1AmplificationKit,permitssensitivedetectionofalteredDNA.
      KeyApplications
      • PCR
      BIOLOGicalInformation
      SpeciesReactivity
      • Human
      EntrezGeneNumber
      EntrezGeneSummaryRetinoblastoma(RB)isanembryonicmalignantneoplasmofretinalorigin.Italmostalwayspresentsinearlychildhoodandisoftenbilateral.Spontaneousregression("cure")occursinsomecases.TheretinoblastomageneRBwasthefirsttumorsuppressorgenecloned,andisanegativeregulatorofthecellcyclethroughitsABIlitytobindthetranscriptionfactorE2F(MIM189971)andrepresstranscriptionofgenesrequiredforSphase(HanahanandWeinberg,2000[PubMed10647931]).[suppliedbyOMIM]
      GeneSymbol
      • RB1
      • P105-RB
      • retinoblastoma-1
      • RB
      • PP110
      • OSRC
      UniProtNumber
      UniProtSummaryFUNCTION:SwissProt:P06400#Keyregulatorofentryintocelldivisionthatactsasatumorsuppressor.Directlyinvolvedinheterochromatinformationbymaintainingoverallchromatinstructureand,inparticular,thatofconstitutiveheterochromatinbystabilizinghistonemethylation.RecruitsandtargetshistonemethyltransferasesSUV39H1,SUV420H1andSUV420H2,leadingtoepigenetictranscriptionalrepression.ControlshistoneH4"Lys-20"trimethylation.AlsoactsasatranscriptionrepressorofE2Ftargetgenesbyrecruitingchromatin-modifyingenzymestopromoters.InhibitstheintrinsickinaseactivityofTAF1.FormsacomplexwithadenovirusE1AandwithSV40largeTantigen.MaybindandmodulatefunctionallycertaincellularproteinswithwhichTandE1Acompeteforpocketbinding.
      SIZE:928aminoacids;106159Da
      SUBUNIT:InteractspreferentiallywithtranscriptionfactorE2F1.TheunphosphorylatedforminteractswithARID3B,JARID1A,SUV39H1,MJD2A/JHDM3AandTHOC1.InteractswiththeN-terminaldomainofTAF1.InteractswithAATF,DNMT1,LIN9,LMNA,SUV420H1,SUV420H2,PELP1andTMPO-alpha.MayinteractwithKNTC2.InteractswithEID1andZUBR1.InteractswithARID4AandJARID1B.
      SUBCELLULARLOCATION:Nucleus.
      TISSUESPECIFICITY:Expressedintheretina.
      PTM:PhosphorylatedfromStoMphaseofthecellcycleandisdephosphorylatedinG1.T,butnotE1A,bindsonlytotheunphosphorylatedform.
      DISEASE:SwissProt:P06400#DefectsinRB1arethecauseofchildhoodcancerretinoblastoma(RB)[MIM:180200].RBisacongenitalmalignanttumorthatarisesfromthenuclearlayersoftheretina.Itoccursinabout1:20"000livebirthsandrepresentsabout2%ofchildhoodmalignancies.Itisbilateralinabout30%ofcases.AlthoughmostRBappearsporadically,about20%aretransmittedasanautosomaldominanttraitwithincompletepenetrance.Thediagnosisisusuallymadebeforetheageof2yearswhenstrabismusoragraytoyellowreflexfrompupil(cateye)isinvestigated.&DefectsinRB1areacauseofbladdercancer[MIM:109800].&DefectsinRB1areacauseofosteogenicsarcoma[MIM:259500].
      SIMILARITY:SwissProt:P06400##Belongstotheretinoblastomaprotein(RB)family.
      PhysicochemicalInformation
      Dimensions
      MaterialsInformation
      MaterialsInformation
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