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    當前位置 ： Millipore >>> 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

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

    價格: ￥6228.00 市場價: 10380.00
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    貨號: S7810
    品牌: Millipore
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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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