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

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貨號: S7810

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Description
CatalogueNumber	S7810
BrandFamily	Chemicon®
TradeName	CpGWiz Chemicon
Description	CpGWIZ?RB1-MethylationspecificPCRassay
Overview	MSP,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.
BackgroundInformation	Methylationofcytosineslocated5"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.
MaterialsRequiredbutNotDelivered	EquipmentandSupplies 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

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
Application	MSP,performedusingtheCpGenomeDNAModificationKit&theCpGWIZRB1AmplificationKit,permitssensitivedetectionofalteredDNA.
KeyApplications	PCR

BIOLOGicalInformation
SpeciesReactivity	Human
EntrezGeneNumber	NM_000321.2
EntrezGeneSummary	Retinoblastoma(RB)isanembryonicmalignantneoplasmofretinalorigin.Italmostalwayspresentsinearlychildhoodandisoftenbilateral.Spontaneousregression("cure")occursinsomecases.TheretinoblastomageneRBwasthefirsttumorsuppressorgenecloned,andisanegativeregulatorofthecellcyclethroughitsABIlitytobindthetranscriptionfactorE2F(MIM189971)andrepresstranscriptionofgenesrequiredforSphase(HanahanandWeinberg,2000[PubMed10647931]).[suppliedbyOMIM]
GeneSymbol	RB1 P105-RB retinoblastoma-1 RB PP110 OSRC
UniProtNumber	P06400
UniProtSummary	FUNCTION: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.

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