Methylation-Specific PCR - 百度文库

来自 : 发布时间：2025-01-15

91From:MethodsinMolecularBiology,Vol.192:PCRCloningProtocols,2ndEditionEditedby:B.-Y.ChenandH.W.Janes©HumanaPressInc.,Totowa,NJ10Methylation-SpecificPCRHaruhikoOhashi1.Introduction1.1.SignificanceofDNAMethylationasanEpigeneticPhenomenonMethylationoftheDNAisanimportantepigenetic(i.e.,notassociatedwithalter-ationintheprimarystructureoftheDNA)phenomenon,whichplaysimportantrolesinregulationofgeneexpression,maintenanceofgenomeintegrity,andgenomicimprinting.Althoughnotonlyothernucleotides,butalsoproteinsandlipidscanbemethylated,inthecontextofthepresentdiscussion,\"methylation”designatesonlythatofcytosineresiduesthatarelocated5 toguanines(CpGcytosines).Amethylresidueisaddedtothe5positionofthepyrimidineringofcytosine(5-methylcytosine)inthecourseofDNAreplication,aprocessmediatedbyDNA(cytosine-5)-methyltransferases.1.2.OtherMethodsforDNAMethylationAnalysisInvestigationonDNAmethylationhadlongbeenperformedbySouthernhybridizationwithmethylation-sensitiveendonucleases.Someendonuleasesdigestdouble-strandDNAattheircognitivesequencesonlywhencytosineswithinarefreeofmethylation,whereasothersdoso,regardlessofthepresenceorabsenceof5-methylcytosines.GenomicDNAisdigestedbyamethylation-sensitiveendonucleaseandelectrophoresedonagarosegelandprobedwithspecificDNAprobe.Ifthecyto-sinesintheendonucleaserecognitionsiteismethylated,theDNAisleftuncleavedatthatsite,thusappearsband(s)oflarger-than-expectedsize(s).ThemainproblemswithSouthernblottingwithmethylation-sensitiveendonu-cleasesisthatonlythecytosinesinthecontextofavailablemethylation-sensitiveendo-nucleases[i.e.,HpaII(CCGG),HhaI(GCGC)]couldbeexamined;also,relativelylargeamountofDNA(5–10μg)isrequiredforeachanalysis.Thelatterproblemcouldbecircumventedbytheuseofpolymerasechainreaction(PCR).SincecytosinemethylationshouldnotbeconservedafterPCR(5-methylcytosinesshouldbecon-vertedtocytosines),digestionbymethylation-sensitiveendonucleasesshouldbeper-