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DNAmethylation,ConceptDetectionApplication,DNAmethylationinvolvestheadditionofamethylgrouptothe5positionofcytosine,whichoccursinthecontextofCpG(cytosinefollowedbyguanine)dinucleotides.Thismodificationcanbeinheritedthroughcelldivision.,CpGsitesareregionsofDNAwhereacytosinenucleotideoccursnexttoaguaninenucleotideinthelinearsequenceofbasesalongitslength.CpGisshorthandforCphosphateG,thatis,cytosineandguanineseparatedbyaphosphate,whichlinksthetwonucleosidestogetherinDNA.CpGnotationisusedtodistinguishthislinearsequencefromthebase-pairingofcytosineandguanine.ThefrequencyofCpGdinucleotidesinhumangenomesis1%.,ThereareregionsoftheDNAthathaveahigherconcentrationofCpGsites,knownasCpGislands.ManygenesinmammaliangenomeshaveCpGislandsassociatedwiththestartofthegene.Becauseofthis,thepresenceofaCpGislandisusedtohelpinthepredictionandannotationofgenes.,CpGIslands(CGI)searchOnlineResource,/http:/www.ebi.ac.uk/Tools/emboss/cpgplot/index.html,Methods1Non-methylation-specificPCRbasedmethodsDirectsequencingPyrosequencingMethylation-sensitivesingle-strandconformationanalysis(MS-SSCA)Highresolutionmeltinganalysis(HRM)Methylation-sensitivesinglenucleotideprimerextension(MS-SnuPE)Base-specificcleavage/MALDI-TOF2Methylation-specificPCR(MSP)3Microarray-basedmethods,TreatmentofDNAwithbisulfite*convertscytosineresiduestouracil,butleaves5-methylcytosineresiduesunaffected.Thus,bisulfitetreatmentintroducesspecificchangesintheDNAsequencethatdependonthemethylationstatusofindividualcytosineresidues,yieldingsingle-nucleotideresolutioninformationaboutthemethylationstatusofasegmentofDNA.,*亚硫酸氢盐,Directsequencing,Thefirstreportedmethodofmethylationanalysisusingbisulfite-treatedDNAutilizedPCRandstandarddideoxynucleotideDNAsequencingtodirectlydeterminethenucleotidesresistanttobisulfiteconversion.Primersaredesignedtobestrand-specificaswellasbisulfite-specific(i.e.,primerscontainingnon-CpGcytosinessuchthattheyarenotcomplementarytonon-bisulfite-treatedDNA),flanking(butnotinvolving)themethylationsiteofinterest.,ThistechniquerequiredcloningofthePCRproductpriortosequencingforadequatesensitivity,andthereforewasaverylabour-intensivemethodunsuitableforhigherthroughput.,DirectSequencing,PyrosequencingFollowingPCRamplificationoftheregionofinterest,Pyrosequencingisusedtodeterminethebisulfite-convertedsequenceofspecificCpGsitesintheregion.TheratioofC-to-TatindividualsitescanbedeterminedquantitativelybasedontheamountofCandTincorporationduringthesequenceextension.Themainlimitationofthismethodisthecostofthetechnology.However,Pyrosequencingdoeswellallowforextensiontohigh-throughputscreeningmethods.,Methylation-sensitivesingle-strandconformationanalysis(MS-SSCA)Thismethodisbasedonthesinglestrandconformationpolymorphismanalysis(SSCA)methoddevelopedforsingle-nucleotidepolymorphism(SNP)analysis.,ThismethodisideallydesignedtoassessallCpGsitesasawholeintheregionofinterestratherthanindividualmethylationsites.,Highresolutionmeltinganalysis(HRM)Afurthermethodtodifferentiateconvertedfromunconvertedbisulfite-treatedDNAisusinghighresolutionmeltinganalysis(HRM),areal-timePCR-basedtechniqueinitiallydesignedtodistinguishSNPs.,TheMS-HRMassayforBNIP3methylation.ResultsoftheBNIP3-MS-HRMassayforfiveclinicalsamplescomparedtothedilutionstandards.,Thismethodallowsdirectquantitationinasingle-tubeassay,butagainassessesmethylationintheamplifiedregionasawholeratherthanatspecificCpGsites.,Methylation-sensitivesinglenucleotideprimerextension(MS-SnuPE),Analysisofmethylationbybase-specificcleavageandMALDI-TOFMS,EhrichMetal.PNAS2005;102:15785-15790,2005byNationalAcademyofSciences,Methylation-specificPCR(MSP),Methylation-specificPCRisasensitivemethodtodiscriminatelyamplifyanddetectamethylatedregionofinterestusingmethylated-specificprimersonbisulfite-convertedgenomicDNA.Suchprimerswillonlyannealtosequencesthataremethylated,andthuscontaining5-methylcytosinesthatareresistanttoconversionbybisulfite.Alternatively,unmethylated-specificprimerscanbeused.,Microarray-basedmethodsMicroarray-basedmethodsarealogicalextensionofthetechnologiesavailabletoanalyzebisulfite-treatedDNAtoallowforgenome-wideanalysisofmethylation.,NucleicAcidsResearch200634(11):e82,Copyrightrestrictionsmayapply.,Gebhard,C.etal.Nucl.AcidsRes.200634:e82;doi:10.1093/nar/gkl437,OutlineofMB-PCR,Copyrightrestrictionsmayapply.,Gebhard,C.etal.Nucl.AcidsRes.200634:e82;doi:10.1093/nar/gkl437,MB-PCRdetectsmethylationofCpGislandpromoters,Copyrightrestrictionsmayapply.,Gebhard,C.etal.Nucl.AcidsRes.200634:e82;doi:10.1093/nar/gkl437,DetectingCpGmethylationinleukemiacelllinesbyMB-PCR,Copyrightrestrictionsmayapply.,Gebhard,C.etal.Nucl.AcidsRes.200634:e82;doi:10.1093/nar/gkl437,MethylationoftheICSBPpromoterinverselycorrelateswithICSBPexpressioninleukemiacelllines,Copyrightrestrictionsmayapply.,Gebhard,C.etal.Nucl.AcidsRes.200634:e82;doi:10.1093/nar/gkl437,SensitivityofMB-PCR,Copyrightrestrictionsmayapply.,Gebhard,C.etal.Nucl.AcidsRes.200634:e82;doi:10.1093/nar/gkl437,DetectionofaberrantCpGmethylationinprimaryAMLblasts,FragileXsyndrome脆性X综合征,LocationofFMR1gene,intellectualdisabilityelongatedfacelargeearsflatfeetlargertesteslowmuscletoneclutteredspeechnervousspeech,FMR1(fragileXmentalretardation1)isahumangenethatcodesforaproteincalledfragileXmentalretardationprotein,orFMRP.Thisproteinisnormallymadeinmanytissues,especiallyinthebrainandtestes.Itmayplayaroleinthedevelopmentofsynapticconnectionsbetweennervecellsinthebrain,wherecell-to-cellcommunicationoccurs.Theconnectionsbetweennervecellscanchangeandadaptovertimeinresponsetoexperience(acharacteristiccalledsynapticplasticity).FMRPmayhelpregulatesynapticplasticity,whichisimportantforlearningandmemory.,FMR1hasbeenshowntointeractwithFXR2,CYFIP1,CYFIP2,NUFIP1,FXR1andNUFIP2.,ExpressionoftheFMR1GeneandAssociatedClinicalDisorders,ThefragileXregionwithnormal,premutation,andfullmutationCGGrepea

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