分子診斷原理與技術(shù)

DNAmethylationConceptDetectionApplication第1頁DNAmethylationinvolvestheadditionofamethylgrouptothe5positionofcytosine,whichoccursinthecontextofCpG(cytosinefollowedbyguanine)dinucleotides.Thismodificationcanbeinheritedthroughcelldivision.第2頁第3頁CpGsitesareregionsofDNAwhereacytosine

nucleotideoccursnexttoaguaninenucleotideinthelinearsequenceofbasesalongitslength.CpGisshorthandfor"—C—phosphate—G—",thatis,cytosineandguanineseparatedbyaphosphate,whichlinksthetwonucleosidestogetherinDNA.CpGnotationisusedtodistinguishthislinearsequencefromthebase-pairingofcytosineandguanine.

ThefrequencyofCpG

dinucleotidesinhumangenomesis1%.第4頁ThereareregionsoftheDNAthathaveahigherconcentrationofCpGsites,knownasCpGislands.ManygenesinmammaliangenomeshaveCpGislandsassociatedwiththestartofthegene.Becauseofthis,thepresenceofaCpGislandisusedtohelpinthepredictionandannotationofgenes.第5頁CpGIslands(CGI)searchOnlineResource/http://www.ebi.ac.uk/Tools/emboss/cpgplot/index.html第6頁Methods1Non-methylation-specificPCRbasedmethods

DirectsequencingPyrosequencingMethylation-sensitivesingle-strandconformationanalysis(MS-SSCA)Highresolutionmeltinganalysis(HRM)Methylation-sensitivesinglenucleotideprimerextension(MS-SnuPE)Base-specificcleavage/MALDI-TOF

2Methylation-specificPCR(MSP)

3Microarray-basedmethods第7頁TreatmentofDNAwithbisulfite*convertscytosineresiduestouracil,butleaves5-methylcytosineresiduesunaffected.Thus,bisulfitetreatmentintroducesspecificchangesintheDNAsequencethatdependonthemethylationstatusofindividualcytosineresidues,yieldingsingle-nucleotideresolutioninformationaboutthemethylationstatusofasegmentofDNA.*亞硫酸氫鹽第8頁第9頁第10頁DirectsequencingThefirstreportedmethodofmethylationanalysisusingbisulfite-treatedDNAutilizedPCRandstandarddideoxynucleotideDNAsequencingtodirectlydeterminethenucleotidesresistanttobisulfiteconversion.Primersaredesignedtobestrand-specificaswellasbisulfite-specific(i.e.,primerscontainingnon-CpGcytosinessuchthattheyarenotcomplementarytonon-bisulfite-treatedDNA),flanking(butnotinvolving)themethylationsiteofinterest.第11頁ThistechniquerequiredcloningofthePCRproductpriortosequencingforadequatesensitivity,andthereforewasaverylabour-intensivemethodunsuitableforhigherthroughput.DirectSequencing第12頁PyrosequencingFollowingPCRamplificationoftheregionofinterest,Pyrosequencingisusedtodeterminethebisulfite-convertedsequenceofspecificCpGsitesintheregion.TheratioofC-to-TatindividualsitescanbedeterminedquantitativelybasedontheamountofCandTincorporationduringthesequenceextension.Themainlimitationofthismethodisthecostofthetechnology.However,Pyrosequencingdoeswellallowforextensiontohigh-throughputscreeningmethods.第13頁/DynPage.aspx?id=7454SEEFLASH第14頁Methylation-sensitivesingle-strandconformationanalysis(MS-SSCA)Thismethodisbasedonthesinglestrandconformationpolymorphismanalysis(SSCA)methoddevelopedforsingle-nucleotidepolymorphism(SNP)analysis.ThismethodisideallydesignedtoassessallCpGsitesasawholeintheregionofinterestratherthanindividualmethylationsites.第15頁Highresolutionmeltinganalysis(HRM)Afurthermethodtodifferentiateconvertedfromunconvertedbisulfite-treatedDNAisusinghighresolutionmeltinganalysis(HRM),areal-timePCR-basedtechniqueinitiallydesignedtodistinguishSNPs.TheMS-HRMassayforBNIP3methylation.ResultsoftheBNIP3-MS-HRMassayforfiveclinicalsamplescomparedtothedilutionstandards.Thismethodallowsdirectquantitationinasingle-tubeassay,butagainassessesmethylationintheamplifiedregionasawholeratherthanatspecificCpGsites.第16頁Methylation-sensitivesinglenucleotideprimerextension(MS-SnuPE)第17頁Analysisofmethylationbybase-specificcleavageandMALDI-TOFMSEhrichMetal.PNAS2023;102:15785-15790?2023byNationalAcademyofSciences第18頁Methylation-specificPCR(MSP)Methylation-specificPCRisasensitivemethodtodiscriminatelyamplifyanddetectamethylatedregionofinterestusingmethylated-specificprimersonbisulfite-convertedgenomicDNA.Suchprimerswillonlyannealtosequencesthataremethylated,andthuscontaining5-methylcytosinesthatareresistanttoconversionbybisulfite.Alternatively,unmethylated-specificprimerscanbeused.第19頁Microarray-basedmethodsMicroarray-basedmethodsarealogicalextensionofthetechnologiesavailabletoanalyzebisulfite-treatedDNAtoallowforgenome-wideanalysisofmethylation.第20頁NucleicAcidsResearch202334(11):e82第21頁Copyrightrestrictionsmayapply.Gebhard,C.etal.Nucl.AcidsRes.202334:e82;doi:10.1093/nar/gkl437OutlineofMB-PCR第22頁Copyrightrestrictionsmayapply.Gebhard,C.etal.Nucl.AcidsRes.202334:e82;doi:10.1093/nar/gkl437MB-PCRdetectsmethylationofCpGislandpromoters第23頁Copyrightrestrictionsmayapply.Gebhard,C.etal.Nucl.AcidsRes.202334:e82;doi:10.1093/nar/gkl437DetectingCpGmethylationinleukemiacelllinesbyMB-PCR第24頁Copyrightrestrictionsmayapply.Gebhard,C.etal.Nucl.AcidsRes.202334:e82;doi:10.1093/nar/gkl437MethylationoftheICSBPpromoterinverselycorrelateswithICSBPexpressioninleukemiacelllines第25頁Copyrightrestrictionsmayapply.Gebhard,C.etal.Nucl.AcidsRes.202334:e82;doi:10.1093/nar/gkl437SensitivityofMB-PCR第26頁Copyrightrestrictionsmayapply.Gebhard,C.etal.Nucl.AcidsRes.202334:e82;doi:10.1093/nar/gkl437DetectionofaberrantCpGmethylationinprimaryAMLblasts第27頁FragileXsyndrome脆性X綜合征LocationofFMR1geneintellectualdisabilityelongatedfacelargeearsflatfeetlargertesteslowmuscletoneclutteredspeechnervousspeech第28頁FMR1(fragileXmentalretardation1)isahuman

genethatcodesforaproteincalledfragileXmentalretardationprotein,orFMRP.Thisproteinisnormallymadeinmanytissues,especiallyinthebrainandtestes.Itmayplayaroleinthedevelopmentofsynapticconnectionsbetweennervecellsinthebrain,wherecell-to-cellcommunicationoccurs.Theconnectionsbetweennervecellscanchangeandadaptovertimeinresponsetoexperience(acharacteristiccalledsynapticplasticity).FMRPmayhelpregulatesynapticplasticity,whichisimportantforlearningandmemory.

FMR1hasbeenshowntointeractwithFXR2,CYFIP1,CYFIP2,NUFIP1,FXR1andNUFIP2.第29頁第30頁第31頁ExpressionoftheFMR1GeneandAssociatedClinicalDisorders第32頁ThefragileXregionwithnormal,premutation,andfullmutationCGGrepeats.TheCGGrepea