分子诊断原理与技术.ppt

DNAmethylation ConceptDetectionApplication DNAmethylationinvolvestheadditionofamethylgrouptothe5positionofcytosine whichoccursinthecontextofCpG cytosinefollowedbyguanine dinucleotides Thismodificationcanbeinheritedthroughcelldivision CpGsitesareregionsofDNAwhereacytosinenucleotideoccursnexttoaguaninenucleotideinthelinearsequenceofbasesalongitslength CpGisshorthandfor C phosphate G thatis cytosineandguanineseparatedbyaphosphate whichlinksthetwonucleosidestogetherinDNA CpGnotationisusedtodistinguishthislinearsequencefromthebase pairingofcytosineandguanine ThefrequencyofCpGdinucleotidesinhumangenomesis1 ThereareregionsoftheDNAthathaveahigherconcentrationofCpGsites knownasCpGislands ManygenesinmammaliangenomeshaveCpGislandsassociatedwiththestartofthegene Becauseofthis thepresenceofaCpGislandisusedtohelpinthepredictionandannotationofgenes CpGIslands CGI searchOnlineResource http cpgislands usc edu 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 andfullmutationCGGrepeats TheCGGrepeatregionisrepresentedbyajaggedline RestrictionsitesareindicatedbysolidarrowsforEcoRIandadashedarrowformethylationsensitiveEagI FullmutationallelesaretypicallymethylatedandareresistanttodigestionattheEagIsite RestrictionmapofthefragileXregion ProbesusedinSouthernanalysisoftheregionareshown FragileXSouthernanalysisofgenomicDNAdigestedwithEcoRIandEagI andhybridizedwithprobeStB12 3 Lane1 fullmutationmalewithmethylationmosaicism Lane2 premutationmale Lane3 premutationfemale Lane4 fullmutationmale Lane5 fullmutationfemale Lane6 Fullmutationmale Lanes7to9 normalfemales Lane10 fullmutationmale DNAsizemarkersareindicatedontheright