




已阅读5页,还剩31页未读, 继续免费阅读
版权说明:本文档由用户提供并上传,收益归属内容提供方,若内容存在侵权,请进行举报或认领
文档简介
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
温馨提示
- 1. 本站所有资源如无特殊说明,都需要本地电脑安装OFFICE2007和PDF阅读器。图纸软件为CAD,CAXA,PROE,UG,SolidWorks等.压缩文件请下载最新的WinRAR软件解压。
- 2. 本站的文档不包含任何第三方提供的附件图纸等,如果需要附件,请联系上传者。文件的所有权益归上传用户所有。
- 3. 本站RAR压缩包中若带图纸,网页内容里面会有图纸预览,若没有图纸预览就没有图纸。
- 4. 未经权益所有人同意不得将文件中的内容挪作商业或盈利用途。
- 5. 人人文库网仅提供信息存储空间,仅对用户上传内容的表现方式做保护处理,对用户上传分享的文档内容本身不做任何修改或编辑,并不能对任何下载内容负责。
- 6. 下载文件中如有侵权或不适当内容,请与我们联系,我们立即纠正。
- 7. 本站不保证下载资源的准确性、安全性和完整性, 同时也不承担用户因使用这些下载资源对自己和他人造成任何形式的伤害或损失。
最新文档
- 营销经理发言稿
- 时间控制描述与评价课件
- 班组管理安全培训
- 入场安全教育培训
- 大班颠倒世界课件
- IBM内部咨询培训
- 二零二五年度夫妻离婚协议中共同债务承担与信用修复协议
- 二零二五版电力设施智能化设计及报批合同
- 二零二五年度智能交通系统采购合同及数据共享协议
- 二零二五年度加油站客户关系管理与维护服务合同
- 电缆线路防外破施工方案
- 场地租赁协议场地租赁合同
- 五年级数学(小数乘除法)计算题专项练习及答案汇编
- Module1 Unit I want a hot dog,please(说课稿)-2023-2024学年外研版(三起)英语六年级下册
- DB23/T 3657-2023医养结合机构服务质量评价规范
- 报告审核制度及流程
- 《全球变化》课件
- 山东黄金笔试
- ps教学课件教学课件
- 《铁路轨道维护》课件-钢轨钻孔作业
- 造纸机械用铸铁烘缸定期检验要求
评论
0/150
提交评论