分子診斷學(xué)概論課件

分子診斷學(xué)概論第一章綜說overview疾病發(fā)生原因的影響層次DNA、RNA或蛋白質(zhì)分子診斷的目的偵測這些致病因子是那個層次發(fā)生變化本書著重DNA、RNA的變化蛋白質(zhì)層次由原文書章節(jié)提供TheApplicationofProteomicsToDiseaseDiagnostics分子診斷學(xué)概論第一章綜說overview1分子診斷學(xué)概論課件2遺傳分子的基礎(chǔ)生物巨分子：DNA、RNA、蛋白質(zhì)、糖類、脂質(zhì)遺傳物質(zhì)DNA的發(fā)現(xiàn)1928格里夫茲(Griffith)肺炎雙球菌轉(zhuǎn)形試驗1942艾佛瑞(Avery)研究格里夫茲轉(zhuǎn)形的物質(zhì)為何?1952赫希-卻斯(Hershey-Chase)以放射線標(biāo)示噬菌體的蛋白質(zhì)（S35）和DNA（P32），感染大腸桿菌的實驗1953雙螺旋結(jié)構(gòu)的發(fā)現(xiàn)2003人類基因體計畫的完成遺傳分子的基礎(chǔ)3參考資料：/~cmallery/150/gene/sf11x1b.jpg參考資料：/4參考資料：/~cmallery/150/gene/sf11x1b.jpg參考資料：/5分子診斷學(xué)概論課件6參考資料：http://.tw/advance/a021.asp

參考資料：.t7分子診斷學(xué)概論課件8分子診斷學(xué)概論課件9分子診斷學(xué)概論課件10Presentedhereisagenomesequenceofanindividualhuman.Itwasproducedfrom~32millionrandomDNAfragments,sequencedbySangerdideoxytechnologyandassembledinto4,528scaffolds,comprising2,810millionbases(Mb)ofcontiguoussequencewithapproximately7.5-foldcoverageforanygivenregion.WedevelopedamodifiedversionoftheCeleraassemblertofacilitatetheidentificationandcomparisonofalternatealleleswithinthisindividualdiploidgenome.ComparisonofthisgenomeandtheNationalCenterforBiotechnologyInformationhumanreferenceassemblyrevealedPresentedhereisagenomeseq11morethan4.1millionDNAvariants,encompassing12.3Mb.Thesevariants(ofwhich1,288,319werenovel)included3,213,401singlenucleotidepolymorphisms(SNPs),

53,823blocksubstitutions(2–206bp),292,102heterozygousinsertion/deletionevents(indels)(1–571bp),559,473homozygousindels(1–82,711bp),90inversions,aswellasnumeroussegmentalduplicationsandcopynumbervariationregions.Non-SNPDNAvariationaccountsfor22%ofalleventsidentifiedinthedonor,howevertheyinvolve74%ofallvariantbases.Thissuggestsanimportantrolefornon-SNPgeneticalterationsindefiningthediploidgenomestructure.Moreover,44%ofgeneswereheterozygousforoneormorevariants.Usinganovelhaplotypeassemblystrategy,wewereabletospan1.5Gbofgenomesequenceinsegments.200kb,providingfurtherprecisiontothediploidnatureofthegenome.Thesedatadepictadefinitivemolecularportraitofadiploidhumangenomethatprovidesastartingpointforfuturegenomecomparisonsandenablesaneraofindividualizedgenomicinformation.morethan4.1millionDNAvari12AuthorSummaryWehavegeneratedanindependentlyassembleddiploidhumangenomicDNAsequencefrombothchromosomesofasingleindividual(J.CraigVenter).Ourapproach,basedonwhole-genomeshotgunsequencingandusingenhancedgenomeassemblystrategiesandsoftware,generatedanassembledgenomeoverhalfofwhichisrepresentedinlargediploidsegments(.200kilobases),enablingstudyofthediploidgenome.Comparisonwithpreviousreferencehumangenomesequences,whichwerecompositescomprisingmultiplehumans,revealedthatthemajorityofgenomicalterationsarethewell-studiedclassofvariantsbasedonsinglenucleotides(SNPs).However,theresultsalsorevealthatlesserstudiedgenomicvariants,insertionsanddeletions,whilecomprisingaminority(22%)ofgenomicvariationevents,actuallyaccountforalmost74%ofvariantnucleotides.Inclusionofinsertionanddeletiongeneticvariationintoourestimatesofinterchromosomaldifferencerevealsthatonly99.5%similarityexistsbetweenthetwochromosomalcopiesofanindividualandthatgeneticvariationbetweentwoindividualsisasmuchasfivetimeshigherthanpreviouslyestimated.Theexistenceofawell-characterizeddiploidhumangenomesequenceprovidesastartingpointforfutureindividualgenomecomparisonsandenablestheemergingeraofindividualizedgenomicinformation.AuthorSummary13Identificationandanalysisoffunctionalelementsin1%ofthehumangenomebytheENCODEpilotproject.

Nature.2007Jun14;447(7146):799-816

TheEncyclopediaofDNAElements(ENCODE)Projectprovideamorebiologicallyinformativerepresentationofthehumangenomebyusinghigh-throughputmethodstoidentifyandcataloguethefunctionalelementsencoded.Identificationandanalysisof14First,ourstudiesprovideconvincingevidencethatthegenomeispervasivelytranscribed,suchthatthemajorityofitsbasescanbefoundinprimarytranscripts,includingnon-protein-codingtranscripts,andthosethatextensivelyoverlaponeanother.Second,systematicexaminationoftranscriptionalregulationhasyieldednewunderstandingabouttranscriptionstartsites,includingtheirrelationshiptospecificregulatorysequencesandfeaturesofchromatinaccessibilityandhistonemodification.Third,amoresophisticatedviewofchromatinstructurehasemerged,includingitsinter-relationshipwithDNAreplicationandtranscriptionalregulation.Finally,integrationofthesenewsourcesofinformation,inparticularwithrespecttomammalianevolutionbasedoninter-andintra-speciessequencecomparisons,hasyieldednewmechanisticandevolutionaryinsightsconcerningthefunctionallandscapeofthehumangenome.Together,thesestudiesaredefiningapathforpursuitofamorecomprehensivecharacterizationofhumangenomefunction.First,ourstudiesprovidecon15ThehighlightsofourfindingstodateincludeThehumangenomeispervasivelytranscribed,suchthatthemajorityofitsbasesareassociatedwithatleastoneprimarytranscriptandmanytranscriptslinkdistalregionstoestablishedprotein-codingloci.Many

novelnon-protein-codingtranscriptshavebeenidentified,withmanyoftheseoverlappingprotein-codinglociandotherslocatedinregionsofthegenomepreviouslythoughttobetranscriptionallysilent.Numerouspreviouslyunrecognizedtranscriptionstartsiteshavebeenidentified,manyofwhichshowchromatinstructureandsequence-specificprotein-bindingpropertiessimilartowell-understoodpromoters.Regulatorysequencesthatsurroundtranscriptionstartsitesaresymmetricallydistributed,withnobiastowardsupstreamregions.Thehighlightsofourfindings16Chromatinaccessibilityandhistonemodificationpatternsarehighlypredictiveofboththepresenceandactivityoftranscriptionstartsites.DistalDNaseIhypersensitivesiteshavecharacteristichistonemodificationpatternsthatreliablydistinguishthemfrompromoters;someofthesedistalsitesshowmarksconsistentwithinsulatorfunction.DNA

replication

timingiscorrelatedwithchromatinstructure.Atotalof5%ofthebasesinthegenomecanbeconfidentlyidentifiedasbeingunderevolutionaryconstraintinmammals;forapproximately60%oftheseconstrainedbases,thereisevidenceoffunctiononthebasisoftheresultsoftheexperimentalassaysperformedtodate.Chromatinaccessibilityandhi17Althoughthereisgeneraloverlapbetweengenomicregionsidentifiedasfunctionalbyexperimentalassaysandthoseunderevolutionaryconstraint,notallbaseswithintheseexperimentallydefinedregionsshowevidenceofconstraint.Differentfunctionalelementsvarygreatlyintheirsequencevariabilityacrossthehumanpopulationandintheirlikelihoodofresidingwithinastructurallyvariableregionofthegenome.Surprisingly,manyfunctionalelementsareseeminglyunconstrainedacrossmammalianevolution.Thissuggeststhepossibilityofalargepoolofneutralelementsthatarebiochemicallyactivebutprovidenospecificbenefittotheorganism.Thispoolmayserveasa'warehouse'fornaturalselection,potentiallyactingasthesourceoflineage-specificelementsandfunctionallyconservedbutnon-orthologouselementsbetweenspeciesAlthoughthereisgeneralover18遺傳物質(zhì)：核酸(nucleicacid)核酸：DNA（去氧核醣核酸）、RNA（核醣核酸）核酸基本單位：核苷酸(nucleotide)核苷酸：鹼基(base)、五碳醣(pentosesugar)、磷酸(phosphate)DNA的組成(王文姿等，2003)H遺傳物質(zhì)：核酸(nucleicacid)DNA的組成(王文19HDNA去氧核糖核酸RNA核糖核酸HDNARNA20鹼基鹼基21分子診斷學(xué)概論課件22分子診斷學(xué)概論課件23DNA的結(jié)構(gòu)資料來源：/biology主溝majorgroove小溝minorgrooveDNA的結(jié)構(gòu)資料來源：http://academic.bro24三個氫鍵二個氫鍵二個氫鍵三個氫鍵DNA的結(jié)構(gòu)資料來源：/biology

穩(wěn)定力量來自氫鍵非共價鍵-堆積力量：凡得瓦力、斥水性作用力、親水性作用力糖骨架：磷酸雙酯鍵三個氫鍵二個氫鍵二個氫鍵三個氫鍵DNA的結(jié)構(gòu)資料來源：htt25環(huán)境與序列的影響會形成不同形式結(jié)構(gòu)的DNA較寬與緊密外表成鋸齒狀環(huán)境與序列的影響會形成不同形式結(jié)構(gòu)的DNA較寬與緊密外表成鋸26http://nucleix.mbu.iisc.ernet.in/image/abzDNA.JPGhttp://nucleix.mbu.iisc.ernet.27基因的一般結(jié)構(gòu)基因定義：染色體上一段有功能的特定序列，可轉(zhuǎn)錄成RNA分子或是轉(zhuǎn)譯成多勝肽鏈定義可能會更改：調(diào)控性功能的序列？基因的一般結(jié)構(gòu)基因定義：染色體上一段有功能的特定序列，可轉(zhuǎn)錄28基因的結(jié)構(gòu)表現(xiàn)子（exon外顯子）-可轉(zhuǎn)錄或轉(zhuǎn)譯出產(chǎn)物，稱為編碼區(qū)轉(zhuǎn)錄調(diào)控區(qū)啟動子promoter：其特殊序列與轉(zhuǎn)錄因子結(jié)合，引導(dǎo)RNA聚合酶，產(chǎn)生基因轉(zhuǎn)錄轉(zhuǎn)錄起始點定為+1轉(zhuǎn)錄進(jìn)行的方向稱為下游相反的方向稱為上游保留性序列TATAbox大約在-10~-35處CAATbox大約在-75處影響啟動子效率GCbox由GGGCGG序列組成，也稱為sp1box，為轉(zhuǎn)錄因子sp1結(jié)合位置基因的結(jié)構(gòu)29分子診斷學(xué)概論課件30分子診斷學(xué)概論課件31Sp1分離自人類細(xì)胞由加州大學(xué)柏克萊分校的Tjian教授的實驗室分離可有效地促進(jìn)猿猴濾過性病毒（simianvirus40﹐簡稱SV40）基因的轉(zhuǎn)錄加強(qiáng)子Enhancer加強(qiáng)特殊基因的轉(zhuǎn)錄活性位置不固定與調(diào)節(jié)蛋白結(jié)合，與啟動子間形成圈環(huán)構(gòu)造靜默子silencer與增強(qiáng)子功能相反的調(diào)控Sp1分離自人類細(xì)胞32反應(yīng)元responseelement與轉(zhuǎn)錄因子結(jié)合，作用到啟動子，增強(qiáng)轉(zhuǎn)錄作用。與特異性刺激因子結(jié)合，調(diào)節(jié)基因表現(xiàn)TRE-TPAresponseelement,SRE-serumresponseelement,HSE-heatshockresponseelement,CRE-Campresponseelement,ERE-estrogenresponseelement,GRE-glucocorticoidresponseelement,MRE-metalresponseelement….阻絕子insulator一段0.5~3kb的DNA，阻斷轉(zhuǎn)錄因子散佈（可能增強(qiáng)子或靜默子）。反應(yīng)元responseelement33轉(zhuǎn)錄區(qū)表現(xiàn)子exon（外顯子）具有轉(zhuǎn)譯成多勝肽鏈或轉(zhuǎn)錄成RNA分子的序列，為編碼區(qū)內(nèi)含子intro（插入子）與exon一起被轉(zhuǎn)錄，但在mRNA剪切時會被切除。5端GT-AG3端可能與調(diào)控有關(guān)，基因的穩(wěn)定有相關(guān)聚腺苷酸化訊號polyadenylationsignalmRNA3端的AAUAAA序列下游約15-30bp會被切除，再加上polyAtail。轉(zhuǎn)錄區(qū)34分子診斷學(xué)概論課件35遺傳中心法則DNA→RNA→蛋白質(zhì)mRNA的剪切由核內(nèi)的小分子RNA(snRNA)和snRNA蛋白質(zhì)複合體(snRNPs)及SR蛋白質(zhì)參與，這些分子形成剪接體結(jié)構(gòu)(splicesome)反轉(zhuǎn)錄酶的發(fā)現(xiàn)顛覆了此中心法則1970HowardTermin和DavidBaltimore發(fā)現(xiàn)(1975Nobel)一些RNA病毒能利用反轉(zhuǎn)錄酶將它們的遺傳物質(zhì)(RNA)反轉(zhuǎn)錄為DNA遺傳中心法則36分子診斷學(xué)概論第一章綜說overview疾病發(fā)生原因的影響層次DNA、RNA或蛋白質(zhì)分子診斷的目的偵測這些致病因子是那個層次發(fā)生變化本書著重DNA、RNA的變化蛋白質(zhì)層次由原文書章節(jié)提供TheApplicationofProteomicsToDiseaseDiagnostics分子診斷學(xué)概論第一章綜說overview37分子診斷學(xué)概論課件38遺傳分子的基礎(chǔ)生物巨分子：DNA、RNA、蛋白質(zhì)、糖類、脂質(zhì)遺傳物質(zhì)DNA的發(fā)現(xiàn)1928格里夫茲(Griffith)肺炎雙球菌轉(zhuǎn)形試驗1942艾佛瑞(Avery)研究格里夫茲轉(zhuǎn)形的物質(zhì)為何?1952赫希-卻斯(Hershey-Chase)以放射線標(biāo)示噬菌體的蛋白質(zhì)（S35）和DNA（P32），感染大腸桿菌的實驗1953雙螺旋結(jié)構(gòu)的發(fā)現(xiàn)2003人類基因體計畫的完成遺傳分子的基礎(chǔ)39參考資料：/~cmallery/150/gene/sf11x1b.jpg參考資料：/40參考資料：/~cmallery/150/gene/sf11x1b.jpg參考資料：/41分子診斷學(xué)概論課件42參考資料：http://.tw/advance/a021.asp

參考資料：.t43分子診斷學(xué)概論課件44分子診斷學(xué)概論課件45分子診斷學(xué)概論課件46Presentedhereisagenomesequenceofanindividualhuman.Itwasproducedfrom~32millionrandomDNAfragments,sequencedbySangerdideoxytechnologyandassembledinto4,528scaffolds,comprising2,810millionbases(Mb)ofcontiguoussequencewithapproximately7.5-foldcoverageforanygivenregion.WedevelopedamodifiedversionoftheCeleraassemblertofacilitatetheidentificationandcomparisonofalternatealleleswithinthisindividualdiploidgenome.ComparisonofthisgenomeandtheNationalCenterforBiotechnologyInformationhumanreferenceassemblyrevealedPresentedhereisagenomeseq47morethan4.1millionDNAvariants,encompassing12.3Mb.Thesevariants(ofwhich1,288,319werenovel)included3,213,401singlenucleotidepolymorphisms(SNPs),

53,823blocksubstitutions(2–206bp),292,102heterozygousinsertion/deletionevents(indels)(1–571bp),559,473homozygousindels(1–82,711bp),90inversions,aswellasnumeroussegmentalduplicationsandcopynumbervariationregions.Non-SNPDNAvariationaccountsfor22%ofalleventsidentifiedinthedonor,howevertheyinvolve74%ofallvariantbases.Thissuggestsanimportantrolefornon-SNPgeneticalterationsindefiningthediploidgenomestructure.Moreover,44%ofgeneswereheterozygousforoneormorevariants.Usinganovelhaplotypeassemblystrategy,wewereabletospan1.5Gbofgenomesequenceinsegments.200kb,providingfurtherprecisiontothediploidnatureofthegenome.Thesedatadepictadefinitivemolecularportraitofadiploidhumangenomethatprovidesastartingpointforfuturegenomecomparisonsandenablesaneraofindividualizedgenomicinformation.morethan4.1millionDNAvari48AuthorSummaryWehavegeneratedanindependentlyassembleddiploidhumangenomicDNAsequencefrombothchromosomesofasingleindividual(J.CraigVenter).Ourapproach,basedonwhole-genomeshotgunsequencingandusingenhancedgenomeassemblystrategiesandsoftware,generatedanassembledgenomeoverhalfofwhichisrepresentedinlargediploidsegments(.200kilobases),enablingstudyofthediploidgenome.Comparisonwithpreviousreferencehumangenomesequences,whichwerecompositescomprisingmultiplehumans,revealedthatthemajorityofgenomicalterationsarethewell-studiedclassofvariantsbasedonsinglenucleotides(SNPs).However,theresultsalsorevealthatlesserstudiedgenomicvariants,insertionsanddeletions,whilecomprisingaminority(22%)ofgenomicvariationevents,actuallyaccountforalmost74%ofvariantnucleotides.Inclusionofinsertionanddeletiongeneticvariationintoourestimatesofinterchromosomaldifferencerevealsthatonly99.5%similarityexistsbetweenthetwochromosomalcopiesofanindividualandthatgeneticvariationbetweentwoindividualsisasmuchasfivetimeshigherthanpreviouslyestimated.Theexistenceofawell-characterizeddiploidhumangenomesequenceprovidesastartingpointforfutureindividualgenomecomparisonsandenablestheemergingeraofindividualizedgenomicinformation.AuthorSummary49Identificationandanalysisoffunctionalelementsin1%ofthehumangenomebytheENCODEpilotproject.

Nature.2007Jun14;447(7146):799-816

TheEncyclopediaofDNAElements(ENCODE)Projectprovideamorebiologicallyinformativerepresentationofthehumangenomebyusinghigh-throughputmethodstoidentifyandcataloguethefunctionalelementsencoded.Identificationandanalysisof50First,ourstudiesprovideconvincingevidencethatthegenomeispervasivelytranscribed,suchthatthemajorityofitsbasescanbefoundinprimarytranscripts,includingnon-protein-codingtranscripts,andthosethatextensivelyoverlaponeanother.Second,systematicexaminationoftranscriptionalregulationhasyieldednewunderstandingabouttranscriptionstartsites,includingtheirrelationshiptospecificregulatorysequencesandfeaturesofchromatinaccessibilityandhistonemodification.Third,amoresophisticatedviewofchromatinstructurehasemerged,includingitsinter-relationshipwithDNAreplicationandtranscriptionalregulation.Finally,integrationofthesenewsourcesofinformation,inparticularwithrespecttomammalianevolutionbasedoninter-andintra-speciessequencecomparisons,hasyieldednewmechanisticandevolutionaryinsightsconcerningthefunctionallandscapeofthehumangenome.Together,thesestudiesaredefiningapathforpursuitofamorecomprehensivecharacterizationofhumangenomefunction.First,ourstudiesprovidecon51ThehighlightsofourfindingstodateincludeThehumangenomeispervasivelytranscribed,suchthatthemajorityofitsbasesareassociatedwithatleastoneprimarytranscriptandmanytranscriptslinkdistalregionstoestablishedprotein-codingloci.Many

novelnon-protein-codingtranscriptshavebeenidentified,withmanyoftheseoverlappingprotein-codinglociandotherslocatedinregionsofthegenomepreviouslythoughttobetranscriptionallysilent.Numerouspreviouslyunrecognizedtranscriptionstartsiteshavebeenidentified,manyofwhichshowchromatinstructureandsequence-specificprotein-bindingpropertiessimilartowell-understoodpromoters.Regulatorysequencesthatsurroundtranscriptionstartsitesaresymmetricallydistributed,withnobiastowardsupstreamregions.Thehighlightsofourfindings52Chromatinaccessibilityandhistonemodificationpatternsarehighlypredictiveofboththepresenceandactivityoftranscriptionstartsites.DistalDNaseIhypersensitivesiteshavecharacteristichistonemodificationpatternsthatreliablydistinguishthemfrompromoters;someofthesedistalsitesshowmarksconsistentwithinsulatorfunction.DNA

replication

timingiscorrelatedwithchromatinstructure.Atotalof5%ofthebasesinthegenomecanbeconfidentlyidentifiedasbeingunderevolutionaryconstraintinmammals;forapproximately60%oftheseconstrainedbases,thereisevidenceoffunctiononthebasisoftheresultsoftheexperimentalassaysperformedtodate.Chromatinaccessibilityandhi53Althoughthereisgeneraloverlapbetweengenomicregionsidentifiedasfunctionalbyexperimentalassaysandthoseunderevolutionaryconstraint,notallbaseswithintheseexperimentallydefinedregionsshowevidenceofconstraint.Differentfunctionalelementsvarygreatlyintheirsequencevariabilityacrossthehumanpopulationandintheirlikelihoodofresidingwithinastructurallyvariableregionofthegenome.Surprisingly,manyfunctionalelementsareseeminglyunconstrainedacrossmammalianevolution.Thissuggeststhepossibilityofalargepoolofneutralelementsthatarebiochemicallyactivebutprovidenospecificbenefittotheorganism.Thispoolmayserveasa'warehouse'fornaturalselection,potentiallyactingasthesourceoflineage-specificelementsandfunctionallyconservedbutnon-orthologouselementsbetweenspecies