第三部分基因组和测序原理

第三部分基因组和测序原理TheHumanGenome23chromosomes3,2x109basepairs(3,200,000,000)<5%mRNA(30,000genes)<3%translatedintoprotein>90%unknownfunctionExon1Exon2Exonn、、、protein-codingregion、、、、、、、、、、、、geneStrategies:randomshotgun(HUGO)wholegenomeshotgun(Celera)RandomShotgun:Genomiclibraryproductionwholegenome:3,200,000kbpartiallydigestedDNA:sizing:100-500kbBAClibrariesAdaptedfrom: BaylorCollegeofMedicine- HumanGenomeSequencingCenterMappingpieces:100-500kbMappingMARKERSMARKERS:sequencetaggedsites(STS)restrictionsitepatternknowngenes、、、MappingofBAC/YACClonesmappedBACclone:100-500kbshearedDNA:1、0-2、0kbsequencingtemplates:randomreads(0、6-1kb)Adaptedfrom: BaylorCollegeofMedicine- HumanGenomeSequencingCenterRandomShotgun:Sequencing(randomphase)Celera:WholeGenomeShotgunSequencingwholegenome:3,200,000kbshearedDNA:1、0-2、0kbsequencingtemplatesAdaptedfrom: BaylorCollegeofMedicine- HumanGenomeSequencingCenterrandomreads(0、6-1kb)9大家应该也有点累了，稍作休息大家有疑问的，可以询问和交流ShotgunSequencing:Assembly(both)=Consensus(sequence)gaplowbasequalitysinglestrandedregionmis-assembly(inverted)Adaptedfrom: BaylorCollegeofMedicine- HumanGenomeSequencingCenter„draftquality“ShotgunSequencing:Finishing(both)ThefinalGoalHighAccuracySequence:<1error/10,000basesAdaptedfrom: BaylorCollegeofMedicine- HumanGenomeSequencingCenter„finishedquality“DNA测序原理MilestonesofDNASequencing1953Watson,Crick,Franklin molecularstructureofDNA1976 Maxam,Gilbert DNAsequencingviachemicaldegradation1977 Sanger DNAsequencingviachaintermination1986 Hood;Ansorge automatedDNAsequencingwith

‘dyeprimer’

1989 Murray ‘cyclesequencing’withTaqpolymerase1991 NEN-DuPONT ‘dyeterminator’sequencing1999 ABI/MD 96wellcapillarysequencerHowDNASequenceIsDetermined?PolyacrylamideGelElectrophoresisATC32PAT32PA32PTAGCTACGATCG32PATCGA32PATCGAT32PATCGATC32PATCGATCG32PATCGATCGA32PATCGATCGAT32PDNAfragmentshavingadifferenceofonenucleotidecanbeseparatedongelelectrophoresisButthesebandscan’ttellustheidentityoftheterminalnucleotidesIfthosebandwiththesameterminalnucleotidecanbegrouped,thenitispossibletoreadthewholesequence杂交测序法质谱法单分子测序法原子探针显微镜测序法流式细胞仪测序法大规模平行实测法、DNA芯片法经典方法新技术方法Maxam-GilbertDNA化学降解法(Maxam和Gilbert,1977)Sanger双脱氧链终止法(Sanger和Coulson1977)DNA测序方法:生成互相独立得若干组带放射性标记得寡核苷酸,每组寡核苷酸都有固定得起点,但却随机终止于特定得一种或者多种残基上。由于DNA上得每一个碱基出现在可变终止端得机会均等,因此上述每一组产物都就是一些寡核苷酸混合物,这些寡核苷酸得长度由某一种特定碱基在原DNA全片段上得位置所决定。在可以区分长度仅差一个核苷酸得不同DNA分子得条件下,对各组寡核苷酸进行电泳分析,只要把几组寡核苷酸加样于测序凝胶中若干个相邻得泳道上,即可从凝胶得放射自影片上直接读出DNA上得核苷酸顺序。

方法概述:核酸序列分析得经典方法:化学法:化学裂解法(Maxam-Gillbert法)酶法:DNA链得末端合成终止法(sanger法)Sanger'sMethod:Maxam-Gilbert'sMethod:HowtoObtainDNAFragments32P32PATCGATCG32PATCGATATCGATATCGTAGCTAGCTATAGCTAGCTATAGCTAGCTAATCGA32PSpecificReactiontoG

ATCGSTOPATerminatedKeepongoingBiosyntheticmethodChemicalmethodTemplateorNon-radioactive(invisible)32PA,T,C,GAAnalogueDestroy→CleavageDestroy→CleavageATCGATCGATProducingvariousfragmentsJuangRH(2004)BCbasics化学裂解法(Maxam-Gillbert法)基本原理:基于某些化学试剂可以使DNA链在1种或2种碱基处发生专一性断裂得特性,精确地控制反应强度,使一个断裂点仅存在于少数分子中,不同分子在不同位点断裂,从而获得一系列大小不同得DNA片段,将这些片段经电泳分离。分析前,用同位素标记DNA得5´末端,用4组不同得特异反应,就可以使末端标记得DNA分子切成不同长度得片段,其末端都就是该特异得碱基。经变性胶电泳和放射自显影得到测序图谱。反应得关键:使DNA得4种核苷酸中,只有1-2种发生特异性得化学切割反应;碱基得特异性修饰;被修饰得碱基从核糖环上转移;失去碱基得糖环部位发生DNA链断裂。常用得专门用来对核苷酸作化学修饰,并打断磷酸二酯键得化学试剂有硫酸二甲酯,甲酸和肼。

4组特异得反应如下:(1)G反应用硫酸二甲酯使鸟嘌呤上得N7原子甲基化,加热引起甲基化鸟嘌呤脱落,多核苷酸链可在该处断裂。(2)G+A反应用甲酸使A和G嘌呤环上得N原子质子化,从而使其糖苷键变得不稳定,再用哌啶使键断裂。(3)T+C反应用肼使T和C得嘧啶环断裂,再用哌啶除去碱基。(4)C反应当有盐存在时,只有C与肼反应,并被哌啶除去。哌啶促使修饰碱基脱落,并使去掉碱基得磷酸二酯键断裂。

DNA得化学测序示意图Maxam-Gilbert化学降解测序法先用限制性内切酶把DNA切成10—200bp得测序材料,用碱性磷酸化酶处理该片段,消除5′末端上得磷酸,在5′OH端标记32P,用多核苷酸磷酸激酶催化,标记片段变性为单链,化学试剂在特定碱基位置降解单链DNA,产生一组长度不等得DNA片段,

反应试剂G反应:硫酸二甲酯G+A反应:甲酸T+C反应:肼C反应:NaCl+肼化学试剂在特定碱基位置降解单链DNA,产生一组长度不等得DNA片段,经电泳和放射性自显影后,从4个反应系统统一阅读,待测DNA得全部核苷酸序列就可直接读出。Maxam-Gilbert化学降解测序法不需要进行酶催化反应,因此不会产生由于酶催化反应而带来得误差;对未经克隆得DNA片段可以直接测序;化学降解测序法特别适用于测定含有如5-甲基腺嘌呤A或者G,C含量较高得DNA片段,以及短链得寡核苷酸片段得序列。测定得长度短(如裂解位点得统计学分布等等)Advantages:Disadvantages:Sanger得酶降解测序法(链终止法)这项技术得关键就是在DNA得聚合过程中依赖特殊反应底物(2′3′—双脱氧核苷三磷酸ddNTP)得特异性终止进行DNA测序。

DNAreplication:biochemistryOCNpurineorpyrimidinePOOOHPOOOHPOOOHHOPOOOHOOCNpurineorpyrimidineOH5’3’2',3'ddNTP与普通dNTP不同之处在同她们在脱氧核糖得3'位置缺少一个羟基。她们可以在DNA聚合酶作用下通过其5'三磷酸基团掺入到正在增长得DNA链中,但由于没有3'羟基,她们不能同后续得dNTP形成磷酸二酯链,因此,正在增长得DNA链不可能继续延伸。TheSangermethodrequiresMultiplecopiesofsinglestrandedtemplateDNAAsuitableprimer(asmallpieceofDNAthatcanpairwiththetemplateDNAtoactasastartingpointforreplication)DNApolymerase(anenzymethatcopiesDNA,addingnewnucleotidestothe3’endofthetemplateA‘pool’ofnormalnucleotidesAsmallproportionofdideoxynucleotideslabeledinsomeway(radioactivelyorwithfluorescentdyes)ThetemplateDNApiecesarereplicated,incorporatingnormalnucleotides,butoccasionallyandatrandomdideoxy(DD)nucleotidesaretakenup、ThisstopsreplicationonthatpieceofDNATheresultisamixofDNAlengths,eachendingwithaparticularlabeledDDnucleotide、Becausethedifferentlengths‘travel’atdifferentratesduringelectrophoresis,theirordercanbedetermined、酶法序列分析得原理

酶反应:DNA聚合酶(Klenow)电泳方向模板CCGGTAGCAACT3´5´GG5´3´标记引物dATPdCTPdGTPdTTP+ddATPdATPdCTPdGTPdTTP+ddTTPdATPdCTPdGTPdTTP+ddGTPdATPdCTPdGTPdTTP+ddCTPGGCCAGGCCATCGTTGAGGCGGCCGGCCATCGGCCATCGTTGGGCCATCGGGCCATGGCCATCGTGGCCATCGTTACGTAGTTGCTACC3´5´TCAACGATGG5´3´读出模板互补序列读出模板序列GGCCATCGTTGAGGCCATCGTGGCCATCGTTGGGCCATCGTTGGCCATCGGGCCATCGGCCAGGCCATGGCCGGCDNAtemplateisdenaturedtosinglestrands、DNAprimer(with3’endnearsequenceofinterest)isannealedtothetemplateDNAandextendedwithDNApolymerase、Fourreactionsaresetup,eachcontaining:DNAtemplatePrimerannealedtotemplateDNADNApolymerasedNTPS(dATP,dTTP,dCTP,anddGTP)Next,adifferentradio-labeleddideoxynucleotide(ddATP,ddTTP,ddCTP,orddGTP)isaddedtoeachofthefourreactiontubesat1/100ththeconcentrationofnormaldNTPs、ddNTPspossessa3’-Hinsteadof3’-OH,peteinthereactionwithnormaldNTPS,andproducenophosphodiesterbond、ManualDideoxyDNAsequencing-Howitworks:Whenevertheradio-labeledddNTPsareincorporatedinthechain,DNAsynthesisterminates、EachofthefourreactionmixturesproducesapopulationofDNAmoleculeswithDNAchainsterminatingatallpossiblepositions、Extensionproductsineachofthefourreactionmixutesalsoendwithadifferentradio-labeledddNTP(dependingonthebase)、Next,eachreactionmixtureiselectrophoresedinaseparatelane(4lanes)athighvoltageonapolyacrylamidegel、Electrophoresisat70°C(2h)、FixgelinHAc,drygel(1h)、Autoradiography(24-48h)、PatternofbandsineachofthefourlanesisvisualizedonX-rayfilm、Locationof“bands”ineachofthefourlanesindicatethesizeofthefragmentterminatingwitharespectiveradio-labeledddNTP、DNAsequenceisdeducedfromthepatternofbandsinthe4lanes、取代放射性同位素得方法地高辛,生物素:用地高辛,生物素代替发射性同位素示踪测序反应产物,最后用酶显色反应显示出测序结果、银染方法:把生成DNA分子核苷酸碱基顺序得方法与一种超敏感得银染方法相结合。这一新得技术组合使电泳分离得序列片段不依赖于仪器就直接可见。这种无放射性得系统包括通过用酶促双氧链终止反应形成一系列DNA序列片段,凝胶电泳分离这些片段,并把凝胶浸入超敏感得银染溶液中,观察引物延伸产物得银染条带,从而确定DNA分子得序列。荧光标记物ddGTPddATPddTTPddCTP引物标记法测序(DyePrimer)一个样本,4个反应。4个反应中引物序列相同,但分别标记有不同颜色得荧光。+AmpliTaqFSenzymedCTP,dATP,dGTP,dTTPddATP(terminator)+AmpliTaqFSenzymedCTP,dATP,dGTP,dTTPddCTP+AmpliTaqFSenzymedCTP,dATP,dGTP,dTTPddGTP+AmpliTaqFSenzymedCTP,dATP,dGTP,dTTPddTTP反应结束后,将4管反应液混合,经乙醇沉淀后上样Advantagesofprimer-labels:cleanerDisadvantages:expensivetomanufacturefourreactionslimitedtocertainregions,customoligosorlimitedtoclonedinsertsbehind‘universal’primingsites、Solution:fluorescentdyeterminators(upgrade,seconditeration)终止法测序(DyeTerminator)一个样本,1个反应。反应中包含分别带4色荧光标记得ddNTP(终止子)unlabeledprimertemplateDNA+AmpliTaqFSdATP,dCTP,dGTP,dTTPddCTPddATPddGTPddTTP测序反应结束后,采用乙醇沉淀法进行纯化,除去过量得ddNTP后上样。DideoxyDNAsequencingwastimeconsuming,radioactive,andthroughputwaslow,typically~300bpperrun、AutomatedDNAsequencingemploysthesamegeneralprocedure,butusesddNTPslabeledwithfluorescentdyes、bine4dyesinonereactiontubeandelectrophoresinonelaneonapolyacrylamidegelorcapillarycontainingpolyacrylamide、UVlaserdetectsdyesandreadsthesequence、Sequencedataisdisplayedascoloredpeaks(chromatograms)thatcorrespondtothepositionofeachnucleotideinthesequence、Throughputishigh,upto1,200bpperreactionand96reactionsevery3hourswithcapillarysequencers、MostautomatedDNAsequencerscanloadroboticallyandoperatearoundtheclockforweekswithminimallabor、AutomatedDye-TerminatorDNASequencing:Advantagesofprimer-Terminator:--performedinasingletubeandtheresultingfragmentstobeloadedinasinglewell,resultingingreatersamplecapacityper“lane”、Disadvantages:

--lesscleanerAmountoffluorescentDNAproducedislowandthusalargeamountoftemplateisrequiredtoproducegoodfluorescentsignals、Solution:CyclesequencingCyclesequencingSequenase(T7)polymerasehasbeenawidelyusedenzymeforDNAsequencingwithradioactivenucleotidesbecauseofitshighprocessivityandlowerrorrate、HoweverusingthistypeofenzymeforfluorescentDNAsequencingisnotoptimalbecausetheamountoffluorescentDNAproducedislowandthusalargeamountoftemplateisrequiredtoproducegoodfluorescentsignals、TheenzymecurrentlyingeneraluseforautomatedfluorescentDNAsequencingisavariantofTaqDNApolymerase、ThethermostabilityofTaqpolymeraseallowsthesequencingreactionstobecarriedoutlikePCRreactions,andthereactionsarecalled"cyclesequencing"reactions、TheadvantageofusingthethermostableTaqpolymeraseovertheuseofSequenaseisthatmultipleroundsofsequencingcanbeperformedwithouttheneedtoaddfreshenzyme、PolymeraseChainReaction95°C50-65°C72°C95°C50-65°C72°C95°C50-65°C72°CCyclesequencingreactionsareanalogoustoPCRsexceptonlyasingleprimerisusedandonlysinglestrandedproductsaregenerated、CycleSequencing95°C50-65°C72°C95°C50-65°C72°C95°C50-65°C72°CCyclesequencingThisallowstheuseofmuchlesstemplateDNA,Modifications(pointmutationsaffectingaminoacidresiduesinorneartheactivesite)totheTaqDNApolymerasehaveenabledittoincorporatethefluorescentdye-labeledterminatorsmoreevenlyandefficiently,resultinginveryevenpeakheightsoverasequenceread、AttemptshavebeenmadetodeveloppolymeraseswhichhavesomeoftheadvantageouspropertiesofSequenasebutarethermostablelikeTaq、Cyclesequencing测序技术进展

同位素标记到荧光标记,平板电泳到毛细管电泳

多色荧光标记毛细管电泳单色荧光标记平板电泳同位素标记平板电泳ACGTACGT测序图谱TATTGCATTGTCTGCATTGTCTDNA自动测序采用荧光替代放射性核素标记就是实现DNA序列分析自动化得基础。用不同荧光分子标记四种双脱氧核苷酸,然后进行Sanger测序反应,反应产物经电泳(平板电泳或毛细管电泳)分离后,通过四种激光激发不同大小DNA片段上得荧光分子使之发射出四种不同波长荧光,检测器采集荧光信号,并依此确定DNA碱基得排列顺序。

DNA得测序仪示意图puteranalysis凝胶中DNA移动方向样品槽激光器输入光学系统成象透镜聚焦透镜高灵敏度相机旋光镜/棱镜组件DNA序列分析自动化:用荧光剂标记DNA引物或ddNTP,带有这种荧光剂标记得DNA片段能在激光诱导下发出荧光。按照标准得双脱氧终止法或化学终止法进行测序反应,跑DNA凝胶后用计算机检测。CapillaryelectrophoresisAdvantagescapillaryLongerreadsMorere