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1、Genomics基因组学基因组学基因组学genomicswxj课件生物化学与分子生物学系是是一一个个细细胞胞（或或病病毒毒）所所携携带带的的全全部部遗遗传传信信息息，它它代代表了一种生物所具有的全部遗传信息。表了一种生物所具有的全部遗传信息。真真核核生生物物基基因因组组是是指指一一套套完完整整单单倍倍体体DNA（染染色色体体DNA）及及线线粒粒体体或或叶叶绿绿体体DNA的的全全部部序序列列，既既有有编编码码序序列列，也有大量存在的非编码序列。也有大量存在的非编码序列。细菌基因组包含了拟核和质粒中的细菌基因组包含了拟核和质粒中的DNA序列。序列。病病毒毒基基因因组组有有的的为为DNA（DNA病病
2、毒毒），有有的的则则为为RNA（RNA病毒）。病毒）。基因组基因组 (Genome)基因组学genomicswxj课件生物化学与分子生物学系nGenomicsisadisciplineingeneticsconcerningthestudyofthegenomesoforganisms.ThefieldincludesintensiveeffortstodeterminetheentireDNAsequenceoforganismsandfine-scalegeneticmappingefforts.Thefieldalsoincludesstudiesofintragenomicphenom
3、enasuchasheterosis,epistasis,pleiotropyandotherinteractionsbetweenlociandalleleswithinthegenome.Genomics基因组学genomicswxj课件生物化学与分子生物学系n基因组学（基因组学（genomics）是研究基因组的）是研究基因组的结构结构和和功能功能以及基因间的以及基因间的相互作用相互作用的学科。的学科。n研究内容研究内容结构基因组学（结构基因组学（structural genomicsstructural genomics）: : 遗传图谱、遗传图谱、 物物理图谱、序列图谱以及转录图谱和大
4、规模理图谱、序列图谱以及转录图谱和大规模DNADNA测序。测序。功能基因组学（功能基因组学（functional genomicsfunctional genomics）: : 分析鉴定基因分析鉴定基因组功能。组功能。比较基因组学（比较基因组学（comparative genomicscomparative genomics）：）：基因组之间比基因组之间比较鉴定，研究生物进化，预测新基因。较鉴定，研究生物进化，预测新基因。Genomics基因组学genomicswxj课件生物化学与分子生物学系结构基因组学（structuralgenomics） 结构基因组学着重研究基因组的结构并构建高结构基因
5、组学着重研究基因组的结构并构建高分辨的分辨的遗传图遗传图、物理图物理图、转录图转录图和和序列图；序列图；揭示基揭示基因组的全部序列及其组成。因组的全部序列及其组成。 主要任务主要任务： 基因组作图基因组作图 大规模测序大规模测序 基因组学genomicswxj课件The Human Genome Project (HGP)人类基因组计划人类基因组计划基因组学genomicswxj课件生物化学与分子生物学系7To tackle such an enormous project, 158 genome scientists, representing 67 institutions from 12
6、 countries, gathered in Japan in the summer of 2002. Over the course of a ten-day annotation marathon, the scientists validated, mapped, and annotated the cDNAs. As things stand, the team has been able to assemble the cDNAs into over 20,000 strong candidates for human genes. Several valuable finding
7、s about the human genome have emerged: there are over 5,000 candidates for new genes, including an exciting group of several hundred that do not appear to encode proteins; Up to 4% of the genome appears not to be represented in the current human genome sequence; and Several thousand DNA sequence var
8、iants have been uncovered that will be useful for disease mapping studies.Annotation Marathon Validates 21,037 Human Gene Loci Fukuchi (2004) PLoS Biology基因组学genomicswxj课件生物化学与分子生物学系HGPnHGP发展史n测序策略n关键技术n成果n应用基因组学genomicswxj课件生物化学与分子生物学系1.ThehistoryandthemilestonesofHGP2.TheHGPsequencingstrategyMap-F
9、irst,Sequencing-Later(geneticmap,physicalmap,expressionmap,allwouldcontainthemarkersorsignpoststoallowDNAsequencetobepiecedtogetherinproperorderalongchromosomes).*Hierarchygenomelibrarytosetup.*Hierarchicalgenomesegmentstobesequenced.Assemblyandannotationofhumangenome9TheOutlineofHGP基因组学genomicswxj课
10、件生物化学与分子生物学系3.ThekeymethodsappliedtoHGPi.TheestablishmentofthehierarchicalBACslibraryofthehumangenome.ii.TheimprovementofDNAsequencing(CyDye-labeleddNTPs,capillaryelectrophoresis,laserdetector,pyrosequencing).iii.TheestablishmentofBioinformaticstoannotatetheDNAsegmentssequenced(theassemblyofDNAseque
11、nceinorder,definitionoffunctionalelements),andannotationofhumangenomesequencethroughassays4.TheachievedfromHGP(workingdraft,Chr22,Chr21,thefinaldraft)5.HGPapplication(HGPmeaningstootherscientificfields)TheOutlineofHGP基因组学genomicswxj课件生物化学与分子生物学系1115 Feb 2001, Nature 基因组学genomicswxj课件生物化学与分子生物学系12基因组
12、学genomicswxj课件生物化学与分子生物学系IntroductiontoHumanGenomeProject1.In1990,Americangeneticistsembarkedonanambitiousquest:tomapandultimatelysequencetheentirehumangenome,andquicklythiseffortbecameaninternationalprogram(USA40%,German15%,France15%,UK20%,Japan7%,andChina1%),IHGSC.2.Thehumangenomeprojectisthehuge-
13、morethan3billionbptosequence.Togetanideaofthemagnitudeofthistask,nIfall3billionbpwerewrittendown,itwouldtakeabout500,000pagesoftheJournalNaturetocontainalltheinformation.Ifyoucouldstandtheboredom,itwouldtakeyourabout60years,working8h/day,everyday,at5basesasecond,toreaditall.4.TheoriginalplanforHGPwa
14、ssystematicandconservative(map-then-sequencing).Atfirst,geneticswouldpreparegeneticandphysicalmapsofthegenome,i.e.withmarkersorsignpoststoallowallDNAsequencestobepiecedtogetherintheproperorder.Later,thebulkofsequencingwillbedoneonlyifthemappingwascompletedandclonesrepresentingallpointsonthemapwerein
15、hand(systematicallystoredinfreezersaroundtheworld),scheduledtocompleteby2005.13基因组学genomicswxj课件生物化学与分子生物学系5.In1998,AprivatecompanyCeleracreatedbyCraigVenterannouncedthatCelerawouldcompletetheroughdraftofhumangenomebytheendoftheyear2000,insistingthatthegenesareprotectedbypatent.6.ExcitedbyCeleraanno
16、uncement,FrancisCollins,directorofHGP,promisedtoproducearoughdraftbytheendoftheyear2000,too.7.June26,2000,VenterandCollinsappearedwithPresidentClintonataceremonyintheEastRoomoftheWhiteHousetoannouncethecompletionofaroughdraftofthehumangenome(Celera99%vsHGP85%,publishedinFeb15,2001,HGPinNature&Celera
17、inScience).IntroductiontoHumanGenomeProject基因组学genomicswxj课件生物化学与分子生物学系15Weaver (2001) Molecular Biology 2nd Ed p786In Nov,2010, Nature, 1000 Genome Sequencing Project to be announced for investigation of human genome variations.基因组学genomicswxj课件生物化学与分子生物学系16Human KaryotypeMouse KaryotypeZebrafish K
18、aryotypePig KaryotypeNucleus Karyotypes Determine Species in BiologyCellular Nuclear Karyotype : chromosome number & structure基因组学genomicswxj课件生物化学与分子生物学系1.Inmap-then-sequencingstrategy:(1)themappingofthehumanandmousegenomestoallowthestudyofinheriteddiseaseandprovideacrucialscaffoldforgenomeassembly
19、(2)thesequencingoforganismswithsmaller,simplergenomestoserveasatestbedformethoddevelopmentandassistininterpretingthehumangenome(3)thesequencingofhumangenomeandannotatingofthesegmentssequenced,byIHGSC.2.Inshotgun-sequencingstrategy:omittingmap,creatingaBACclonecollectionofgenome,sequencingBACclones,b
20、yCelera.Q:whyneedtomapforthehumangenome?A:inordertorelatehumangenesequencesalongchromosomes,havetolocateasetoflandmarksalongchromosomes.HGPSequencingStrategy基因组学genomicswxj课件生物化学与分子生物学系基因组学genomicswxj课件生物化学与分子生物学系MapsofHGPn遗传图(GeneticMap)n物理图(PhysicalMap)n转录图(ExpressionMap)n序列图(SequenceMap)基因组学genom
21、icswxj课件生物化学与分子生物学系GeneticMap(LinkageMap)ngenetictraitidentitytobelocatedalongchromosomesnHistorically,themarkersoriginallyusedweredetectablephenotypes(enzymeproduction,eyecolor)derivedfromcodingDNAsequences;eventually,confirmedorassumednoncodingDNAsequencessuchasmicrosatellitesorthosegeneratingrest
22、rictionfragmentlengthpolymorphisms(RFLPs)havebeenused.基因组学genomicswxj课件生物化学与分子生物学系遗遗传传作作图图（geneticmapping）:就就是是确确定定连连锁锁的的遗遗传传标标志志位位点点在在一一条条染染色色体体上上的的排排列列顺顺序序及及它它们们之之间间的的相相对对遗遗传传距距离离，用用厘厘摩摩尔尔根根（centi-Morgan，cM）表表示示，当当两两个个遗遗传传标标记记之之间间的的重重组组值为值为1%时，图距即为时，图距即为1cM。遗传图遗传图(geneticmap)连锁图连锁图(linkagemap)基因组学
23、genomicswxj课件生物化学与分子生物学系DNA标志n限限制制性性片片段段长长度度多多态态性性（restrictionfragmentlengthpolymorphism，RFLP）n可可变变数数目目串串联联重重复复序序列列（variable number oftandemrepeats，VNTRs）n单单 核核 苷苷 酸酸 多多 态态 性性 （ singlenucleotidepolymorphism,SNP）基因组学genomicswxj课件生物化学与分子生物学系RestrictionFragmentLengthPolymorphisms(RFLPs)nEachpersondiffe
24、rsgeneticallyfromeveryother,thesequencesoftheirDNAwilldiffersomewhataswillthepatternofcuttingbyrestrictionenzymes.nIncaseofHindIIIsite(AAGCTT),forexample,onepersonwith3HindIIIsitesseparatedby4kband2kbinagivenregionofachromosome(ifchromosometocutwithHindIII,ittoproduce4kband2kb),anotherwith2HindIIIsi
25、teslackingthemiddleHindIIIsite(ifcut,toproducea6kbsegment),thisphenomena,duetothedifferenceoftherestrictionenzymesiteamongpopulation,iscalledRFLPs.基因组学genomicswxj课件生物化学与分子生物学系 在群体中生物个体在群体中生物个体之间，由于之间，由于DNA DNA 某一位某一位点上的变异有可能引起点上的变异有可能引起该位点特异性的限制性该位点特异性的限制性内切酶识别位点的改变，内切酶识别位点的改变，包括原有位点的消失或包括原有位点的消失或出现
26、新的酶切位点。当出现新的酶切位点。当用限制性内切酶处理不用限制性内切酶处理不同生物个体的同生物个体的DNADNA时，时，致使酶切片段长度发生致使酶切片段长度发生变化，个体之间出现限变化，个体之间出现限制性片段长度的差异，制性片段长度的差异，这称为限制性片段长度这称为限制性片段长度多态性。多态性。限制性片段长度多态性基因组学genomicswxj课件生物化学与分子生物学系AmplifiedFragmentLengthPolymorphism(AFLP)nAFLPisaPCR-basedtoolusedingeneticsresearch,DNAfingerprinting,andinthepra
27、cticeofgeneticengineering.Developedintheearly1990sbyKeygene,AFLPusesrestrictionenzymestodigestgenomicDNA,followedbyligationofadaptorstothestickyendsoftherestrictionfragments.Asubsetoftherestrictionfragmentsisthenselectedtobeamplified.Thisselectionisachievedbyusingprimerscomplementarytotheadaptorsequ
28、ence,therestrictionsitesequenceandafewnucleotidesinsidetherestrictionsitefragments.基因组学genomicswxj课件生物化学与分子生物学系variablenumberoftandemrepeats(VNTRs)nVNTRisalocationinagenomewhereashortnucleotidesequenceisorganizedasatandemrepeat.Thesecanbefoundonmanychromosomes,andoftenshowvariationsinlengthbetweenin
29、dividuals.Eachvariantactsasaninheritedallele,allowingthemtobeusedforpersonalorparentalidentification.nTherearetwoprincipalfamiliesofVNTRs:microsatellitesandminisatellites.Theformerarerepeatsofsequenceslessthanabout6bpinlength(2-6BP),whilethelatterinvolvelongerblocks(10-100BP).基因组学genomicswxj课件生物化学与分
30、子生物学系基因组中存在的一种重复基因组中存在的一种重复DNA短序列。可分为两种短序列。可分为两种微微卫星卫星DNA(microsatellites)和小卫星和小卫星DNA(minisatellites)。其基本原理与其基本原理与RFLP大致相同，通过限制性内切酶的大致相同，通过限制性内切酶的酶切和酶切和/或或PCR，可一次性检测到众多微卫星位点，得到，可一次性检测到众多微卫星位点，得到个体特异性的个体特异性的DNA指纹图谱。指纹图谱。可变数目串联重复序列（VNTR）基因组学genomicswxj课件生物化学与分子生物学系SNPnASNPisaDNAsequencevariationoccurr
31、ingwhenasinglenucleotideinthegenomediffersbetweenmembersofabiologicalspeciesorpairedchromosomesinanindividual.nVariationsintheDNAsequencesofhumanscanaffecthowhumansdevelopdiseasesandrespondtopathogens,chemicals,drugs,vaccines,andotheragents.SNPsarealsothoughttobekeyenablersinrealizingtheconceptofper
32、sonalizedmedicine.However,theirgreatestimportanceinbiomedicalresearchisforcomparingregionsofthegenomebetweencohorts(suchaswithmatchedcohortswithandwithoutadisease)ingenome-wideassociationstudies.基因组学genomicswxj课件生物化学与分子生物学系SNP不以“长度”的差异作为检测手段，而是直接以序列的变异作为标记。SNP是指在基因组水平上由单个核苷酸变异所造成的DNA序列多态性。SNP是人类可遗传的
33、变异中最常见的一种，也是基因组中最为稳定的变异。SNP最大限度地代表了不同个体之间的遗传差异，因而成为研究多基因疾病、药物遗传学及人类进化的重要遗传标记。单核苷酸多态性单核苷酸多态性（SNP）基因组学genomicswxj课件生物化学与分子生物学系物理作图（物理作图（physicalmapping）是在遗传作图基）是在遗传作图基础上制作的更详细的人类基因组图谱。包括：础上制作的更详细的人类基因组图谱。包括：荧光原位杂交图（荧光原位杂交图（FISHmap）限制性酶切图（限制性酶切图（restrictionmap）连续克隆系图（连续克隆系图（clonecontigmap）物理图物理图 (Physi
34、calMap)基因组学genomicswxj课件生物化学与分子生物学系荧光原位杂交图荧光原位杂交图（fluorescentin situ hybridizationmap，FISHmap）：）：将荧光标记的探将荧光标记的探针与染色体杂交确定分子针与染色体杂交确定分子标记所在的位置。探针常标记所在的位置。探针常选用已知基因的大片段序选用已知基因的大片段序列列基因组学genomicswxj课件生物化学与分子生物学系限制性酶切图（限制性酶切图（restriction map）：）：将限制将限制性酶切位点标定在性酶切位点标定在DNA分子的相对位置。分子的相对位置。基因组学genomicswxj课件生物
35、化学与分子生物学系基因组学genomicswxj课件生物化学与分子生物学系连续克隆系图（连续克隆系图（clonecontigmap）：）：采用酶采用酶切位点稀有的限制性内切酶或高频超声破碎技术切位点稀有的限制性内切酶或高频超声破碎技术将将DNA分解成大片段后，再通过构建酵母人工分解成大片段后，再通过构建酵母人工染色体（染色体（yeastartificialchromosome，YAC）或）或细菌人工染色体（细菌人工染色体（bacterialartificialchromosome，BAC）获取含已知基因组序列标）获取含已知基因组序列标签位点（签位点（sequencetaggedsite，STS
36、）的）的DNA大大片段。片段。基因组学genomicswxj课件生物化学与分子生物学系nSequence-taggedsites(orSTSs)areshort(200to500basepair)DNAsequencesthathavesingleoccurrenceinthegenomeandwhoselocationandbasesequenceareknown.STSsweredonebyHGPon1998.nSTSscanbeeasilydetectedbyPCRusingspecificprimers.Forthisreasontheyareusefulforconstructing
37、geneticandphysicalmapsfromsequencedatareportedfrommanydifferentlaboratories.Theyserveaslandmarksonthedevelopingphysicalmapofagenome.Sequence-TaggedSites基因组学genomicswxj课件生物化学与分子生物学系*STS（sequencetaggedsite，基因组序列标签位，基因组序列标签位点）：点）：是指染色体定位明确，并且可用是指染色体定位明确，并且可用PCR扩增扩增的单拷贝序列，每隔的单拷贝序列，每隔100kb距离就有一个标志。距离就有一个
38、标志。在在STS基础上构建能够覆盖每条染色体的大片段基础上构建能够覆盖每条染色体的大片段DNA连续克隆系就可绘制精细物理图谱，为大规连续克隆系就可绘制精细物理图谱，为大规模模DNA测序做好了准备。测序做好了准备。基因组学genomicswxj课件生物化学与分子生物学系nbasedoncDNAlibraryandESTs(expressedsequencetags)tolocategenesalongchromosomes.nESTisashortsub-sequenceofatranscribedcDNAsequence.Theymaybeusedtoidentifygenetranscrip
39、ts,andareinstrumentalingenediscoveryandgenesequencedetermination.Thereareapproximately65.9millionESTsnowavailableinpublicdatabases(e.g.GenBank18June2010,allspecies).nAnESTisproducedbyone-shotsequencingofaclonedmRNA(i.e.sequencingseveralhundredbasepairsfromanendofacDNAclonetakenfromacDNAlibrary).Expr
40、essionMap(transcriptionalmap)基因组学genomicswxj课件Sequence MapThe Goal !基因组学genomicswxj课件生物化学与分子生物学系VectorsforHGP No matter which sequencing strategy is used, one must first clone fragments of the genome in appropriate vectors, and proper large fragments are particular valuable.nVectorfeaturesnanorigino
41、freplicationnaselectablemarkernamulticloningsite基因组学genomicswxj课件生物化学与分子生物学系VectorTypenYAC(0.2-2Mb)nBAC(100-300kb)nCosmid(50kb)nPhage(15kb)nPlasmid(10kb)基因组学genomicswxj课件生物化学与分子生物学系YeastArtificialChromosome(YAC)alineards-DNA,withtelomereslocatedatitsterminus,originsiteandcentromereclosertolefttelome
42、re,allowedofinsertionofforeignDNAwithover500kbatthesitebetweencentromereandrighttelomere.ButYACtobeunstable,inefficientandhardtoisolate.BacterialArtificialChromosome(BAC)unlikethelinearYAC,BACiscircularsupercoiledresistancetobreakage,withinsertionofforeignDNAaveragewith150kb(100-300kb).Forexample,pB
43、AC108Lchloramphenicolresistancegene(CmR)whichisbasedonanaturalplasmidF-plasmidthatinhabitsinE.colicells.Plasmid:withitsbackbonesizeofaround6kbandallowanceofinsertionofforeignDNA10kb,circularsupercoiled,andinhabitedinE.coli.41基因组学genomicswxj课件生物化学与分子生物学系HGPSequencingStrategyMap-then-SequencingStrateg
44、y(CloneContigMap)Uptodown,byIHGSC.WholeGenomeShotgunSequencingStrategyBottomtoup,byCelera.基因组学genomicswxj课件IHGSCStrategy:Hierarchical-GenomeShotgunSequencing(HGSS)Isolation & digestion of chromosomes Establish of hierarchical BAC library along every chromosomes with BAC clone fingerprint & STCGenome
45、 Source : blood cells & sperm cells*Hierarchical BAC Library of Every Single Chromosome of Human GenomeHierarchical plasmid library of every single BAC clone for sequencing*DNA Sequencing : clone-by-clone (plasmids)Assembly of DNA sequences via merging STCs of BAC clones*Annotation of DNA Sequences
46、as Function ElementsIsolation of blood cells from the peripheral blood of people and isolation of sperm cellsWith virus transformation, cells to be immortal Establish of hierarchical plasmid library from every single BAC clone for sequencingSanger DNA sequencing (chain termination)CyDye labeled dNTP
47、s, laser detector, capillary electrophoresisPyro-sequencingBioinformatics analysis (ORF, CpG island, cDNA seq, Gene Ontology, Sequence homology, etc.)Function confirmation assay With BAC clone fingerprint & STC, genetic marker, seq markers, expression tag to place clones in order along each chromoso
48、mes 43STCs : sequence-tagged connectors via both end segments sequenced of 500 bp of genome insert in every BAC clones基因组学genomicswxj课件生物化学与分子生物学系44Hierarchical BAC Library of Human Genome IHGSC (2001) Nature p863Organized BAC Clone Contigs along chromosome Via overlap seq & BAC-FingerprintsHierarch
49、ical BAC Library (170 kb) of Shotgun ChromosomesHuman Genomic DNA Plasmid Libraries (4-6 kb) of Shotgun Insert of BAC ClonesDNA Sequencing of Plasmid ClonesAssembly of Sequenced DNA SegmentsVia End-to-End, Physical Maps, BAC-STCsContig : a set of segments with overlapped stretches among them.基因组学gen
50、omicswxj课件生物化学与分子生物学系45Workflow of Human Genome Sequencing Wikipedia 基因组学genomicswxj课件生物化学与分子生物学系CeleraStrategy:Whole-GenomeShotgunSequencing(WGSS)Weaver(2001)MoleculeBiology,46a. Chromosomes are cloned into a BAC vector, yielding a collection of 300,000 BAC clones, but at random.b.Every seed BAC is
51、 selected for next sequencing. c.The seed BAC is subcloned into a plasmid vector, yielding a plasmid library. d.Three thousand of the plasmid clones are sequenced, and the sequences are ordered by their overlaps, producing the sequence of the whole 150-kb BAC. e.Find the BACs (about 30) with overlap
52、ping STCs, then compare them by fingerprinting to find those with minimal overlaps, and sequence them. This process, known as BAC walking, can in principle create a contig covering the whole chromosome.A random collection Great risk of long-range mis-assembly 基因组学genomicswxj课件生物化学与分子生物学系47Celera Flo
53、w Diagram for Genome Sequencing Venter (2001) Science基因组学genomicswxj课件生物化学与分子生物学系48Distribution of Molecular Functions of 26,383 Human Genes By Celera Venter (2001) Science p1335基因组学genomicswxj课件生物化学与分子生物学系49Celera Anatomy of Whole Genome Assembly Venter (2001) Science基因组学genomicswxj课件生物化学与分子生物学系50I
54、HGSC Hierarchical Shotgun Vs Celera Whole-Genome Shotgun Waterston (2002) PNASHierarchical Genome BAC Library Hierarchical Organization1.Genome to chromosomes2.Chromosome to BAC clones3.BAC clone to plasmid clones基因组学genomicswxj课件生物化学与分子生物学系DNASequencingnChaintermination(Sanger)nPyrosequencing基因组学ge
55、nomicswxj课件reactionmechanism基因组学genomicswxj课件生物化学与分子生物学系53DNAtoSynthesize:formationofphosphodiesterbondsNew phosphodiester bondSynthesis Reaction PPiDirection 53 Resource components : dNTPs, NTPsPrimer (RNA or DNA oligo 10-60 nt) with 3-OH freeTemplate DNA DNA polymerase : catalyze new phosphodieste
56、r bond formation between 3-OH of primer with the alpha phosphate of incoming nucleotide基因组学genomicswxj课件生物化学与分子生物学系54TheMechanismofDNAPolymeraseforDNASynthesisNew phosphodiester bondThese two Mg2+ ions to coordinate to tri-phosphate of incoming dNTP and to three Asp residues in enzyme in site.The up
57、per Mg2 ion to facilitate an attack of 3-OH at 3-end of growing DNA onto the alpha-phosphate of incoming dNTP.The lower Mg2 ion to facilitate displacement of pyrophosphate with new phosphodiester bond.Both Mg2 ions to stabilize the pentacovalent transition state.基因组学genomicswxj课件生物化学与分子生物学系55Kambara
58、 (2010) Chemical Record A.Principle of Sanger sequencing is based on the nascent chain termination of DNA synthesis due to the last incorporated ddNTP being 2-& 3-dideoxyribonucleotide B.Improvements on 4-color -tagged-ddNTPs & laser detector, capillary electrophoresis, luminescence detector53 Targe
59、t DNA SequenceSangers Principle of DNA Sequencing on Chain Termination of DNA Synthesis 1231020:1基因组学genomicswxj课件生物化学与分子生物学系56Thinner of gel, less width of DNA band (more accuracy) could be obtained even in range of high electric field (volt/cm).In fixed gel thickness, less width of DNA band could
60、be obtained only in range of lower electric field (volt/cm).Less width of DNA bands improve the sharp discrimination among a variety of DNA length.Kambara (2010) The Chemical Record Schematic View of Capillary Array DNA Sequencer1DNA Band Width vs Electric Field in Known Gel Thickness2Schematic for
61、Automatic System of Pyrosequencing3基因组学genomicswxj课件生物化学与分子生物学系Explanationfor:PrincipleforPyrosequencing57PyrogramPrinciple for Pyrosequencing Kambara (2010) Chemical Record1.Pyrosequencing Principle2.Adding of dNTP one-by-one3.E Km (DNA Pol, ATP sulfurylase, luciferase, Apyrase)4.Pyrogram read基因组学g
62、enomicswxj课件生物化学与分子生物学系PrincipleforDNAPyrosequencingKambara(2010)ChemicalRecord581.Pyrosequencing is based on detection of released PPi during DNA synthesis.2.In real practice, PPi is first converted to ATP by ATP sulfurylase in presence of APS, then ATP provides energy to luciferase to oxidize luci
63、ferin and generate bioluminescent light, detected by detector.3.Pyrosequencing is a continuous cycle of polymerization upon addition of 4 individual nucleotides stepwise, and companying with degradation of unincorporated nucleotides to prevent their interference with proper polymerization.4.Pyrosequ
64、encing system includes ssDNA as template & its primer, 4 enzymes (apyrase, luciferase, ATP sulfurylase, DNA polymerase), luminescence detector (560 nm), individual dNTP addition stepwise.Improvement in Pyrosequencing1.Replace dATP with dATPalphaS, low reaction noise.2.Add apyrase, improve 4-dNTP to
65、be incorporated sequentially due to removal of unincorporated nucleotide.3.Add ssDNA-binding protein, for long reading.1.PPi generated due to proper dNTP polymerization 2.ATP generated due to PPi supplied 3.Luminescent light generated due to ATP supplied, then detected for pyrogram基因组学genomicswxj课件生
66、物化学与分子生物学系PyrogramofPyrosequencinginLiquid-Phase59Proportional signals are obtained for one, two, three, and four base incorporations. Nucleotide addition, according to the order of nucleotides, is indicated below the pyrogram and the readout of pyrosequencing is indicated above pyrogram.To obtain r
67、apid polymerization, the nucleotide concentration added must be above the Km of DNA polymerase Based on the Km of 4 enzymes (Km from small to large), the sequential reaction could occur along first polymerization through ATP generation to luminescence light to final removal of unincorporated nucleot
68、ideThe key reactions during pyrosequencing are polymerization and removal of unincorporated nucleotide.Kinetic Constant of 4 Enzymes in PyrosequencingRonaghi (2001) Genome Res 基因组学genomicswxj课件生物化学与分子生物学系Solid&LiquidPyrosequencingRonaghi(2001)GenomeRes601.Primed DNA template and four enzymes involve
69、d in liquid-phase pyrosequencing are placed in a well of a microtiter plate. 2.The four different nucleotides are added stepwise. 3.The incorporated nucleotide is detected via capturing of the bioluminescent light by using the enzyme ATP sulfurylase and luciferase. 4.The unincorporated nucleotides a
70、re continuously degraded by the enzyme apyrase allowing addition of subsequent nucleotide. Liquid Pyrosequencing (4-E)1.The four different nucleotides are added stepwise to the immobilized primed DNA template2.The incorporation event is followed using the enzyme ATP sulfurylase and luciferase 3.Afte
71、r each nucleotide addition, a washing step is performed to allow iterative additionSolid Pyrosequencing (3-E)基因组学genomicswxj课件生物化学与分子生物学系61Schematic Finishing of ClonesBy IHGSC (2004) NatureAssembly of Sequenced DNA Fragments into Chromosomes, whole Genome Sequence基因组学genomicswxj课件生物化学与分子生物学系Bioinfo
72、rmaticsandComputerAnalysis(Websitestablep691,Clark2005)nThefieldofbioinformatics(programcollectionsondatabases)dealswiththecomputerizedanalysisoflargeamountsofsequencedata(nucleicacid,protein,SNP,etc.).nGenomeminingmeanstheapplicationofDatamining(viaprogramstofindusefulinformationbyfilteringorsiftin
73、gthroughthedata)togenomicdatabank.62基因组学genomicswxj课件生物化学与分子生物学系生物信息学n数据库的建设：数据库的建设：汇集了大量汇集了大量DNA序列、蛋白质信息序列、蛋白质信息n预测基因、基因产物的结构、功能；分析基因预测基因、基因产物的结构、功能；分析基因-基因、基因基因、基因-产物、产物产物、产物-产物之间的相互作用或联系；描述细胞或整产物之间的相互作用或联系；描述细胞或整体水平的基因表达谱；推测系统发生关系。体水平的基因表达谱；推测系统发生关系。n主要的生物信息中心主要的生物信息中心n美国国家生物技术信息中心（美国国家生物技术信息中心（N
74、CBI，http:/）n欧洲生物信息研究所（欧洲生物信息研究所（EBI，http:/ebi.ac.uk）n日本日本DNA数据库（数据库（DDBJ，http:/）nGenBank（http:/）基因组学genomicswxj课件生物化学与分子生物学系64Clark (2005) Molecular Biology p691基因组学genomicswxj课件生物化学与分子生物学系功能基因组学（functionalgenomics）n功能基因组学：功能基因组学：利用结构基因组学研究所得到的利用结构基因组学研究所得到的各种信息研究所有基因的生物学功能的学科。即各种信息研究所有基因的生物学功能的学科。即
75、从整体水平上研究一种组织或细胞在同一时间或从整体水平上研究一种组织或细胞在同一时间或同一条件下所表达基因的种类、数量、功能及在同一条件下所表达基因的种类、数量、功能及在基因组中的定位，或同一细胞在不同状态下基因基因组中的定位，或同一细胞在不同状态下基因表达的差异。表达的差异。n主要研究内容包括主要研究内容包括n基因组的表达基因组的表达n基因组功能注释基因组功能注释n基因组表达调控网络及机制基因组表达调控网络及机制基因组学genomicswxj课件生物化学与分子生物学系（一）通过全基因组扫描鉴定（一）通过全基因组扫描鉴定DNA序列中的基因序列中的基因n对测得的基因组序列进行对测得的基因组序列进行
76、“注释注释”，包括鉴定和描，包括鉴定和描述推测的编码序列、非编码序列及其功能。述推测的编码序列、非编码序列及其功能。n技术支持：人类基因组技术支持：人类基因组DNA序列数据库；高性能计序列数据库；高性能计算机。算机。n改进的十进制计算机进行全基因组扫描，鉴定内含改进的十进制计算机进行全基因组扫描，鉴定内含子与外显子之间的衔接，寻找全长子与外显子之间的衔接，寻找全长ORFs，确定多肽，确定多肽链编码序列。链编码序列。基因组学genomicswxj课件生物化学与分子生物学系StrategiestoGenomeMiningwithBioinformatics1.SelectDataofInteres
77、t2.Pre-processDataorDataCleaningremoveunnecessaryinformation3.TransformDataintoaFormatconvenientforanalysis4.ExtractPatternsandRelationshipfromdata5.Interpret&EvaluatePatternsandRelationshipviaapplicationofthemtopractice基因组学genomicswxj课件生物化学与分子生物学系n同源基因：同源基因：在进化过程来自共同的祖先的基因，通在进化过程来自共同的祖先的基因，通过核苷酸或氨基
78、酸序列的同源性比较，可以推测基过核苷酸或氨基酸序列的同源性比较，可以推测基因组内相似基因的功能。因组内相似基因的功能。n有效工具：有效工具：NCBI的序列局部相似性查询（的序列局部相似性查询（BasicLocalAlignmentSearch，BLAST）程序。）程序。n操作流程：操作流程：GenBank中查找基因序列访问号码中查找基因序列访问号码（accessionnumber），在），在BLAST界面上输入界面上输入2条或条或多条访问号码，就可实现两两或多对序列的比对。多条访问号码，就可实现两两或多对序列的比对。（二）通过（二）通过BLAST等程序搜索同源基因等程序搜索同源基因基因组学ge
79、nomicswxj课件生物化学与分子生物学系AnalysisPerformedonDNASequenceswithBioinformaticsA.HomologsAnalysisviacomparisonofsequenceofinterestwiththerelatedavailableindatabasetoinferitsfunctionortracetheevolution.B.CodonBiasAnalysistolocatecodingregions.Theideaistruethattheremustbedifferenceincodonfrequencybetweencodin
80、g-andnon-coding-DNAdueto3rdbaseredundancyandpreferentialuseofsomecodonsoverothers(withincodingregions,butnotinrandom,intergenicDNA).C.SearchforKnownConsensusSequences,suchasmotifandconsensusdomain,etc.基因组学genomicswxj课件生物化学与分子生物学系（三）通过实验设计验证基因功能n转基因（转基因（transgene）n基因过表达（基因过表达（overexpression）n基因敲除（基因敲
81、除（knock-out）n基因敲减（基因敲减（knock-down）或基因沉默）或基因沉默（genesilencing）n合适的模式生物替代人体进行实验合适的模式生物替代人体进行实验基因组学genomicswxj课件生物化学与分子生物学系（四）通过转录组和蛋白质组描述基因表达模式n基因表达：基因表达：RNA的转录和蛋白质的翻译的转录和蛋白质的翻译n基因的表达模式及调控描述：借助转录组学基因的表达模式及调控描述：借助转录组学和蛋白质组学相关技术与方法和蛋白质组学相关技术与方法基因组学genomicswxj课件生物化学与分子生物学系DNAMicroarrays&Microchips(chip)i.
82、Aserialsequencesofknowngenesarrayedonchip.ii.SamplemRNAsconvertedtocDNAs,labeledwithCyDyes,mixedinequal,hybridizedtogenechip.iii.laser-scannedwithdifferentwavelength,individual&overlappedimageanalyzedwithsoftwareforgeneID&geneabundancebasedonthearraypositionandcolordensityofspotsofsamples.Functional
83、GenomicsTechniquestheEraofthepost-genome-sequencing基因组学genomicswxj课件生物化学与分子生物学系731.A serial of known genes spotted on chip.2.Sample mRNA1 & mRNA2 labeled with different color CyDye, mixed in equal, hybridized together to chip.3.Laser-scanned of chip with different wavelength, individual & overlapped image analyzed with program for gene relative abundance (spot color) and gene ID (spot position).Weaver (2001) Molecular Biology 2nd p Functional Genomics : DNA MicroarrayABCD基因组学genomicswxj课件
