基因体计画及应用

IntroductionofGenomicsandProteomicsHumangenomeproject基因體計畫及應用

Outline:Humangenomeproject(HGP)geneticmapandphysicalmapBioinformaticsandgenomicsdatabase:NCBI/ENSEMBLcomparativegenomicsapplicationsFromhumangenomeprojectTheHumanGenomeProject(HGP)isaninternational13-yeareffortformallybeguninOctober1990.Theprojectwasplannedtolast15years,butrapidtechnologicaladvancesacceleratedthecompletionto2003.Projectgoalsweretodeterminethecompletesequenceofthe3billionDNAbases,identifyallhumangenes,andmakethemaccessibleforfurtherbiologicalstudy./sci/techresources/Human_Genome/project/about.shtmlHumangenomeproject(HGP)1988年，美國國家衛生研究院(NIH)和能源部(DOE)規劃組成一個委員會，提出”人類基因體計劃”humangenomeproject一個五年整合型的研究計劃。1990年執行[1990.11.01]StructuralGenomics:Thestudyofgenomesincludingnucleotidesequencegenecontentorganization(chromatinremodeling,DNApacking.....)genenumberFunctionalGenomics:Thestudyofthefunctionofsequencesidentifiedbystructuralgenomics.Structuralvs.FunctionalGenomicsGeneticMaps:

measure“genetic”distanceBasedonrecombinationfrequenciesbetweenlociDistancesarecMormapunits(%recombination)Lowresolution(distancesmaybeinaccurate)

遺傳連鎖圖譜大約的距離，非絕對!!PhysicalMaps:實體圖譜

measuredistanceinbasepairsBasedondirectanalysisofDNA.Methodsinclude:-restrictionmapping

(RFLPrestrictionframentlengthpolymorphism)-FISH(Fluorescenceinsituhybridization)-DNAsequencingmoreaccurateanddetailed,eveninunknownfunctionalsequenceStructuralGenomicsConnectionbetweenthesetwomaps,----1cM≈1MbAbridgeofphysicalandgeneticmaps-cytologicalmapsTofindthecorrelationbetweenphysicalandgeneticmaps

[it’seasytodistinguishthechromosomebythestainedbands]GoalsofHGPCreatephysicalmapofthe24humanchromosomes(22autosomes,X&Y)Identifytheentiresetofgenes&mapthemalltotheirchromosomesDeterminethenucleotidesequenceoftheestimated3billionbasepairsAnalyzegeneticvariationamonghumansMapandsequencethegenomesofmodelorganisms(模式生物)U.S.DepartmentofEnergyGenomePrograms,GenomicsandItsImpactonScienceandSociety,2003GeneticvsphysicalmapsThegenomedraftwasannouceedbytwoinstitutesin2001：Celeragenomics(foundedin1998)(NCHGR)NationalCenterforHumanGenomeResearch,NIH

TechnologiesusedintheHGPHGPwasaidedbyseveralmajortechnologiesbelow:Geneticmarkers(forphysicalmap)ThePCRSangerDNAsequencingLarge-insertcloningsystems(cosmid,YAC,BAC)Capillary-basedsequencing(毛細管定序)andmethodsforgenotypingSNP(single-nucleotidepolymorphisms)Vectorsusedinthehumangenomeproject

建立基因體圖書館(genomiclibrary)Vectorsneeded:Yeast&bacterialartificialchromosomes Cloningcapacity;cosmid~50Kb(cossite-carryingplasmid)Yeastartificialchromosomes(YAC) Largecapacity&selfreplicating 1,000,000+ntcapacity

Limitions:Inefficient;Isolation;Unstable(linear);CrypticBacterialartificialchromosome(BAC) BasedonFandF’plasmidsthatconjugatebetweenbacterialcells Mobilizethewholehostchromosomeafterinsertionbetweencells 300,000ntcapacity“MolecularBiology”byRobertF.Weaver2ndEdition,McGrawHillPublishing,2002“MolecularBiology”byRobertF.Weaver2ndEdition,McGrawHillPublishing,2002Yeastartificalchromosomes(YAC)AdvantageofYAC.Inplasmidvectors,sequencesupto10-15kb,inλphagevectorssequencesupto22kbandincosmidvectorssequencesupto40kbcanbecloned.Sequencesthatareseveralhundredkilobasepairs(upto1Mb)inYAC.

However,twomajordisadvantageoftheYACcloningsystemCloningefficiencyislow(1000clones/mgDNAasagainst106-107clones/mgDNAforcosmids

Oftenproduceschimerashttp://www.studentsguide.in/biotechnology-genomics/cloning-expression-vectors/yeast-artificial-chromosomes-yacs.html“MolecularBiology”byRobertF.Weaver2ndEdition,McGrawHillPublishing,2002Bacterialartificalchromosomes(BAC)台灣–榮陽團隊(107bases)107/3x109

約0.3%chromosome4Wholegenomeshotgunmethod(散彈槍策略法)

Top-down:先建基因圖譜(map)-clonebycloneBotton-up:霰彈槍法(wholegenomeshotgunmethod)StrategiesforgenomesequencingCelera主要採取的方法HierarchicalSequencingandWholeGenomeShotgunSequencing©Gibson&MuseAPrimerofGenomeSciencefromGibson&Muse,APrimerofGenomeSciencehttp:///genomics/[Clonebyclonestrategy]直接定序!!先將每個clone的相對序列決定出來，再決定每一段序列(HGP)SequencingofGenomes

TheClone-by-CloneStrategyMapping(genetically&physically)thewholegenome UseoverlappingclonesClone-by-Clonesequencingstrategy Lookingfor“flagposts”Toolsformappingofgenes:1.RestrictionFragmentLengthPolymorphisms(RFLPs) Usetodeterminetheposition/locationofageneorastretchofDNA HowtolookforRFLPs?2.VariableNumberofTandemRepeats(VNTRs) Repeatedsequencesintandemderivedfromminisatellites3.SequenceTaggedSites(STSs) Short(60-1000bp)sequencesdetectablebyPCR4.Microsatellites:repeatsofveryshortsequences Highlypolymorphic,thusgeneticmappingispossible Usefulinphysicalmappingorlocatingspecificsequenceinthegenome“MolecularBiology”byRobertF.Weaver2ndEdition,McGrawHillPublishing,2002PrimersforPCRweredesignedfromsequencesofsmallareasofDNAthatwerealreadyknown

“MolecularBiology”byRobertF.Weaver2ndEdition,McGrawHillPublishing,2002GenerationsofGeneticMarkers1st:RestrictionFragmentLengthPolymorphism(RFLP) BotsteinD.etal AJHG32:314(1980)

Donis-KellerH.etal Cell51:319(1987)2nd

MicrosatelliteMarkers NIH/CEPHcollaborativeMappinggroup. Science258:67(1992)

WeissenbachJ,etal Nature359:794(1992)

Buetowetal Nat.Gent.6:391(1994)

Gyapayetal Nat.Gent.7:246(1994) Murrayetal Science265:2049(1994) TheUtahMarkerDevelopmentGroup AJHG57:619(1995) Dib,C.etal Nature380:152(1996)

Broman,KWetal, AJHG63:861(1998) 3rd:SingleNucleotidePolymorphism(SNP)Markers Wang,DGetal Science280:1077(1998)

Altshuler,D.etal Nature407:513(2000)http:///portal/server.pt/gatewayTraditionalDNAsequencingCapillaryElectrophoresisSmallinnerdiameterofcapillarytubereducesJouleheatingtonegligiblelevels,allowingtheuseofextremelyhighvoltage,forrapidsequencingProvidesa2-foldimprovementoverslabgelsequencingmethodshttp://elchem.kaist.ac.kr/BK21_SMS.web/2001_instanal/FIG/20011029/0003_ANG_2000_04463_DNA_CAE.pdfCapillaryElectrophoresisProvidesa2-foldimprovementoverslabgelsequencingmethodshttp://elchem.kaist.ac.kr/BK21_SMS.web/2001_instanal/FIG/20011029/0003_ANG_2000_04463_DNA_CAE.pdf/sci/techresources/Human_Genome/education/images.shtmlMITWhiteheadInstituteforBiomedicalResearchsince1990大約8百萬元!Large-scaleDNAsequencing,SangerInstitute,UKhttp://www.sanger.ac.ukShorttaggedtractsofDNAsequence(200to500basepairs)Uniqueandhasasingleoccurrenceinthehumangenome.Theirlocationandbasesequenceareknownthereforetheycanbeusedaslandmarksingenomemapping.

Canbedetectedbythepolymerasechainreaction(PCR)inthepresenceofallothergenomicsequences.Usefulformappingthesequencedatareportedfromdifferentlaboratoriesandprovideaframeworkforthephysicalmapofthehumangenome.TheoverwhelmingadvantageofSTSovermappinglandmarksdefinedthroughothermethodsisthatthemethodcanbecompletelydescribedasinformationinadatabase.http:///dbSTS/index.htmlWhat’sSequenceTaggedSite(STS?)Olsonetal,1989最常被使用的界標(landmark)STS-contentmapofaHumanchromosomeOlsonetal,1989

YACisolationandcontigassemblyYeastArtificalChromosome(YAC)可任意選取STS為固定位置的界標利用STS篩選有重疊序列的YAC，建立基因圖譜(physicalmap)。CeleraScaffolds

URLlinksfromtheslide:/cgi/content/full/291/5507/1304NationalCenterforBiotechnologyInformation(NCBI)NCBIestablishedin1988asadivisionoftheNationalLibraryofMedicine(NLM)attheNationalInstitutesofHealth(NIH)TheGenBanksequencedatabase-->isanannotatedcollectionofallpubliclyavailablenucleotidesequencesandtheirproteintranslations.http://UniSTS

ontheGenBankDatabaseattheNCBIproviding"aunified,non-redundantviewofSTS's.UniSTSintegratesmarkerandmappingdatafromavarietyofpublicresources."UsedineGenomeasasourceofSTS’s,andtocollectandmanageelementnames.http:///entrez/query.fcgi?db=unistsDevelopedby(EMBL-EBI)and(SangerCenter

)AddressedontheautomaticannotationforeukaryoticgenomeEnsembl歐洲分子生物實驗室Ensemblgenomebrowser()http:///Homo_sapiens/searchview?_q=D7S1760http:///Homo_sapiens/markerview?marker=sWSS2009http:///Homo_sapiens/contigview?l=7:105348185-105348493;context=10000MethodologyResolution

cytogeneticchromosomebandingseveralMbchromosomebreakpointmapsRadiationhybrid>=0.5MbrestrictionmapNotImapsseveralhundredkbforrare-cuttersclonecontigmapoverlappingYACorcosmidclones40~sveralhundredKbSTSmaporderedSTSstensofKbESTmapmappingcDNAbacktootherphysicalmaps

~90KbDNAsequencingmapCompleteDNAsequence1bpTypeofmapDifferenttypesofphysicalmapsforhumangenomePredictionofnumberofhumangenesHomologsearchesagainstknowndatabases(GenBank,EMBL….)(ht/;http://www.ebi.ac.uk)2.Exonprediction(GENSCAN),http:///GENSCAN.html3.gene-findingsoftwarepackages:GENSCAN,GRAIL,BLASTandetc.(NIXanalysis)overprediction:“geneclusters”inaverylargegeneorartifactual

cDNAs*underprediction:verysmallexonsandrestrictedexpressionoruntranslatedRNA20000-30000protein-codinggenestherepeatsequencesmayinfluencethedynamicsandstructureofchromosomes.significantvariationsbetweenlengthofgenesinbalanceddistributionofgenesonthechromosome