廣州市番禺區(qū)番禺大道一期工程施工課件

TheHumanGenomeProjectTheHumanGenomeProject1IntroductionUntiltheearly1970’s,DNAwasthemostdifficultcellularmoleculeforbiochemiststoanalyze.DNAisnowtheeasiestmoleculetoanalyze–wecannowisolateaspecificregionofthegenome,produceavirtuallyunlimitednumberofcopiesofit,anddetermineitsnucleotidesequenceovernight.MolecularBiologyOfTheCell.Albertsetal.491-495IntroductionUntiltheearly192IntroductionAttheheightoftheHumanGenomeProject,sequencingfactoriesweregeneratingDNAsequencesatarateof1000nucleotidespersecond24/7.TechnicalbreakthroughsthatallowedtheHumanGenomeProjecttobecompletedhavehadanenormousimpactonallofbiology…..MolecularBiologyOfTheCell.Albertsetal.491-495IntroductionAttheheightoft3HumanGenomeProjectGoals:

■identifyalltheapproximate30,000genesinhumanDNA,■determinethesequencesofthe3billionchemicalbasepairsthatmakeuphumanDNA,■storethisinformationindatabases,■improvetoolsfordataanalysis,■transferrelatedtechnologiestotheprivatesector,and■addresstheethical,legal,andsocialissues(ELSI)thatmayarisefromtheproject.

Milestones:■1990:ProjectinitiatedasjointeffortofU.S.DepartmentofEnergyandtheNationalInstitutesofHealth

■June2000:Completionofaworkingdraftoftheentirehumangenome(covers>90%ofthegenometoadepthof3-4xredundantsequence)■February2001:Analysesoftheworkingdraftarepublished■April2003:HGPsequencingiscompletedandProjectisdeclaredfinishedtwoyearsaheadofscheduleU.S.DepartmentofEnergyGenomePrograms,GenomicsandItsImpactonScienceandSociety,2003HumanGenomeProjectGoals:

U.S4Whatdoesthedrafthumangenomesequencetellus?

BytheNumbers?Thehumangenomecontains3billionchemicalnucleotidebases(A,C,T,andG).

?Theaveragegeneconsistsof3000bases,butsizesvarygreatly,withthelargestknownhumangenebeingdystrophinat2.4millionbases.

?Thetotalnumberofgenesisestimatedataround30,000--muchlowerthanpreviousestimatesof80,000to140,000.

?Almostall(99.9%)nucleotidebasesareexactlythesameinallpeople.

?Thefunctionsareunknownforover50%ofdiscoveredgenes.U.S.DepartmentofEnergyGenomePrograms,GenomicsandItsImpactonScienceandSociety,2003Whatdoesthedrafthumangeno5Whatdoesthedrafthumangenomesequencetellus?HowIt'sArranged?Thehumangenome'sgene-dense"urbancenters"arepredominantlycomposedoftheDNAbuildingblocksGandC.

?Incontrast,thegene-poor"deserts"arerichintheDNAbuildingblocksAandT.GC-andAT-richregionsusuallycanbeseenthroughamicroscopeaslightanddarkbandsonchromosomes.

?Genesappeartobeconcentratedinrandomareasalongthegenome,withvastexpansesofnoncodingDNAbetween.

?Stretchesofupto30,000CandGbasesrepeatingoverandoveroftenoccuradjacenttogene-richareas,formingabarrierbetweenthegenesandthe"junkDNA."TheseCpGislandsarebelievedtohelpregulategeneactivity.

?Chromosome1hasthemostgenes(2968),andtheYchromosomehasthefewest(231).

U.S.DepartmentofEnergyGenomePrograms,GenomicsandItsImpactonScienceandSociety,2003Whatdoesthedrafthumangeno6Whatdoesthedrafthumangenomesequencetellus?

TheWheatfromtheChaff?Lessthan2%ofthegenomecodesforproteins.

?Repeatedsequencesthatdonotcodeforproteins("junkDNA")makeupatleast50%ofthehumangenome.

?Repetitivesequencesarethoughttohavenodirectfunctions,buttheyshedlightonchromosomestructureanddynamics.Overtime,theserepeatsreshapethegenomebyrearrangingit,creatingentirelynewgenes,andmodifyingandreshufflingexistinggenes.

?Thehumangenomehasamuchgreaterportion(50%)ofrepeatsequencesthanthemustardweed(11%),theworm(7%),andthefly(3%).U.S.DepartmentofEnergyGenomePrograms,GenomicsandItsImpactonScienceandSociety,2003Whatdoesthedrafthumangeno7Whatdoesthedrafthumangenomesequencetellus?

HowtheHumanCompareswithOtherOrganisms

?Unlikethehuman'sseeminglyrandomdistributionofgene-richareas,manyotherorganisms'genomesaremoreuniform,withgenesevenlyspacedthroughout.

?HumanshaveonaveragethreetimesasmanykindsofproteinsastheflyorwormbecauseofmRNAtranscript"alternativesplicing"andchemicalmodificationstotheproteins.Thisprocesscanyielddifferentproteinproductsfromthesamegene.

?Humanssharemostofthesameproteinfamilieswithworms,flies,andplants;butthenumberofgenefamilymembershasexpandedinhumans,especiallyinproteinsinvolvedindevelopmentandimmunity.

?AlthoughhumansappeartohavestoppedaccumulatingrepeatedDNAover50millionyearsago,thereseemstobenosuchdeclineinrodents.Thismayaccountforsomeofthefundamentaldifferencesbetweenhominidsandrodents,althoughgeneestimatesaresimilarinthesespecies.Scientistshaveproposedmanytheoriestoexplainevolutionarycontrastsbetweenhumansandotherorganisms,includingthoseoflifespan,littersizes,inbreeding,andgeneticdrift.

U.S.DepartmentofEnergyGenomePrograms,GenomicsandItsImpactonScienceandSociety,2003Whatdoesthedrafthumangeno8Whatdoesthedrafthumangenomesequencetellus?

VariationsandMutations?Scientistshaveidentifiedabout3millionlocationswheresingle-baseDNAdifferences(SNPs)occurinhumans.Thisinformationpromisestorevolutionizetheprocessesoffindingchromosomallocationsfordisease-associatedsequencesandtracinghumanhistory.

?Theratioofgermline(spermoreggcell)mutationsis2:1inmalesvsfemales.Researcherspointtoseveralreasonsforthehighermutationrateinthemalegermline,includingthegreaternumberofcelldivisionsrequiredforspermformationthanforeggs.

U.S.DepartmentofEnergyGenomePrograms,GenomicsandItsImpactonScienceandSociety,2003Whatdoesthedrafthumangeno9Whatdoesthedrafthumangenomesequencetellus?Ledtothediscoveryofwholenewclassesofproteinsandgenes,whilerevealingthatmanyproteinshavebeenmuchmorehighlyconservedinevolutionthanhadbeensuspected.Providednewtoolsfordeterminingthefunctionsofproteinsandofindividualdomainswithinproteins,revealingahostofunexpectedrelationshipsbetweenthem.MolecularBiologyOfTheCell.Albertsetal.491-495Whatdoesthedrafthumangeno10Whatdoesthedrafthumangenomesequencetellus?Bymakinglargeamountsofproteinavailable,ithasyieldedanefficientwaytomassproduceproteinhormonesandvaccinesDissectionofregulatorygeneshasprovidedanimportanttoolforunravelingthecomplexregulatorynetworksbywhicheukaryoticgeneexpressioniscontrolled.MolecularBiologyOfTheCell.Albertsetal.491-495Whatdoesthedrafthumangeno11Howdoesthehumangenomestackup?Howdoesthehumangenomestac12?Genenumber,exactlocations,andfunctions?Generegulation?DNAsequenceorganization?Chromosomalstructureandorganization?NoncodingDNAtypes,amount,distribution,informationcontent,andfunctions?Coordinationofgeneexpression,proteinsynthesis,andpost-translationalevents?Interactionofproteinsincomplexmolecularmachines?Predictedvsexperimentallydeterminedgenefunction?Evolutionaryconservationamongorganisms?Proteinconservation(structureandfunction)?Proteomes(totalproteincontentandfunction)inorganisms?CorrelationofSNPs(single-baseDNAvariationsamongindividuals)withhealthanddisease?Disease-susceptibilitypredictionbasedongenesequencevariation?Genesinvolvedincomplextraitsandmultigenediseases?Complexsystemsbiologyincludingmicrobialconsortiausefulforenvironmentalrestoration?Developmentalgenetics,genomics

FutureChallenges:WhatWeStillDon’tKnowU.S.DepartmentofEnergyGenomePrograms,GenomicsandItsImpactonScienceandSociety,2003?Genenumber,exactlocations13AnticipatedBenefitsofGenomeResearchMolecularMedicine

?improvediagnosisofdisease

?detectgeneticpredispositionstodisease

?createdrugsbasedonmolecularinformation

?usegenetherapyandcontrolsystemsasdrugs

?design“customdrugs”(pharmacogenomics)basedonindividualgeneticprofiles

MicrobialGenomics?rapidlydetectandtreatpathogens(disease-causingmicrobes)inclinicalpractice

?developnewenergysources(biofuels)

?monitorenvironmentstodetectpollutants

?protectcitizenryfrombiologicalandchemicalwarfare

?cleanuptoxicwastesafelyandefficientlyU.S.DepartmentofEnergyGenomePrograms,GenomicsandItsImpactonScienceandSociety,2003AnticipatedBenefitsofGenome14RiskAssessment?evaluatethehealthrisksfacedbyindividualswhomaybeexposedtoradiation(includinglowlevelsinindustrialareas)andtocancer-causingchemicalsandtoxinsBioarchaeology,Anthropology,Evolution,andHumanMigration?studyevolutionthroughgermlinemutationsinlineages

?studymigrationofdifferentpopulationgroupsbasedonmaternalinheritance

?studymutationsontheYchromosometotracelineageandmigrationofmales

?comparebreakpointsintheevolutionofmutationswithagesofpopulationsandhistoricalevents

U.S.DepartmentofEnergyGenomePrograms,GenomicsandItsImpactonScienceandSociety,2003AnticipatedBenefitsofGenomeResearch-cont.RiskAssessmentU.S.Departmen15DNAIdentification(Forensics)?identifypotentialsuspectswhoseDNAmaymatchevidenceleftatcrimescenes

?exoneratepersonswronglyaccusedofcrimes

?identifycrimeandcatastrophevictims

?establishpaternityandotherfamilyrelationships

?identifyendangeredandprotectedspeciesasanaidtowildlifeofficials(couldbeusedforprosecutingpoachers)

?detectbacteriaandotherorganismsthatmaypolluteair,water,soil,andfood

?matchorgandonorswithrecipientsintransplantprograms

?determinepedigreeforseedorlivestockbreeds

?authenticateconsumablessuchascaviarandwine

U.S.DepartmentofEnergyGenomePrograms,GenomicsandItsImpactonScienceandSociety,2003AnticipatedBenefitsofGenomeResearch-cont.DNAIdentification(Forensics)16Agriculture,LivestockBreeding,andBioprocessing

?growdisease-,insect-,anddrought-resistantcrops

?breedhealthier,moreproductive,disease-resistantfarmanimals

?growmorenutritiousproduce

?developbiopesticides

?incorporateediblevaccinesincorporatedintofoodproducts

?developnewenvironmentalcleanupusesforplantsliketobacco

U.S.DepartmentofEnergyGenomePrograms,GenomicsandItsImpactonScienceandSociety,2003AnticipatedBenefitsofGenomeResearch-cont.Agriculture,LivestockBreedin17AnticipatedBenefits:?improveddiagnosisofdiseaseearlierdetectionofgeneticpredispositionstodiseaserationaldrugdesigngenetherapyandcontrolsystemsfordrugspersonalized,customdrugs

MedicineandtheNewGeneticsU.S.DepartmentofEnergyGenomePrograms,GenomicsandItsImpactonScienceandSociety,2003GeneTestingPharmacogenomicsGeneTherapyAnticipatedBenefits:Medicine18ELSI:Ethical,Legal,

andSocialIssues?Privacyandconfidentialityofgeneticinformation.?Fairnessintheuseofgeneticinformationbyinsurers,employers,courts,schools,adoptionagencies,andthemilitary,amongothers.?Psychologicalimpact,stigmatization,anddiscriminationduetoanindividual’sgeneticdifferences.?Reproductiveissuesincludingadequateandinformedconsentanduseofgeneticinformationinreproductivedecisionmaking.

?Clinicalissuesincludingtheeducationofdoctorsandotherhealth-serviceproviders,peopleidentifiedwithgeneticconditions,andthegeneralpublicaboutcapabilities,limitations,andsocialrisks;andimplementationofstandardsandquality?controlmeasures.U.S.DepartmentofEnergyGenomePrograms,GenomicsandItsImpactonScienceandSociety,2003ELSI:Ethical,Legal,

andSoc19ELSIIssues(cont.)?Uncertaintiesassociatedwithgenetestsforsusceptibilitiesandcomplexconditions(e.g.,heartdisease,diabetes,andAlzheimer’sdisease).?Fairnessinaccesstoadvancedgenomictechnologies.?Conceptualandphilosophicalimplicationsregardinghumanresponsibility,freewillvsgeneticdeterminism,andconceptsofhealthanddisease.?Healthandenvironmentalissuesconcerninggeneticallymodified(GM)foodsandmicrobes.?Commercializationofproductsincludingpropertyrights(patents,copyrights,andtradesecrets)andaccessibilityofdataandmaterials.U.S.DepartmentofEnergyGenomePrograms,GenomicsandItsImpactonScienceandSociety,2003ELSIIssues(cont.)?Uncertain20BeyondtheHGP:What’sNext?HapMapSystemsBiologyExploringMicrobialGenomesforEnergyandtheEnvironmentChartgeneticvariation

withinthehumangenomeBeyondtheHGP:What’sNext?Ha21GenomestoLife:

ADOESystemsBiologyProgramExploringMicrobialGenomesforEnergyandtheEnvironmentGoals?identifytheproteinmachinesthatcarryoutcriticallifefunctions

?characterizethegeneregulatorynetworksthatcontrolthesemachines

?characterizethefunctionalrepertoireofcomplexmicrobialcommunitiesintheirnaturalenvironments

?developthecomputationalcapabilitiestointegrateandunderstandthesedataandbegintomodelcomplexbiologicalsystemsGenomestoLife:

ADOESystem22HapMapAnNIHprogramtochartgeneticvariation

withinthehumangenome?Begunin2002,theprojectisa3-yearefforttoconstructamapofthepatternsofSNPs(singlenucleotidepolymorphisms)thatoccuracrosspopulationsinAfrica,Asia,andtheUnitedStates.

?Consortiumofresearchersfromsixcountries?ResearchershopethatdramaticallydecreasingthenumberofindividualSNPstobescannedwillprovideashortcutforidentifyingtheDNAregionsassociatedwithcommoncomplexdiseases?MapmayalsobeusefulinunderstandinghowgeneticvariationcontributestoresponsesinenvironmentalfactorsHapMapAnNIHprogramtochart23DideoxySequencingDideoxysequencing(alsocalledchain-terminationorSangermethod)usesanenzymaticproceduretosynthesizeDNAchainsofvaryinglengths,stoppingDNAreplicationatoneofthefourbasesandthendeterminingtheresultingfragmentlengths.Eachsequencingreactiontube(T,C,G,andA)inthediagramcontainsaDNAtemplate,aprimersequence,andaDNApolymerasetoinitiatesynthesisofanewstrandofDNAatthepointwheretheprimerishybridizedtothetemplate;thefourdeoxynucleotidetriphosphates(dATP,dTTP,dCTP,anddGTP)toextendtheDNAstrand;onelabeleddeoxynucleotidetriphosphate(usingaradioactiveelementordye);andonedideoxynucleotidetriphosphate,whichterminatesthegrowingchainwhereveritisincorporated.TubeAhasdidATP,tubeChasdidCTP,etc.DideoxySequencingDideoxysequ24DideoxySequencingForexample,intheAreactiontubetheratioofthedATPtodidATPisadjustedsothateachtubewillhaveacollectionofDNAfragmentswithadidATPincorporatedforeachadeninepositiononthetemplateDNAfragments.Thefragmentsofvaryinglengtharethenseparatedbyelectrophoresisandthepositionsofthenucleotidesanalyzedtodeterminesequence.Thefragmentsareseparatedonthebasisofsize,withtheshorterfragmentsmovingfasterandappearingatthebottomofthegel.SequenceisreadfrombottomtotopDideoxySequencingForexample,25DideoxySequencingPolyacrylamidegelwithsmallporesisusedtofractionatesingle-strandedDNA.Inthesizerange10to500nucleotides,DNAmoleculesthatdifferinsizebyonlyasinglenucleotidecanbeseparatedLane1-partialreplicasterminatinginGLane2-partialreplicasterminatinginALane3-partialreplicasterminatinginTLane4-partialreplicasterminatinginCAlbertsetal.MolecularBiologyoftheCell.DideoxySequencingPolyacrylami26Brown.Genomes2Brown.Genomes227CapillaryElectrophoresisTechniquecombinestheuseofgel-filledcapillarytubeswithauniquelaserscanningsystemtosequenceeachofthefourdifferenttypesofbases--adenine,cytosine,guanine,andthymine--inasampleofDNA.CapillaryarrayelectrophoresiscutsthetimeoftheSangerdideoxymethoddownbyreplacingtheslabwithhundredsoftinygel-filledcapillaries,about100microns(fourthousandthsofaninch)ininternaldiameter,thatcanbebundledintoasinglearrayforautomateddetection.CapillaryElectrophoresisTechn28CapillaryElectrophoresisThedideoxymethodwasimprovedbytheuseoffluorescentlabels.Theprimerissynthesizedandsplitintofourbatches,eachofwhichislabeledwithadifferentfluorescentdye.Eachdyelabeledprimerisusedinasequencingreactionwithoneofthedideoxynucleotides.Thereactionproductsarepooledandanalysedinasinglelaneofasequencinggel.Afour-colourfluorescencedetectormonitorstheDNAasitmigratestothebottomofthegel.Thefluorescencesignatureisusedtoidentifytheterminalnucleotide.CapillaryElectrophoresisThed29CapillaryElectrophoresisAmethodwasthendevelopedbasedonthiswherefluorescentdideoxynucleotideswereusedinasinglesequencingreaction.Thefragmentsarethenseparatedinasinglelaneofasequencinggelandidentifiedbythefluorescentsignature.Thelimitationsofgelelectrophoresissoonbecameapparent.Gelstakealongtimetorunandhavealimitedreproducibility.Anautomatedmethodwasmuchmoredesirable,buttheautomationofgelsrequirescomplexrobotichandling.Theuseofcapillariesallowsmuchhigherelectricalfieldstobeusedmakingtheseparationsfaster.Flexiblecapillariesarealsoeasilyincorporatedintoanautomatedinstrumentmakingsequencingcheap,fastandefficient.Howeverasinglecapillaryisstillabottleneckinthesequencingprocess.Ageliseasilycapableofrunningupto96samplessimultaneously.Toovercomethisainstrumentfittedwithanarrayofcapillarieswasdeveloped.Inthefirstinstrumentsthedetectormovedacrossthearray,butthetimelagmeanssomeinformationcanbemissed.Nowallthecapillariesaresimultaneouslymonitoredusinganarrayofphotodiodes.CapillaryElectrophoresisAmet30CapillaryElectrophoresishttp://elchem.kaist.ac.kr/BK21_SMS.web/2001_instanal/FIG/20011029/0003_ANG_2000_04463_DNA_CAE.pdfCapillaryElectrophoresishttp:31CapillaryElectrophoresisCapillaryholdsasievingmedium,whichallowsseparationofDNAfragmentsbasedontheirsizeSampleisinjectedintothecapillarybyplacingtheendofthecapillaryinthesamplesolution,andapplyingelectriccurrent.Afterinjectioniscomplete,thesampleisreplacedbyrunningbufferandelectricfieldisreappliedtodrivethesamplesthroughthecapillary.Laser-inducedfluorescencedetectorneartheendofthecapillaryrecordsthefluorescentsignalinfourdifferentchannelstoresolvethefluorescentsignalfromthefourdyes.http://elchem.kaist.ac.kr/BK21_SMS.web/2001_instanal/FIG/20011029/0003_ANG_2000_04463_DNA_CAE.pdfCapillaryElectrophoresisCapil32CapillaryElectrophoresisSmallinnerdiameterofcapillarytubereducesJouleheatingtonegligiblelevels,allowingtheuseofextremelyhighvoltage,forrapidsequencingProvidesa2-foldimprovementoverslabgelsequencingmethodshttp://elchem.kaist.ac.kr/BK21_SMS.web/2001_instanal/FIG/20011029/0003_ANG_2000_04463_DNA_CAE.pdfCapillaryElectrophoresisSmall33CapillaryElectrophoresis/ce/ce.htmlCapillaryElectrophoresishttp:34AutomatedSequencingAutomatedSequencing35廣州市番禺區(qū)番禺大道一期工程施工課件36Brown.Genomes2Brown.Genomes237ShotgunSequencingBacteriophagefX174,wasthefirstgenometobesequenced,aviralgenomewithonly5,368basepairs(bp).FredericSanger,inanotherrevolutionarydiscovery,inventedthemethodof"shotgun"sequencing,astrategybasedontheisolationofrandompiecesofDNAfromthehostgenometobeusedasprimersforthePCRamplificationoftheentiregenome.TheamplifiedportionsofDNAarethenassembledbytheiroverlappingregionstoformcontiguoustranscripts(otherwiseknownascontigs).Thefinalstepinvolvedtheutilizationofcustomprimerstoelucidatethegapsbetweenthecontigsthusgivingthecompletelysequencedgenome.Sangerfirstused"shotgun"sequencingfiveyearslatertocompletethebacteriophagelsequencethatwassignificantlylarger,48,502bp.Thismethodallowedsequencingprojectstoproceedatamuchfasterratethusexpandingthescopeofrealisticsequencingventure.Sangerfirstused"shotgun"sequencingfiveyearslatertocompletethebacteriophagelsequencethatwassignificantlylarger,48,502bp./course/projects/final-4/ShotgunSequencingBacteriophag38ShotgunSequencingAteamheadedbyJ.CraigVenterfromtheInstituteforGenomicResearch(TIGR)andNobellaureateHamiltonSmithofJohnsHopkinsUniversity,sequencedthe1.8MbbacteriumwithnewcomputationalmethodsdevelopedatTIGR'sfacilityinGaithersburg,Maryland.Previoussequencingprojectshadbeenlimitedbythelackofadequatecomputationalapproachestoassemblethelargeamountofrandomsequencesproducedby"shotgun"sequencing.Inconventionalsequencing,thegenomeisbrokendownlaboriouslyintoordered,overlappingsegments,eachcontainingupto40KbofDNA.Thesesegmentsare"shotgunned"intosmallerpiecesandthensequencedtoreconstructthegenome.Venter'steamutilizedamorecomprehensiveapproachby"shotgunning"theentire1.8MbH.Influenzaegenome./course/projects/final-4/ShotgunSequencingAteamheade39ShotgunSequencingPreviously,suchanapproachwouldhavefailedbecausethesoftwaredidnotexisttoassemblesuchamassiveamountofinformationaccurately.Software,developedbyTIGR,calledtheTIGRAssemblerwasuptothetask,reassemblingtheapproximately24,000DNAfragmentsintothewholegenome.AftertheH.Influenzaegenomewas"shotgunned"andtheclonespurifiedsufficientlytheTIGRAssemblersoftwarerequiredapproximately30hoursofcentralprocessingunittimeonaSPARCenter2000containinghalfagigabyteofRAM./course/projects/final-4/ShotgunSequencingPreviously,40ShotgunSequencingVenter'sH.InfluenzaeprojecthadfailedtowinfundingfromtheNationalInstituteofHealthindicatingtheseriousdoubtssurroundinghisambitiousproposal.Itsimplywasnotbelievedthatsuchanapproachcouldsequencethelarge1.8Mbsequenceofthebacteriumaccurately.Venterprovedeveryonewrongandsucceededinsequencingthegenomein13monthsatacostof50centsperbasewhichwashalfthecostanddrasticallyfasterthanconventionalsequencing./course/projects/final-4/ShotgunSequencingVenter'sH.41Dr.J.CraigVenterEarnedhisPh.D.inPhysiologyandPharmacologyfromtheUniversityofCaliforniaatSanDiegoandbecamearesearcherattheNationalInstitutesofHealth.WhileservingfirstasaSectionChiefandthenasaLabChiefintheNationalInstituteofNeurologicalDisordersandStroke,hedevelopedexpressedsequencetagsorEST’s,arevolutionarynewstrategyforgenediscovery.In1992,heandhiswife,Dr.ClaireFraser,foundedTheInstituteforGenomicResearchknownasTIGR,whereheservedasPresidentandChiefScientificOfficeruntil1998.Dr.VenterandhisteamatTIGRdecodedthegenomeofthebacteriumHaemophilusinfluenzae,makingitthefirstfree-livingorganismtohaveitsfullDNAdecipheredandtodatehavesequencedover30genomes.HenowservesasChairmanoftheBoardofTrusteesofTIGR.In1998,hefoundedCeleraGenomicsandannouncedthatCelerawoulddecodethehumangenomefasterandmoreeconomicallythanthepubliclyfundedconsortiumofscientists.Thatchallengeisnowcreditedwithcreatingtheclimateofurgencythatspurredcompetitionandsubstantiallyacceleratedtheproject’ssuccessfulconclusion.AttheWhiteHousepressconferenceannouncingthesequencingofthehumangenome,PresidentBillClintoncalledit“themostimportant,mostwondrousmapeverproducedbymankind.”Dr.J.CraigVenterEarnedhis42mRNASequencingAnalternativestrategyforsequencingwaspresentedduringtheearly90’sbyCraigVenter.ThefunctionalportionofthehumanDNAsupposedlyaccountsforlessthan10%(perhapslessthan5%)oftheentirehumangenome.TheHGPstrategy,i.e.sequencingeverything,couldbeconsideredaslavishnesswithresourcessincethemainpartoftheinformationlacksrelevance.ThescopeofthestrategybyVenterandco-workersistofocustheinvestigationstomessengerribonucleicacid(mRNA)insteadofDNA.ThepointofusingmRNAisthatitdoesnotincludeanynon-codingDNA.ThemRNAmoleculecanbeisolatedandusedasatemplatetosynthesizeacomplementaryDNA(cDNA)strand,whichcanthenbeusedtolocatethecorrespondinggenesonachromosomemap.ByusingVenter′smethodanincompletecopyofthegene,calledexpressedsequencetag(EST),isacquired.ESTsareusefulforlocalizingandorientingthemappingandsequencedatareportedfrommanydifferentlaboratoriesandserveaslandmarksonthedevelopingphysicalmapofthehumangenome.http://wcn.ntech.se/history.htmlmRNASequencingAnalternative43WholeGenomeShotgunSequencingVenterandco-workerssuccessin1995ofsequencingtheH.influenzaebacteriumintroducedamethodcalled"wholegenomeshotgunsequencing".Theshotgunmethodinvolvesrandomlysequencingtinyclonedsectionsofthegenome,withnoforeknowledgeofwhereonachromosomethesectionoriginallycamefrom.Thepartialsequencesobtainedarethenreassembledtoacompletesequencebyuseofcomputers.Theadvantagewiththismethodisthatiteliminatestheneedfortime-consumingmapping.Bycompeting(andcooperating)thegovernmentallyfinancedhumangenomeproject(HGP)andtheprivatebiotechnologycompanyCelerahascompletedareferenceDNAsequenceofthehumangenome.BothpartiesmadetheirinformationsimultaneouslyavailableinFebruary2001,bypublishingitinontheInternetandinthescientificjournalsNatureandScience.http://wcn.ntech.se/history.htmlWholeGenomeShotgunSequencin44WholeGenomeShotgunSequencing

Afterthesequenceisshotgunnedthe10millionfragmentsofthegenomicjigsawpuzzleneedtoberecompiledintothereadablebasepairsintheproperorder.Thismethod