生物化學英文版課件：Chapter 9 DNA-based Information Technologies

Chapter9DNA-basedInformationTechnologiesStudyinggenesandtheirproductsUsingDNA-basedmethodstounderstandproteinfunctionGenomicstohumanstoryPaulBergwasawardedone-halfofthe1980NobelPrizeinChemistry,for"hisfundamentalstudiesofthebiochemistryofnucleicacids,withparticularregardtorecombinantDNA“.HerbertBoyerandStanleyCohencombinedtheireffortsinbiotechnologytoinventamethodofcloninggeneticallyengineeredmoleculesinforeigncells.StudyinggenesandtheirproductsDNAcloning

Aclone

isanidenticalcopy

howtoperformDNAcloning

(themethod:recombinantDNAtechnologyofgeneticengineering)DNAcloningprocedures

CuttingDNAatpreciselocations,byusingrestrictionendonucleases;Selectvectors(plasmid):capableofself-replications;JoiningtargetDNAandvectorcovalently(recombinantDNA)byusingDNAligase;TransformrecombinantDNAtohostcells;IdentifyhostcellscontainingrecombinantDNA;Mostcommonusedhostcells:bacterium

Escherichiacoli(E.coli).

ThreetypesofrestrictionendonucleasesTypeI:TheycleavetheDNAatnon-specificsitesthatare~1000bpormorefromtherecognitionsequence.TypeII:TheycleavetheDNAwithinorveryclosetotherecognitionsequences.TypeIII:TheycleavetheDNAabout25to27bpfromtherecognitionsequence.

People

Richardson,CharlesC.

Professor

DNAligaseDNAkinaseDNAexonuclease

DNAligaseDNAkinaseRNApolymeraseCharlesC.RichardsonHarvardMedicalSchoolJerardHurwitzTheMemorialSloan-KetteringInstituteDiscoverersofDNAligaseRestrictionendonucleases(restrictionenzymes)Foundindifferentbacteriaspecies;RecognizeandcleaveDNAatspecificsites;TypeIIismostlyused(noATPisrequired);

Therecognitionsequencesare4-6bplongandpalindromic;Cloningvector(plasmid)

circularDNA,replicateseparatelyfromhostchromosome;anoriginofreplication:10-20copiespercell;antibioticsmaker.(tetR,ampR)severalREsitesforcloning;smallsize4,362bp:easytransformationandmanipulation.pBR322:pBR322usedforcloningofforeignDNAinE.coliBacteriophage

cloningvector

About1/3of

genomeisnotessential;ForeignDNAsizecanbeupto23kblong;Bacterialartificialchromosomes(BACs)

cloningofverylongDNAfragments(100-200kb);containsantibioticsmarkerandreplicationorigin;1-2copypercell;transformationmethod:electroporation;Hostcells:havingmutationsthatfacilitateuptakingoflargeDNA.Yeastartificialchromosome(YAC)

yeast(S.cerevisiae)genome:14x106bp(eukaryoticcell)

shuttlevector:replicateincellsoftwoormorespecies;

YAC:ori,selectivemarkers;

CENandTELsequences(requiredforchromosomemaintainence)DNAtransformationmethodsChemicals:CaCl22)Electrophoration3)GeneGun4)MicroInjectionThehostcellsmustbepreparedtobecompetenttotakeupforeignDNA.Cell-freesystemBacterialsystemYeastInsectMammalian

ProteinexpressionsystemsClonedgenescanbeexpressedtoamplifyproteinproductionAtypicalE.coliexpressionvector

usedforexpressionof

recombinantproteinsinbacteria;

Promoter:instructRNApolymerasetobind;

overexpression:recombinantproteinsrepresent10%oftotalcellularproteins.Advantages

FastgrowthCheapmediumandequipmentforgrowingDisadvantages

differentfrequencieswithwhichthedifferentcodonsappearingenesoftheseorganisms

E.g.CGT,CGC,CGG,AGG,AGA,CGAcodeforarginine,butthelast3(AGG,AGA,CGA)arerarelyusedinE.coliandithaslowamountsofrespectivetRNAs.differencesinpost-translationalmodificationsBaculovirusexpressionsystem

Afamilyoflargerod-shapedviruses(桿狀病毒)Circulardouble-strandedgenomerangingfrom

80-180kb.AcMNPV:Autographacalifonicamultiplenuclearpolyhedrosisvirus

（多核型多角體病毒）:thebestcharacterizedandundergoesasuccessionofearly,lateandverylategeneexpressionduringitsinfectioncycle.:thestrongpolyhedrin

（多角蛋白）

promoter->thetranscriptionalcontrol.HomologousrecombinationbetweentransfervectorandbaculovirusgenomerecombinantbaculovirusgenomeGofIGofIbac.transfervectorbaculovirusgenomepolyhedringenehomologousrecombinationTransfectinsectcellsHarvestrecombinantbaculovirusTransfectinsectcellsforProteinproduction

RapidgenerationofmultigeneinbaculovirusesTwotransfervectors,eachexpressingtwogenesrecombinewiththebaculovirusgenomemaintainedinE.coliasabacmid.Twogeneticlociinthebaculovirusgenomeareused,recombinationiscontrolledbyTn7transpositionthebacmidDNAistransfectedintoinsectcells,whereitdirectsexpressionofallfourgenesathighlevels.Usedforassemblingofcorrectlyprocessedandfoldedtetramericproteincomplexes.

Site-directedmutagenesis

tostudythefunctionofproteintwoapproaches:changeaDNAfragment;pointmutation;Tags(fusedtotargetprotein)areusedinproteinpurificationTag:ageneencodingapeptideorproteinthatbindstoaknownligand.GSH(tag)(ligand)Non-covalentlybindingUsingGSTtaginProteinpurification

ElutewithhighsaltbufferorbuffercontainingfreeGSHGenesequencescanbeamplifiedwithpolymerasechainreaction(PCR)KaryB.Mullis

PCRreactionrequires:primers,DNApolymerase;dNTPs;PCRcandetectandamplifyaslittleasoneDNAmolecule;PCRProcessDNAamplifiedbyPCRcanbeclonedQuantitativePCR(實時定量PCR,qPCR)qPCRisalaboratorytechniqueofmolecularbiologybasedonthePCR,whichisusedtoamplifyandsimultaneouslyquantifyatargetedDNAmolecule.

Double-strandedDNA-bindingdyesasreporters5’3’5’3’ExcitationEmissionSGSGSGSGSGReporterQuenchingmol

TargetDNA(doublestranded)ProbesandthecomplementaryDNAstrandarehybridized.DuringPCR,theprobeisdegradedbytheTaqpolymeraseandthefluorescentreporterrelease.ReporterreleasedFluorescentreporterprobes(eg.TaqMan)detectonlytheDNAcontainingtheprobesequence,significantlyincreasingspecificityThreshold(閾值)：3-15個PCR循環(huán)的熒光信號標準偏差的10倍Thenumberofcyclesatwhichthefluorescenceexceedsthethresholdiscalledthethresholdcycle(Ct)典型PCR擴增曲線

AnalysismethodofaclonedgeneSouthernBlot:thedetectionofageneofinterestbyprobingDNA;2.NorthernBlot:probeRNAonagelwithaDNAprobe;3.WesternBlot:probeproteinsonagelwithanantibody.UsingDNA-basedmethodstounderstandproteinfunctionConstructionofacDNAlibraryfrommRNA

Reversetranscriptase:synthesizeDNAonanRNAorDNAtemplate;cDNA:complementaryDNAs;EST:expressedsequencetagDuplexDNAsareclonedintovector.SequenceorstructuralrelationsprovideinformationonproteinfunctionThegenesinhumanchromosome9andmousechromosome2exhibitaveryhighdegreeofhomologyandthesamegeneorder.

MethodstodetermineaproteinfunctionSequencesimilarity,orstructuralrelationshipprovidesinformationforproteinfunction.1)Iftheaminoacidsequencesoftwoproteinsfromtwodifferentorganismsarehomologous,forexample,30%identityand55%similarity,thetwoproteinsverypossiblyhavesimilarbiologicalfunctionincells.2)Iftwoproteinsdonothavesimilaraminoacidsequences,butdosharesimilarthreedimensionalstructureandhaveknownfunctionaldomainssuchas

ATPbindingandhydrolysisdomains.

Itispossiblethatthetwoproteinshavesimilarfunction.Inmanycases,anewlyidentifiedproteindoesnothaveevidentsequentialorstructuralrelationshiptoknownproteins.Wewillusethefollowingmethodstodetermineitsfunction.Determinewhenandwherethisproteinappears,orinwhichtissue,oratwhatstageofdevelopment.Ingeneral,particularlyfortheseproteinswhosefunctionsarestrictlyregulated,proteinsareproducedjustbeforetheirfunctionsarerequiredforcomingbiologicalevents.

1).Biochemicalreactions,subcellularlocalization,WesternBlottinganalysiscanbeusedtoexaminetheexistenceofaprotein.2).Protein-proteininteraction(invivo,andinvitro)3).Proteomics(two-dimensionalelectrophoresis)ispopulartodetermineexistenceandamountofproteinsatlargescale.GreenFluorescentProtein(GFP)Composedof238aminoacidsThemonomercomposedofacentral

-helixsurroundedbyanelevenstrandedcylinderofanti-parallel

-sheetsFluorophorelocatedoncentralhelixTheactivesiteofGFPSer65-Tyr66-Gly67DeprotonatedphenolateofTyr66iscauseoffluorescenceFluorophore（熒光發(fā)色團）formation咪唑酮nmFluorescenceexcitationandemissionspectraofnativeGFPfromAequoreavictoria(Tsienetal.,1998)Excitation（激發(fā)）Emission（發(fā)射）GFP吸收的光譜：最大峰值為395nm（紫外），并有一個峰值為470nm的副峰（藍光）；發(fā)射光譜最大峰值為509nm（綠光），并帶有峰值為540nm的側(cè)峰（Shouder).2008NobelPrizeinChemistry

(forthediscoveryanddevelopmentofthegreenfluorescentprotein,GFP")OsamuShimomuraJellyfish水母/bioluminescencePurifiedandcharacterizedGFP(1974)MartyChalfieObtainedtheGFPgene(gfp)clonefromPrasherin1992.Promoter::GFPPromoter::GFP::GeneexpressionvectorstoshowtheWhenandwheregenesareexpressed.RogerTsienFiguredouthowGFPglowsMadevariantswithdifferentcolorsGFP(greenfluorescentproteins)isusedinbiologicalresearchCloningofcDNAnexttoageneforGFPcreatesareporterconstruct,andthemRNAtranscriptisthenexpressedasafusionprotein.TheGFPpartoftheproteinisvisibleinthefluorescencemicroscope.Theproteinsexpressedonlyinthefour"touch"neurons.Directimmunofluorescence

Indirectimmunofluorescence:Indirecttestisadouble-layertechniqueTheunlabelledantibodyisapplieddirectlytothetissuesubstrateTreatedwithafluorochrome-conjugatedanti-immunoglobulinserum

Advantage:Becauseseveralfluorescentanti-immunoglobulinscanbindtoeachantibodypresentinthefirstlayer,thefluorescenceisbrighterthanthedirecttest.Itismoretime-efficientsinceitisonlyonesignallabelledreagent,theanti-immunoglobulin,ispreparedduringthelengthyconjugationprocessIndirectimmunofluorescenceofiron-regulatedcellwallmannoproteinFIT1ofS.cerevisiae

Usingepitopetagtostudyprotein-proteininteraction

Epitopetag:shortproteinsequencethatisboundbymonoclonalantibody;Newproteinscanbeidentifiedbymassspectrometry.PrincipleYeastTwo-hybridSystem

(studyprotein-proteininteraction)YeastTwo-hybridSystem

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Thetwofusionsarecreatedinseparateyeaststrains,whicharethenmated.Selection:Onlyreportergeneisexpressed,theyeastcansurvive.haploidHowtostudycellularexpressionlevelsofgenes?Photolithography

（光刻）techniqueforpreparingaDNAmicroarray;nucleotideprecursorsaresynthesizedinaphotoreaction.DNAmicroarray

TheDNA(PCRamplifiedorsynthesized)ispositionedonasolidsurface(usuallyspeciallytreatedglassslides);

Toexaminechangesingeneexpressionduringdifferentgrowthstagesorothercircumstances.Whatisagenome?Genome:AlloftheDNAforanorganismHumanGenome:Nucleus:3.2billionbasepairspackagedintochromosomesMitochondrion:16,600basepairspackagedinonecircularchromosomeSizeofDNAmoleculesandgenomesOrganismNumberofbasepairs(kb)Totallength(

m)

VirusPolyoma,SV405.11.7T739.913

16655BacteriaMycoplasmahominis760260Escherichiacoli4,7001,360EukaryotesYeast(S.cerevisiae)13,5004,600Drosophila165,00056,000Human2,900,000990,000SouthAmericanlungfish102,000,00034,770,000

Initiated1990Completionoriginallyplannedfor2005FinishedsequenceanticipatedSpring,2003,tocommemoratethe50thAnniversaryofWatsonandCrickpublication(Nature171:737-738,April25,1953);HumanGenomeProject

(J.CraigVenterandFrancisS.Collins)GenomicsequencingtimelineThehumangenomeprojectstrategy(shotgunmethod)

EachcontigcontainedSTSs(sequence-tagged

site)atadistanceofvery100kb;SequencingmappedBACorYACclones;Identifyoverlappingregion.Sequence-taggedsite(STS，序列標簽位點

):arelativelyshortsequence(200to500bp)thathasasingleoccurrenceinthegenomeandwhoselocationandbasesequenceareknown(mapped).Contig(重疊群):asetofoverlappingclonesTheproportionsofhumangenomemadeupofvarioustypesofsequencesSaccharomycescerevisiae~5570–5651Schizosaccharomycespombe~4940Fruitfly~13000Nematodeworm~18000Human~30000-35000OrganismsNumberofproteinsNumberofproteinsindifferentorganisms

Late1980s,Slabgelsequencersusingradioactiveisotopes(10Kb/4hrrun);Late1990s,Cappillarysequencers(50Kb/hrrun);2005,Massiveparallelpyrosequencing(20Mb/5hrrun);2007,Sequencingbysynthesis(IIIlumina)(1Gb/5dayrun)2010,Singlemoleculesequencing(100Gb/5dayrun);DevelopmentofDNAsequencingtechnologiesNextgenerationsequencingplatformsPyrosequencing

(454GSFLXsequencing)Basicidea:VisiblelightisgeneratedandisproportionaltothenumberofincorporatednucleotidesDNAPolymeraseIpyrophospateFromfireflies,oxidiz