生理化學課件：Recombinant DNA Technology

RecombinantDNATechnologyFactorVIIIandHemophiliaAThrombosisandThrombolysisβ-glucocerebrosidaseandGaucherDiseaseRecombinantDNAisaformofsyntheticDNAthatisengineeredthroughthecombinationorinsertionofoneormoreDNAstrands.

Clone:Agroupoforganismsorcellsproducedasexuallyfromoneancestororstock,towhichtheyaregeneticallyidentical.Cloning:PropagationofaDNAsequencebyincorporatingitintoahybridconstructthatcanbereplicatedinahostcell.Reproductivecloning:Processofmakingageneticallyidenticalcopyofanorganism.Therapeuticcloning:Processofmakingmultiplecopiesofacelltotreatadisease.RecombinantDNAWhatisClonedDNAUsedFor?Workoutthefunctionofanormalgene.Lookathowmutationsmayaffectagene’sfunction.Makelargeconcentrationsoftheproteincodedforbythegene.GenetherapyRestrictionEndonuclease

GGATCCCCTAGGGCCTAGGATCCG+BamHⅠMolecularToolsinRecombinantDNATechnologyArestrictionendonucleaseisanenzymethatcutsdouble-strandedorsinglestrandedDNAatspecificrecognitionnucleotidesequencesknownasrestrictionsites.AlongwithWernerArberandHamiltonSmith,DanielNathansreceivedtheNobelPrizeinPhysiologyorMedicinein1978forthediscoveryofrestrictionenzymes.Nomenclature

ofRestrictionEndonucleaseHindⅢ

Genus

Species

Strain

OrderHaemophilusinfluenzaedstrainIIISincetheirdiscoveryinthe1970s,morethan100differentrestrictionenzymeshavebeenidentifiedindifferentbacteria.Eachenzymeisnamedafterthebacteriumfromwhichitwasisolatedusinganamingsystembasedonbacterialgenus,speciesandstrain.CharacteristicsofRestrictionEndonuclease

GGATCCCCTAGGPalindromeApalindromeisaword,phrase,numberorothersequenceofunitsthatcanbereadthesamewayineitherdirectionExamplesofRestrictionEnzymesDNALigaseDNAligasecanlinktogethertwoDNAstrandsthathavesingle-strandbreaks.Thealternative,adouble-strandbreak,isfixedbyadifferenttypeofDNAligaseusingthecomplementarystrandasatemplatebutstillrequiresDNAligasetocreatethefinalphosphodiesterbondtofullyrepairtheDNA.ThemechanismofDNAligaseistoformtwocovalentphosphodiesterbondsbetween3'hydroxylendsofonenucleotidewiththe5'phosphateendofanother.ATPisrequiredfortheligasereaction.DNAligaserepairingchromosomaldamage3

GCTTAA

OH3

5

3

5

GAATTCTTAA5

3

5

DNAPolyEcoRⅠCTGCACG3

Exonuclease

5

3

5

3

G

5

3

5

PstⅠ

5

3

5

5

3

3

5

3

OHGGGGGdGTPTDTTerminalDeoxynucleotideTransferaseReverseTranscriptasereversetranscriptase

RNARNA5′3′AAAAAAAAAAAATTTTTT5′3′cDNAcDNArandomprimerOligo-dTPreparationofInterestedDNAArtificialsynthesizingDNAorcDNAlibraryPCRorRT-PCRFromDNALibraryAlibraryisacollectionofmoleculesinastableformthatrepresentssomeaspectofanorganism.TwocommontypesoflibrariesarecDNAlibraries(formedfromComplementaryDNA)andgenomiclibraries.Thenucleotidesequencesofinterestarepreservedasinsertstoaplasmidorthegenomeofabacteriophagethathasbeenusedtoinfectbacterialcells.PolymeraseChainReactionThe

(PCR)isusedto

amplify

asinglecopyorafewcopiesofapieceof

DNA

acrossseveralordersofmagnitude,generatingthousandstomillionsofcopiesofaparticular

DNAsequence.RT-PCRRT-PCR:TheRNAstrandisfirstreversetranscribedintoitscomplementaryDNA,followedbyamplificationoftheresultingDNAusingPCR.SelectControlElementsDNAVectorsＡ

vectorisanyvehicleusedtotransferforeigngeneticmaterialintoacell.Allvectorshaveanoriginofreplication,amulticloningsite,andaselectablemarker.

Vectorcanbedividedtocloningvectorandexpressionvector.

CloningVector：PlasmidCloninglimit:0.1-10kilobases(kb)Aplasmidisanextra-chromosomalDNAmoleculeseparatefromthechromosomalDNAinbacteriawhichiscapableofreplicatingindependentlyofthechromosomalDNA.Inmanycases,itiscircularanddouble-stranded.Therearetwotypesofplasmidintegrationintoahostbacteria:Non-integratingplasmidsreplicateaswiththetopinstance;whereasepisomes,theleftexample,integrateintothehostchromosome.ComponentofPlasmidVectorInsertionofforiegnDNAintotheMCSlocatedwithinthelacZgenecausesinsertionalinactivationofthisgeneattheN-terminalfragmentofbeta-galactosidaseandabolishesintra-alleliccomplementation.ThusbacteriacarryingrecombinantplasmidsintheMCScannothydrolyseX-gal,givingrisetowhitecolonies,whichcanbedistinguishedonculturemediafromnon-recombinantcells,whichareblue.CloningVector：ShuttleVectorAshuttlevectorisavector(usuallyaplasmid)constructedsothatitcanpropagateintwodifferenthostspeciesFusionExpressionVectorSecretedExpressionVectorSignalpeptideompaEukaryoticExpressionVectorReportGeneVectorTissueSpecificityVectorGeneorRNATransferPhysicalChemicalBiologicalPhysicalTransferofNakedDNAMicroinjection:bigcells,25%Electroporation:150kbDNABallistictransfer(genegun)Usedfortissuesthatareeasilyaccessibletodirectinjectionsuchasskinandmusclecells.

TheexpressionhasbeenverylowAntisensecomplementsmRNA(sense)andpreventsproteinexpression.SmallinterferingRNA(siRNA)molecules.DoublestrandedoligodeoxynucleotidesasadecoyforthetranscriptionfactorsOligonucleotidesNakedDNA-calciumphosphateco-precipitationLipoplexes:Liposome-DNAPolyplexes:PLA(poly-lacticacid),PLGA(poly-lactide-co-glycolide)ChemicalTransferLiposome/Polycation/DNAComplexes

CompetentCellTransformationTransfectionInfectionRetroviralVectorHIV,Simianimmunodeficiencyvirus,andFelineinterfusionVirusLongincubationperiod2005,clinicaltrialHemophillia,diabeticwithPDGF(platelet-derivedgrowthfactor)LentiviralVectorsAdenovirusVectorsDouble-strandedlinearDNAVirusResponsiblefor5–10%ofupperrespiratoryinfectionsinchildrenDNAmoleculeisleftfreeinthenucleusofthehostcellThedescendantsofthecellwillnothavetheextragenegenerationDeletedregionInsertedsizeTime(days)Hostimmuneresponse1stE1/E37.5kb7~10strongest2ndE1/E3,E210kb20~403rd(gutlessvector)All,expectITR&packagingseq36kb84leastSelectionSouthernBlot

ASouthernblotistocheckforthepresenceofaDNAsequenceinaDNAsample.SouthernblottingcombinesagarosegelelectrophoresisforsizeseparationofDNAwithmethodstotransferthesize-separatedDNAtoafiltermembraneforprobehybridization.FISHisacytogenetictechniquethatcanbeusedtodetectandlocalizethepresenceorabsenceofspecificDNAsequencesonchromosomes.Itusesfluorescentprobesthatbindtoonlythosepartsofthechromosomewithwhichtheyshowahighdegreeofsequencesimilarity.FluorescenceInSituHybridizationNorthernBlotThenorthernblotistostudygeneexpression.IttakesitsnamefromitssimilaritytotheSouthernblottechnique.ThemajordifferenceisthatRNA,ratherthanDNA,isanalyzedinthenorthernblot.WesternBlot

Thewesternblotistodetectspecificproteinsinagivensampleoftissuehomogenateorextract.Itusesgelelectrophoresistoseparatenativeordenaturedproteinsbythelengthofthepolypeptideorbythe3-Dstructureoftheprotein.Theproteinsarethentransferredtoamembrane,wheretheyaredetectedusingantibodiesspecifictothetargetprotein

DNASequencing

DNAsequencingencompassesbiochemicalmethodsfordeterminingtheorderofthenucleotidebasesinaDNAoligonucleotide.Sequencingmethodshave