分子遗传学理论与技术基础 Chapter IV.DNA recombinant technology

IV.DNArecombinanttechnology1.conceptDNArecombinant.Genecloneormolecularclone.Geneengineering.*Thatmeanstocut,modifyandrejointhedifferentDNAmoleculeswithtoolenzyme,toreformanewreconmolecular,therecontransfertargetgenefragmentintotherecipient(host)celltoclonalamplify,so,wecangetmuchOftargetgenecopies,thatiscalledgenecloning.ifthetargetgenecanexpressinhostcellinordertoreformthehostcell’strait,thatiscalledgeneengineering.Abovetechnologyprocessisalsocalledacommunityname---DNArecombinant.2.TechnologystepsofDNArecombinant.鄂p341,p348图15-5.(1)toisolatethetargetgeneDNAfragmentandthevectorDNA,thentomodifywithsametoolenzyme.(2)ligasereactionligatethetargetgeneDNAandvectorDNAintorecon.(3)therecontransformatethehostcell.thereisonlyonereconinonehostcell.(4)thetransformatedhostcellsareculturedinplatemedium,thentoscreenthetargetgenecellcloneforcloneamplify.3.Thekeyknowledgepoints:1)ToolEnzyme.鄂P299.[concept]:therearemanytypesenzymes,Theirfunctionaredifferent,thataregotfrombiont’scells(muchfrombacteria).Theseenzymesworkasusefultoolsingeneoperationexperiment,so,becalledtoolenzyme.*typesandfunctionoftoolenzyme.(1)RestrictionEndonuclease(RE).*REcanidentifyaspecificbasesequenceofdsDNAmolecular,thentohydrolysisthephosphodiesterbondofdsDNAinendodigetionway.*thetypesofRE:TypeIspecificityunstrength.TypeIIITypeIIdigetionpointisspecific,veryuseful.therearenear100kinds.鄂p304表。*thebioactivityofREtypeII

鄂P301.[reactionenvironment]~temperature:37℃generally,stockat-20℃and-70℃forlongtime.~reactionpH7.2-7.6~helperfactors:[Mg]++,BSA(牛血清白蛋白)[reactionactivity]REactivityunit(u):undercomfortablecon-dition,the1uenzymevolumecandigest1ugDNAin50ulreactionsolutionin1htime.[reactionresults]Bluntend:HapIdigested:5’GTTAAC3’3’CAATTG5’Cohesiveend,EcoRIdigested:5’GAATTC3’3’CTTAAG5’*thedifferentREcanidentifythedifferentseque-ncesinDNA,andhavethedifferentdigestedresult.

(2)DNApolymerase。鄂p305.DNApolymerasecancatalysisthenucleo-tides(dNTP)polymerazatioinobeytheDNAtemplatestrandtosynthesisanewDNAstrand.TherearedifferenttypesofDNApolymerase,thathavedifferentfunction.Forexample:E.coliDNApolymeraseI.TaqDNApolymerase.(3)Ligase鄂p306.Ligasecancatalysistheligation（连接反应）ofDNA’sorRNA’snick.thatdividedtoDNAligaseandRNAligase.forexample,thefunctionofT4DNAligasearetoligate:*1]singlestrandnickofdsDNA.2]cohesiveendoftwodsDNA.3]bluntendoftwodsDNA.4]singlestrandnickofDNA/RNAhybridmolecular.(4)Reversetrascriptase(RTase)

鄂p306.RTasealsocanbecalledRNAdependentDNApolymerase,tocatalysiscDNAstrandpolymeraztionobeymRNAtemplate.mRNA5’AAA—AAA3’cDNA3’TTT----TTT5’(5)Methylase(甲基化酶)鄂p307ThemethylaseactataspecificDNAsequenceofsomeREidentifiedtocatalysisDNAbemethylatedatthespecificsite.ThemethylatedDNAcandefensetobedigestedbyrelativeRE.Forexample:the

EcoRI.MethylaseactattheEcoRI.REidentifysequencesspecifically,tocatalysisDNAbemethylatedatthespecificsite.5’-------GAA*TCC---------3’

CH3(鄂p308表14-6)(6)Exonuclease鄂p308Theexonucleasecancatabolic(降解)theterminalnucleotideofDNAmolecularselectively.5’3’3’5’5’3’3’5’(7)RNase.RNasecancatalysistheRNAmolecularcatabolicing.*RNaseAtype:underhigherdensity,itcancutanypointinRNAmolecular.but,underlowerdensity,itcanonlycut‘c’and‘u’points.*RNaseT1type:beusedtoRNAsequencingandtodeleteRNAmixtureinDNAsample.*RNaseHtype:beusedtocatabolicRNAstrandofRNA/DNAhybridmolecules.(8)DNaseI(现代分子生物学手册p169)DNasecancatalysisDNAmolecularcatabolicing.Ifwecontrolthereactiondensityandthereactiontime,thenickfrequencyofDNAmolecularcanbecontrolled.2)Howtogetthetargetgenefragments?(1)toscreenthetargetgenecellclonefromgenomelibrarywithprobe,thentoCloneamplify.(2)toisolategenomeDNA-->southernblottinghybridization-->totakethetargetgenefragmentsfromblottingfilm.(3)toscreenthetargetcDNAcellclonefromcDNAlibrary,thentocloneamplify.(4)toamplifytargetgenefragmentswithPCRtechnology.3)VectorDNA鄂P309.[concept]:vectorDNAisatoolDNAmole-cular,thatcancarryexogenousDNAintothehostcell,thentoselfduplicationinsomeway.[characterofvectorDNA]:(1)thatisasmallDNAfragment(3—10kb),andcanduplicateinhostcell.(2)thereareseveralsinglerestrictionpoint,thatisoutofreplicon(复制子)regionforexogenousDNAinsertion.(3)afterexogenousDNAinsertion,thevec-torduplicationcan’tbeinfluenced.(4)thereareselectivemarkersforscreenthepositivecellclone.Forexample:theAprandtheTetresistancegene.(5)thereisaregulateregiontopromotethetargetgeneexpression.*鄂P311图。

*forexample:pBR322.aplasmidvector.[thetypesofvector]:expressionvector.clonevector.insertionvector.replacementvector.*usualapplicablevectors:plasmidDNA.(tocarry<15kb)phage入DNA.(tocarry10—100kb)artificialchromosomevector:PAC(>100kb)BAC(>300kb)YAC(2Mb)4)Joiningreactionofthetarget

DNAandthevectorDNA.鄂p346.[principle]:afterREcutandmodify,thetargetDNAfragmentandthevectorDNAarejoinedtogetherintotherecon(重组子)bysomeway.[resultevaluate]:(1)highrecombinantfreq