Lecture 12 - molecular genetic analysis_FA-1

Lecture 12 - molecular genetic analysis_FA-1 - Lecture 12...

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Unformatted text preview: Lecture 12 05-25-11 Chapter 19 MolecularGeneticAnalysisand GeneticEngineering 1. PCRandGelElectrophoresis 2. MolecularHybridization 3. RestrictionEnzymesandGeneCloning 4. PlantTransformation MolecularTechniques MolecularanalysisofDNAorgeneweareinterested IsolateaspecificDNAfragmentfromtheremainderof theDNA  ­Humangenome3.2billionbp.  ­Thegeneweareinterestedis3000bp  ­Targetonemillionthofthegenome(needleinthehaystack)  Makemanycopiesofitforfurtheranalysis  ­amplifyinvitro polymerasechainreaction(PCR)inmachine  ­amplifyinvivo(Cloning) recombination/vectorconstruction andtransformationinhostcell PolymeraseChainReaction(PCR) Moleculartechnique,developedbyKaryMullisin1983,toamplify theDNAfragments ThebasisofPCRisreplicationofDNAfragmentcatalyzedbya DNApolymerase. TwoessentialrequirementsforPCR TemplateDNA,fromwhichanewDNAstrandcanbe synthesized. Apairofprimers(15 ­40bp  ­OHgrouptowhich newnucleotidescanbeadded.  ­Theprimershaveaknownsequenceandcanhybridizeor annealtothetemplateDNAattheregionwith complementarysequences  ­Theprimersaredesignedbytheresearcherand synthesizedbyspecializedcompanies PCRSteps Threerecurringsteps(generally30 ­40timesorcycles): 1)denaturation meltingthedsDNAtossDNAbyheating to94 C 2)primerannealing coolthereactiontoatemperature between35and65 C 3)primerextensionat72 Cusingtheheat ­stable polymerase  Thishappensinaprogrammablemachineknownasa thermalcycler   PCRforAmplification Theamplificationisexponential http://media.pearsoncmg.com/bc/bc_russell_igenetics_1/media/ianimations/a08b/index.html GelElectrophoresis AstandardmoleculartechniqueforseparatingDNAfragments(or othermolecules)onthebasisoftheirsizeandelectricalcharge.  ­DNAisnegativelychargedasaresultofthephosphategroups  ­Whenplacedunderanelectricfielditwillmigratetotheanode(+).  ­ Porousmedium(oftenmade fromagarose,likeJell ­O)is usedtoseparatefragmentsof DNA,RNAorproteinsasthey passthroughtheelectricfield.  ­ Thegelissubmersedinalow ­ saltbuffertoallowelectricity togothroughthegel,andto maintainpH Gel Electrophoresis Procedure Smallerfragments travelfasterthanlarger fragments. http://www.dnalc.org/resources/animations/gelelectrophoresis.html VisualizetheDNAFragmentsinGel Afterelectrophoresis,theDNAfragmentsareseparatedonthegel Ethidiumbromide(orothernucleicacids ­specificdye)intercalates betweenthebasesofDNA,whichproducebrightorange fluorescesunderUVlight. ApplicationsofPCR DetectthepresenceofaparticularDNAsequenceinasample geneisolation Example:detectthepresenceofvirusesinblood DesignprimerfromHIVvirus,ifvirusinbloodtheHIV primerwillannealtotheDNAtemplatefromtheblood sample,andamplifyit.TheamplifiedHIVDNAfragment  Identifygeneticvariation Plantandanimalbreeding Identifyindividualplantsthathaveadiseaseresistantgene Selectindividualsbasedongenotypeinsteadofphenotype, especiallywhenthetraithardtovisualize. Forensicapplications(CSI!)  Paternitytesting Whoisthefather? GeneticVariationDetectedbyPCR toidentify suspects PCRissensitiveenough toallowamplificationof DNAfromfingerprints andcigarettebutts.  MolecularHybridization DenaturationandRenaturationofNucleicAcids Heatingoralkalinecauseshydrogenbondstobreak(denature) dsDNAssDNA Withcoolingcomplementarybasepairsre ­form(renature) ssDNAdsDNA Molecular Hybridization Basedonconceptsof denaturation/renaturation, ssDNA(orRNA)canbe renatured(hybridized)thatdo notoriginatefromthesame nucleicacidsource. RestrictionEnzymes Restrictionenzymesrecognizeandmakedouble ­ strandedcutsinDNAatspecificnucleotide sequences EnzymescutDNAatspecificrecognition sequences   Morethan800differentrestrictionenzymes thatrecognizeandcutmorethan100 differentsequences Recognitionsequencesare4 ­8bplong SomeenzymescutDNAresultingincohesive endsorstickyendsbecausetheyare complementarytoeachother  ­twocohesiveendsofsameofdifferentDNA moleculescanjoinandpair Someenzymesproducingblunt ­ended fragments Restriction Enzymes Restriction Enzymes SouthernBlotting Atechniqueutilizesmolecularhybridization. NamedafterEdwinM.Southern. TheblottingisusedtotransfertheseparatedssDNAfragments fromgeltoapermanentsolidmedium(suchasnitrocelluloseor nylonmembrane),whichthenbehybridizedtotheprobefor identifingthetargetgeneorDNAfragments  Southern Blotting Procedure SouthernBlotting Procedure Application: Detectthecopynumber ofthegeneofinterestin thegenome Detectthegenome variance http://highered.mcgraw ­hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120078/bio_g.swf::SouthernBlot Only restriction fragments containing DNA regions that form complementary base pairing to the probe will be present on the autoradiograph Modified from http://www.biology.ualberta.ca FluorescentinsituHybridization(FISH) Atechniqueutilizesmolecular hybridizationtoidentifygeneor chromosomalregions DNAorRNAprobesthatarelabeled withafluorescentdye Targetchromosomesarespreadon slide Hybridizethedenaturedprobe(ss) tocomplementaryregionsofthe chromosome(denatured)onthe slide. Highlysensitive Detectthechromosomelocationof thegeneofinterestoritsco ­ localizationwiththereference GeneCloning Whatisit? TheprocedureofamplificationofaspecificpieceofDNA inabacterialcellandallowingthecelltoreplicatethe DNA. Identicalcopies(clones)oftheoriginalpieceofDNAare produced Why? ToisolationaspecificDNAsequence(typicallyageneor genefragment)inlargeamountthatmakesitpossibleto manipulateitinthelab(sequencing,invitrotranscription andtranslation,transformation,etc.)   Invitro=inglass=inatesttube CloningVectors keyforgenecloning Stable,replicatingcircularDNA,towhichaforeignDNA(target DNAfragmentorgene)canbeattachedfrointroductionintocell foramplification. CarrierofrecombinantDNA,andisnormallyaplasmid  Threeimportant characteristics: 1.Originofreplication 2.Selectablemarker 3.Uniquerestriction enzymesites PlasmidVectorConstruction Theplasmidcanbeopened(linearized)withrestriction enzymes ADNAfragmentofinterestcanbeinsertedintheplasmid. Ligasethensealsthegap TheplasmidisthenintroducedintoE.coliusingaheatshock (45secat37 electroporation Thisis transformation! Bacteriathathavetakenuptheplasmidwillbeabletogrowon mediumwithantibiotic VectorConstruction Recombinantmoleculeor vector Asthehostcellreplicatesthevectoris replicatedresultinginclonesofthe insertedgene. http://www.sumanasinc.com/webcontent/animations/content/plasmidcloning.html Fig:18.12;K&C ExampleofaCommercialPlasmidVector lacZgene:beta galactosidase Ampicillin resistance  T7andT3promoters forinvitro transcription Multiple cloningsiteor polylinker; insertgoes here Originof replication Methods for selection and/or screening allow recovery of cells with the desired constructs. Selection refers to applying conditions that allow the desired cells or phages (containing vector or vector and insert) to replicate while preventing others from replicating. Typical selections include: antibiotic sensitivity and resistance; nutrient requirements. Screeningfor recombinantplasmids X ­Galisconvertedbybeta ­ galactosidase(encodedbylacZ) forabiglongcompoundname).  Bacteriawithoutplasmid(not resistanttoantibiotic)die no colony insertplasmidsarebluebecause theyhaveafunctionallacZ Bacteriawiththerecombinant lacZhasbeeninterrupted BasicStepsinGeneCloning 1.Thevectoractsasavehiclethattransportsthegeneintoahost cell,usuallyabacterium. 2. Withinthehostcellthevectormultiplies,producingnumerous identicalcopiesofitselfandtheinsertedgenethatitcarries. 3. Whenthehostcelldivides,copiesoftherecombinantDNA moleculesarepassedtotheprogenyandfurthervector replicationtakesplace. 4. Afteralargenumberofcelldivisions,acolony/cloneofidentical hostcellsisproduced. Eachcellintheclonecontainsoneormorecopiesofthe recombinantDNAmolecule(orvector). Thegeneinsertedinvectorisnowsaidtobecloned. 5. Clones/colonieswithaninsertintheplasmidwillbewhite, whereasplasmidsw/oinsertwillbebluewhenthemediumis suppliedwithaspecialsubstrate(X ­Gal)andaninducerofthe lacoperon 6. Theadditivestothemediumfacilitatethedetectionoflow ­ probabilityeventssuchasproperligationanduptakeofthe plasmid.  Tiplasmid Alargetumorinducing(Ti)plasmidinsoilbacterium: Agrobacteriumtumefaciens TheTiplasmidcontainstransferableDNA(T ­DNA) A.tumefaciensinfectsplantsandpartofTiplasmidDNA integratesintoplantchromosome.  Current Opinion in Biotechnology 2006, 17:147 154 TiplasmidforPlantTransformation TheTiplasmidhasbeenengineeredasvectorforplant transformation SavethegenesinTiplasmidneededfortransferringtheT ­DNA Addrestrictionsitesandselectablemarkers Thetumor ­inducinggenesinTiplasmidcanberemoved interest TiplasmidforPlantTransformation TheengineeredplasmidtransformsA.tumefaciens Iftissueexplantsorflowersaremixedwiththebacteriumthat carriesarecombinantTi ­plasmid,aproportionoftheplantcells willbetransformed With proper selection (antibiotics, herbicides), the transformed cells can be selected and regenerated into full plants through tissue culture or by planting transformed seeds BiolisticGenetransfer(GeneGun) M i c r o- p a r t i c l e s i z e M i c r o- p a r t i c l e d e n s i t y P a r t i c l e a c c e l e r a t ion Ta r ge t t i ssu e http://www.biology.ualberta.ca/facilities/mbsu/uploads/sop_pdf//Biolistic_Particle_Delivery_System.pdf ...
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