Basics of Molecular Cloning

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1、BasicsofMolecularCloningPromegaCorporationEducationResourcesUnit006DefiningCloning“Cloning”isaloadedtermthatcanbeusedtomeanverydifferentthings.CuttingapieceofDNAfromoneorganismandinsertingitintoavectorwhereitcanbereplicatedbyahostorganism.(Sometimescalledsubcloning,becauseonlypartoftheorgani

2、sm’sDNAisbeingcloned.)UsingnuclearDNAfromoneorganismtocreateasecondorganismwiththesamenuclearDNARestrictionEnzymesRestrictionEnzymes(alsocalledRestrictionEndonucleases)areproteinsthatcleaveDNAmoleculesatspecificsites,producingdiscretefragmentsofDNA.RestrictionEnzymes(RE)werefirstisolatedbyNa

3、thansandSmithin1970.WhyRestrictionEnzymes?WhywouldbacterialcellscontainproteinsthatcleaveDNAatspecificsequences?Generallyrestrictionenzymesarethoughttoprotectbacterialcellsfromphage(bacterialvirus)infection.Bacterialcellsthatcontainrestrictionenzymescan“cutup”invasiveviralDNAwithoutdamagingt

4、heirownDNA.JoiningDNAFragmentsIn1972,PaulBergandcolleaguesmadethefirst“artificial”recombinantDNAmolecule.DemonstratedthattheDNAofSimianvirus40couldbelinearizedbyEcoR1CreatedacircularDIMERofSimianvirusDNAbyjoiningtwolinearizedfragmentsAlsoinsertedpiecesofLambdaphageDNAintolinearizedSimian40vi

5、rusmolecule.IsolatingGenesHerbertBoyerandStanleyCohenbuiltontheworkofBerg,NathansandSmithtouserestrictionenzymestoisolateasinglegene,placeitintoaplasmidvector.Bacterialcellswerethentransformedwiththerecombinantplasmid.Thebacteriahostcellsreplicatedtheplasmid,producingmanycopiesofthegene,thus

6、amplifyingit.Thepracticalapplicationwasthatexpensivehumanproteinproducts,likeinsulin,whichwereusedtotreatdisease,couldeventuallybeproducedfromrecombinantmoleculesinthelaboratoryusingbacteriaoranotherhost.Humanproteinproductslikeinsulincouldbeusedinverylargequantitiesfromtherecombinantmolecul

7、e.Patientsnolongerhadtouseinsulinisolatedfrompigsorcows.PlasmidVectorsPlasmidsarecircularpiecesofDNAfoundnaturallyinbacteria.Plasmidscancarryantibioticresistancegenes,genesforreceptors,toxinsorotherproteins.Plasmidsreplicateseparatelyfromthegenomeo