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时间:2019-10-08
《Basics of Molecular Cloning》由会员上传分享,免费在线阅读,更多相关内容在行业资料-天天文库。
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
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