分子生物学分子生物学 (21).pdf

Chapter 8Gene Cloning and Expression8.3 The 8.3 The principal principal process process of of gene cloninggene cloning Preparation of target gene and appropriate vector Inserting target DNA into vector Introduction of recombinant DNA into host cell Screening and identification for recombinant DNA Amplification,expression and other researches oftarget DNAPrincipal process Principal process of gene cloningof gene cloning（1）Isolation of the target gene:8.3.1 Preparation of target gene and related vector Genomic DNA library cDNA library Amplified by PCR Chemical synthesized genomic DNA library cDNA library Amplified by PCR Chemical synthesizedChoice of Choice of vector:vector:Compared contentPlasmidLambda phageCohesive plasmidM13 phageCloning capacity10 kb22 kb4050 kb1 kbGenomic DNA library-+-cDNA library+-Subcloning+-+Sequence analysis+-+E.coli expression+-Principal features and application of different cloning vector systems+:available；-:unavailable Sticky end Artificial linker Ahomopolymer tail Blunt end8.3.2 Inserting target gene into vector（1）Sticky end（2）Artificial linker（3）Ahomopolymer tail（4）Blunt end Transformation Infection Transfection8.3.3 Introduction of recombinant DNA into host cells Genetics method8.3.4 Screening and identification for recombinant DNA Immunological screening Nucleic acid hybridization PCR DNA restriction endonuclease digestionInsertion inactivationBlue-white screening(-complementation)Insertion inactivation（1）Genetics method Blue-white screening(-complementation)（2）Immunological screening（3）Nucleic acid hybridization(4)PCR(5)DNA restriction endonuclease digestionThank you!