Genetic Engineering and Biotechnology Genetic engineering: Isolation, manipulation and expression of genetic materials Gene cloning: Isolation and purification of specilic genes In vitro recombination=recombinant dNa
Genetic Engineering and Biotechnology Genetic engineering: Isolation, manipulation and expression of genetic materials. Gene cloning: Isolation and purification of specific genes In vitro recombination=recombinant DNA
Gene Cloning Procedures Isolation and fragmentation of the source DNA Joining the dna fragments to a cloning vector with DNA ligase Incorporation into a host Detection and purification of the desired clone Production of large numbers of cells or bacteriophage containing the desired clone for isolation and study of the clone dna
Gene Cloning Procedures Isolation and fragmentation of the source DNA Joining the DNA fragments to a cloning vector with DNA ligase Incorporation into a host Detection and purification of the desired clone Production of large numbers of cells or bacteriophage containing the desired clone for isolation and study of the clone DNA
Plasmids as Cloning Vectors Clal Small, only 4361 BP. EcoRI Hind Ill Stable in E coli BamHI High copy number(1000-3000 copies per cell) Easily isolated in the Ampicillin Sa/I esistance supercoiled form Plasmid pBR322 Foreign DNA can be inserted in good amount Restriction sites are known Single cleavage sites for several Tetracycline resistance restriction enzymes Two antibiotic resistance Origin of DNA replication markers Transformation easy
Plasmids as Cloning Vectors Small, only 4361 BP, Stable in E. coli, High copy number (1000-3000 copies per cell) Easily isolated in the supercoiled form Foreign DNA can be inserted in good amount Restriction sites are known Single cleavage sites for several restriction enzymes Two antibiotic resistance markers Transformation easy Plasmid pBR322
BamHI site Gene cloning and Expression Tc pBR322 using Plasmid pBr322 BamHI sites BamHI digestion Foreign DNA BamHI digestion DNA ligase Recyclized Hybrid containing foreign DNA pBR322 Transformation of E coli Transformants Transformants resistant to both resistant to ampicillin ampicillin and tetracycline but sensitive to tetracycline
Gene cloning and Expression using Plasmid pBR322
Bacteriophages as Cloning Vectors Modified lambda hages as cloning vectors pl 80 100 Capsid components J b region att int xis N P c POP OSR anoo Replaceable region Charon 4A B-Gal gene Another substitution Charon 16
Bacteriophages as Cloning Vectors Modified lambda phages as cloning vectors