chapter15基因重组与基因工程.ppt

vector * plasmid * cosmid * ? phage * M13 phage * insect virus DNA (autograph californica virus , ACNPV) * yeast artificial chromosome DNA * vaccinia virus DNA * simian virus 40 DNA 3-10kb 40kb 29-48.5kb 5.243kb 180kb 6.407kb 20kb 4-8kb 15kb 0.3-1.0kb 2.5kb 128kb 100kb 25kb * bovine papilloma virus DNA 8.0kb 10kb 0.2-2.2Mb 0.3-1.2Mb * retrovirus DNA * fowlpox virus DNA * adenovirus DNA * herpes simplex virus DNA * cytomegalovirus DNA * Epstein-Barr virus DNA 240kb 170kb 6.407kb 233-238kb 8-10kb 24-36kb Xmn I 3966 2034 Xmn I Pst I 3612 2067 Pvu II 1424 Ava I 650 Sal I 375 BamH I plasmid pBR322 4.36kb 29 Hind III EcoR I 0 A origin A screening gene A single restriction site condition tetr ampr ori plasmid pUC19 2.69kb Eco R I Sac I Kpn I Sma I Bam H I Xba I Hinc II Pst I Sph I Hind III ampr ori polylinker 52bp Plac lac I lac Z’ 2.2 the basic principle of DNA recombination technology the procedure of gene cloning separate target gene as well as vector 1 cut target gene and vector restrictedly 2 join target gene and vector 3 recombinant transformation 4 separate target gene as well as vector 1 cut target gene and vector restrictedly 2 ligate target gene and vector 3 recombinant screening 5 recombinant screening 5 recombinant transformation 4 recombinant screening 5 go a step further... target gene amplify 6 incomplete 1 2 3 1+3 incomplete digestion 1 2 3 Sma I complete 1+2+3 1 2, 2 2 5, 3 3 9, 4 4 14, 5 5 6 n n+n(n+1)/2 n+1 separate target gene cut and ligate target gene and vector CGG C C GGC Hpa II CCGG GGCC CCGG GGCC Hpa II genome DNA CGG C C GGC Hpa II CCGG GGCC CCGG GGCC Ligase recombinant CCGG GGCC plasmid Hpa II recombinant transformation vectors and recombinants competent cells recombinant screening amp or tet etc + plasmid extraction digist with restriction enzeme 1 2 1 2 marker - + 1 2 marker - + target gene amplification