基因工程基因文库的构建.pptxVIP

Gene library (Genomic library) ： A collection of independent clones representing the entire genome of an organism. First step to isolate a target DNA sequence from an organism’s genome. ;1976 L. Clark, J. Carbon n=ln(1-p)/ln(1-f) n: numbers of recombinant required in full library p: probability of target gene in library f: rate of average insertion DNA sizes to genome;Mammal 3×109bp If p=99% cloned fragment 20kb f=20kb/3×109 n=690773.2;cDNA library: A collection of independent clones representing the entire cDNA reversely transcripted from mRNA in a specific tissue of an organism in a specific phage.;Bacterial colonies consisting of genomic DNA library;Choosing vector;SUP4 基因：酵母细胞Trp-tRNA 基因的赭色突变抑制基因，含有克隆位点。 在发生Ade2-赭色突变的宿主细胞中，如果没有外源基因的插入，则突变基因表达受抑制受体菌为Ade+，形成白色菌落；当有外源基因插入时，SUP4表达将被阻断，抑制作用被消除，受体菌为Ade-，菌落为红色。;Cosmid Half of ? phage. If ? 700000, cosmid 350000 (菌落数) Cosmid is usually applied in large size DNA cloning, but it is much more difficult than ? phage，because there is no much experience. （pJB8 and c2RB are more used in gene library construction);? phage vector 1970’s, like Charon 4A and ?g t-WES，15～20kb Presently, EMBL lines, like ?2001，?DASH， Charon 38-40，?GEM-11 etc. (replacement vectors), 20-24kb;Basic principles in choosing ? vector Easily recover foreign DNA from ?; Easily prepare 2 arms; Availability of vector sequence, RE mapping; Amber mutation (琥珀突变) in vector arms as special screening model; Active gam gene in recombinants； Chi sequence in vector arms.;Enter lytic cycle ? replication give rise to 50 ? genome, then rolling cycle replicating to produce concatemer, and finally it is cut and packaged into particles. recB/recC/recD products, controlling recombination, and have activities of Exnulease V, inhibiting DNA ? replication transfer to rolling cycle. gam gene products bind and inactivate exnuclease V. ;If gam-, recA product is required to package ?, but form small plaques (小噬