基因工程第5章基因文库的构建-1.ppt

* Chapter 5 Construction of DNA libraries 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 E. coli 4639221bp p=99% f = 20kb/4600kb n=1056.9 p=99% f =10kb/4600kb n=2116 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 Steps of library construction Plasmid 10kb ? Phage 0-23kb Cosmid ~45kb BAC ~ 100kb（250-300kb?） YAC ~ 300kb-1.2Mb 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. Gam蛋白与核酸外切酶(Exonuclease V) 结合，抑制了宿主的RecBCD 的核酸外切酶V活性，使得外来DNA分子不会被切割降解。 Enter lytic cycle ? replication give ri