基因制备与克隆策略管理知识分析.pptx

《Princple and Technology of Genetic Engineering》《基因工程原理》; 第五章 基因工程工具酶 The Tool of the Gene Engineering ;第一节 目的基因的制备 ;;;;;;;;;;;;;;;;;;;第二节 目的基因克隆;Chapter 6;cDNA synthesis;★看家基因（Housekeeping genes）：又称管家基因或持家基因：在所有细胞中均要表达的一类基因，其产物对维持细胞的基本结构和代谢功能是必不可少（for basic cellular metabolism），表达水平受环境因素影响较小，在各生长阶段的大多数、或几乎全部组织中持续表达，或变化很小。表达只受启动序列或启动子与RNA聚合酶相互作用的影响，而不受其他机制调节。如微管蛋白基因、糖酵解酶系基因与核糖体蛋白基因等 Housekeeping genes: for basic cellular metabolism ★每一个细胞都会生产大量的特殊的mRNA Poly(A)+RNA: each cell type produce different abundance classes of particular mRNAs.;★ cDNA: complementary DNA (copy DNA), poly(A)+RNA as temple Reverse transcriptase= RTase Oligo(dT) primer Alkaline hydrolysis (or RNase H) to remove mRNA strand T4 DNA polymerase, klenow fragment S1 nuclease to produce a blunt-ended;mRNA;mRNA;★ blunt-end ligation: S1 nuclease destroy the protruding ends of DNA; DNA polymerase (klenow fragment) filling the protruding ends of DNA. Main disadvantage: 1) is an inefficient process, require high concentration DNA for ligation; 2) vector self-ligate to produce circular molecules or the insert/vector DNAs may form concatemers instead of bimolecular recombinants, use phoshpatase (either BAP or CIP) to prevent self-ligation; 3) cloning site disappear in the recombinant DNA;CCGAATTCGG GGCTTAAGCC;Adaptors: are single-stranded non-complementary oligomers that may be used in conjunction with linkers, when annealed together and be added to the cDNA to provide sticky-end cloning without digestion of the linkers.;Homopolymer tailing: the enzyme terminal transferase is used to add homopolymers of dA, dT, dG or dC to a DNA molecule.;★Main advantage: 1) packaging in vitro may generate the recombinant phage, which increases the efficiency of the cloning process. 2) much easier to store and handle large numbers of phage clones than plasmids.;Cloning cDNA in λvectors using linkers:;★genomic library: is often used to describe a s