基因工的程技术.pptVIP

第九章 基 因 工 程Gene Engineering Genetic Engineering 遗传工程 gene cloning 基因克隆 recombination DNA technique 重组DNA技术 Clone（克隆） are identical organisms, cells, or molecules descended from a single ancestor General process of gene engineering 第一节 限制性内切酶与连接酶 Restriction Enzymes and DNA Ligase Cleavage produces sticky ends（粘性末端）and blunt ends（平整末端） The 5’-end is phosphate（5’- P ） and the 3’-end is hydroxyl（3’-OH） Different restriction enzymes produce fragments of different lengths The timing of digestion determine fragment size Restriction map（限制酶图谱） Compile the number, order, and distance between restriction sites along a DNA fragment 2. DNA ligase（DNA连接酶） Catalyzes formation of a covalent bond（共价键） between adjacent 5’-P and 3’-OH termini in a broken double-strand DNA 第二节 分子克隆载体Vectors To serve as a vector, a DNA molecule must have several properties: Autonomously replicate（自主复制） Contain convenient restriction sites Carry a selector marker gene（选择标记基因） Easy to recover from the host cell 1. Plasmid vectors（质粒载体） The characteristics of plasmid ◇Extrachromosome double-stranded DNA ◇Autonomously replicate ◇Incompatibility（不相容性）: the inability of certain plasmids to coexist in the same cell. It is a cause of plasmid immunity. pBR322 pUC18 Common plasmid vectors can carry up to 15kb foreign DNA 2. Lambda vector（λ载体） λ vectors can carry DNA fragments up to 15kb 3. Cosmid （cos 质粒） constructed by inserting the cos sequence of λ, which is necessary for packing phage DNA into phage protein coats, into a plasmid vector Cosmids can carry up to 45kb of insert DNA 4. Bacterial artificial chromosome ( BAC ) 细菌人工染色体 Derived from F plasmid, can carry inserts of about 300kb 5. Yeast artificial chromosome (YAC) 酵母人工染色体 carry 1~2 Mb DNA 1Mb = 1,000,000 bp 6. Ti plasmid and plant vectors Agrobacterium tumifaciens 根癌农杆菌 Tumor inducing (Ti) plasmid 第三节 DNA文库 DNA libraries Library