Genetic Engineering 遗传工程 gene cloning 基因克隆 recombination DNA technique 重组 DNA 技术

1. 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

3. 第一节 限制性内切酶与连接酶Restriction Enzymes and DNA Ligase 1. Restriction Enzymes An endonuclease（核酸内切酶） that recognizes specific nucleotide sequences in DNA and breaks the DNA chain at that sites

4. Specific nucleotide sequences Palindrome 回文结构

5. 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）

8. Fragments with identical sticky ends can be joined to form recombinant DNA molecule

9. Different restriction enzymes produce fragments of different lengths

11. The timing of digestion determine fragment size

12. Restriction map（限制酶图谱） Compile the number, order, and distance between restriction sites along a DNA fragment The DNA fragments of different lengths can be separated by gel electrophoresis（凝胶电泳）

14. 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

16. 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

17. 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.

18. pBR322

19. pUC18

20. Common plasmid vectors can carry up to 15kb foreign DNA 2. Lambda vector（λ载体）

23. 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

27. 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

29. 5. Yeast artificial chromosome (YAC) 酵母人工染色体 carry 1~2 Mb DNA 1Mb = 1,000,000 bp

30. YAC载体的特点： • 两个TEL位点，一个CEN，一个ARS（自主复制序列） • 三个酵母菌遗传标记基因 • 克隆片段大小约1Mb • YAc载体缺点：易出现嵌合体，某些克隆不稳定

32. 6. Ti plasmid and plant vectors Agrobacterium tumifaciens 根癌农杆菌 Tumor inducing (Ti) plasmid

34. 第三节 DNA文库DNA libraries • Library: a collection of DNA clones that contains multiple copies of nearly every fragment in the whole genome inserted into a suitable vector and placed into storage. 1. Genomic library （基因组文库） • Making a genomic library

37. Calculate the number of clones in a library N: number of required clones P: the probability of recovering a given sequence f: the fraction of the genome in each clone

38. 2. cDNA library （cDNA文库）

39. cDNA libraries represent only the expressed genes in a given cell type, tissue, or stage of embryonic development.

40. 第四节 目的DNA的分离Identifying and Isolating of Interested DNA 1. Screening by functional complementation 利用功能互补进行筛选 Isolate Gal gene of yeast 2. Screening with probe （探针） • DNA probes: short single-stranded DNA, from 10 to several thousand nucleotides in length, and are usually labeled by radiation or fluorescent dye（荧光燃料）

41. 利用核酸探针筛选目的DNA

42. See movie

43. Antibody can be used as probes

45. 3. PCR can replace the cell-based cloning Polymerase chain reaction, PCR 聚合酶链式反应 A method for amplifying specific DNA segments that exploits certain features of DNA replication.

48. Problems and discussion questions • The recognition site for the enzyme Sau3A is GATC. The recognition site for the enzyme BamHI is GGATCC, where the four internal bases are identical to the Sau3A site. This means that the single-stranded ends produced by the two enzymes are identical. • Suppose you have a cloning vector that contains a BamHI site and foreign DNA that you have cut with Sau3A. • 1. Can this DNA be ligated into the BamHI site of the vector? • 2. Can the DNA fragment cloned into this site be cut from the vector with Sau3A? With BamHI? • What is the difference between genes isolated by functional complementation and by hybridization?