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1 Gene Cloning and DNA Analysis Terry A Brown 2001 Department of Biochemistry and Applied Molecular Biology University of Manchester Institute of Science and Technology (UMIST) 曼彻斯特科技理工大学 UK, UMIST was found in 1824 Manchester: International music center, famous football club Why choose this textbook?

5 学习方式 Preface 1(1986), 2(1990), 3(1995), 4(2001) Gene Cloning, Molecular Cloning, Recombinant DNA Technology, Gene Manipulation, Gene Engineering, Genetic Engineering,

6 本课程的地位 : 现代科技革命高新技术生物技术基因工程基因克隆

7 Part 1 基因克隆和 DNA 分析的基本原理 ( The basic principles ) Part 2 基因克隆和 DNA 分析的应用 ( The Application in Research) Part 3 基因克隆和 DNA 分析与生物技术 ( The Application in Biotechnology) 共 16 章 Chapter 1 Why gene cloning is important

8 1 The early development of genetics 2 The advent of gene cloning and PCR 3 What is gene cloning? 4 What is PCR? 5 Why gene cloning and PCR are so important 6 How to find your way through this book

The early development of genetics Gregor Mendel: 1822-1884, 1884, 奥地利遗传学家 1843,, 大学毕业, 修道院传教士豌豆杂交实验 (1856( 1856-1865),

9 The early development of genetics Gregor Mendel: , 1884, 奥地利遗传学家 1843,, 大学毕业, 修道院传教士豌豆杂交实验 (1856( ), 株 1865 年 2 月, 在奥地利布尔诺自然科学会议上宣读研究成果, 没有人理解没有人提问没有人感兴趣 1865 年发表植物杂交实验论文, 布尔诺自然科学会杂志第 4 卷, 该杂志尽管分送 120 个欧美图书馆, 但没人重视

1900 年春天, 荷兰的德弗里斯, 德国的科伦斯, 奥地利的切马克, Thomas H Morgan (1866-1945) 1945) Fruit fly, Drosophila, 果蝇哥伦比亚大学果蝇实验室, 20 年 1922 年将 2000 个基因定位在 4 条染色体上 1933 年 Nobel Prize 谈家桢 For his discoveries concerning

10 1900 年春天, 荷兰的德弗里斯, 德国的科伦斯, 奥地利的切马克, Thomas H Morgan ( ) 1945) Fruit fly, Drosophila, 果蝇哥伦比亚大学果蝇实验室, 20 年 1922 年将 2000 个基因定位在 4 条染色体上 1933 年 Nobel Prize 谈家桢 For his discoveries concerning the role played by the chromosome in heredity, he was awarded the Nobel Prize in Among his collaborators at Columbia may be mentioned H. J. Muller,, who was awarded the Nobel Prize in 1946 for his production of mutations by means of X-rays. X Morgan married Lilian V Sampson, in 1904, who had been a student at Bryn Mawr College, and who often assisted him in his research. They had one son n and three daughters.

11 James Watson (1928- ) 美国生物学家 Francis H Crick (1916- ) 英国生物物理学家 1952 年双螺旋结构 1962 年 Nobel Prize Cold Spring Harbor Laboratory From 1977 he held the position of distinguished professor at the Salk Institute for Biological Studies in San Diego, Calif.

Jacques Monod (1910-1976) 1976) 法国生物化学家 1961 年建立操纵子理论 1960 年三联体密码 1965

12 Jacques Monod ( ) 1976) 法国生物化学家 1961 年建立操纵子理论 1960 年三联体密码 1965 年 Nobel Prize 1971 he was appointed Director of the Institute Pasteur. 等

13 1 The early development of genetics 2 The advent of gene cloning and PCR 3 What is gene cloning? 4 What is PCR 5 Why gene cloning and PCR are so important 6 How to find your way through this book

14 The advent of gene cloning and PCR 1967 年世界上 5 个实验室几乎同时发现了 DNA Ligase 1970 Wisconsin 大学 H.G. Khorana 发现 T4 DNA Ligase 1970 年 Hopkins 大学 H.O. Smith 等分离到第一个限制性内切酶 (1978 Nobel Prize ) 1972 年 Stanford 大学 P. Berg 等, DNA 体外重组 1973 年美国加州大学旧金山分校 H. Boyer 和 Stanford 大学 Cohen 等完成了真正意义上的 DNA 体外重组实验.

15 Kanamycin R6-5 EcoR I DNA Ligase Tetracycline psc101 Kan r and Tet r

16 1985, Kary Mullis (1944-) Cetus The Nobel Prize in Chemistry 1993 Kary B. Mullis, La Jolla, CA, USA Michael Smith ( ) 2000) University of British Columbia Vancouver, Canada

18 1 The early development of genetics 2 The advent of gene cloning and PCR 3 What is gene cloning? 4 What is PCR 5 Why gene cloning and PCR are so important 6 How to find your way through this book

19 What is gene cloning? 5 Steps Figure1.1 The basic steps in gene cloning

20 1 The early development of genetics 2 The advent of gene cloning and PCR 3 What is gene cloning? 4 What is PCR 5 Why gene cloning and PCR are so important 6 How to find your way through this book

21 4 steps 3 reactions Figure1.2 The basic steps in PCR

22 1 The early development of genetics 2 The advent of gene cloning and PCR 3 What is gene cloning? 4 What is PCR 5 Why gene cloning and PCR are so important 6 How to find your way through this book

23 Why gene cloning and PCR are so important Gene isolation by cloning Separated an individual gene from hundred thousands other genes Then we can study the structure, expression and function of the gene

24 Figure1.3 Cloning allows individual fragments of DNA to be purified But the problem of selection still exists

25 Figure1.4 The problem of selection

26 Gene isolation by PCR Figure 1.5 Two limitations: The sequences of the annealing sites The length

27 1 The early development of genetics 2 The advent of gene cloning and PCR 3 What is gene cloning? 4 What is PCR 5 Why gene cloning and PCR are so important 6 How to find your way through this book

28 Chapter 2 Vehicles for Gene Cloning: plasmids and bacteriophages Plasmids: 1. Basic features of plasmids 2. Size and copy number 3. Conjugation and compatibility 4. Plasmid classification 5. Plasmids in organisms other than bacteria

29 Basic features of plasmids : Replication within the host cells Small size, ideally < 10 kb Selective marker, resistant gene Multiple cloning sites(mcs)

30 Figure2.1 Plasmids: independent genetic elements found in bacterial cells

31 Figure2.2 The use of antibiotic resistance as a selectable marker for a plasmid.

32 Firgure2.3 Replication strategies for (a) a non- integrative plasmid, and (b) an episome (integrative plasmid) ( 附加体 or 游离基因 : 能可逆地整合进入宿主染色体的质粒 )

33 Plasmids: 1. Basic features of plasmids 2. Size and copy number 3. Conjugation and compatibility 4. Plasmid classification 5. Plasmids in organisms other than bacteria

34 Table2.1 Sizes of representative palsmids Copy number: 1~several hundreds

35 Plasmids: 1. Basic features of plasmids 2. Size and copy number 3. Conjugation and compatibility 4. Plasmid classification 5. Plasmids in organisms other than bacteria Conjugation: 接合 Compatibility: : 相容性 (Non-conjugative) Incompatibility: 不相容性

36 Figure2.4 Plasmid transfer by conjugation between bacterial cells.

37 Plasmids: 1. Basic features of plasmids 2. Size and copy number 3. Conjugation and compatibility 4. Plasmid classification 5. Plasmids in organisms other than bacteria

38 Plasmid classification: Fertility or F plasmids( 致育质粒 ): 只带转移基因, 没有其它基因 ; Resistance or R plasmids( 抗性质粒 ): 带有抗性基因, 如 Chloramphenicol,ampicillin,mercury ect. Col plasmids( Col 质粒 ): 带有编码大肠杆菌素基因, 能杀死其它细菌 ; Degradative plasmids( 降解质粒 ): 能降解一些大分子, 如甲苯水杨酸等 ; Virulence plasmids( 致病质粒 ): 使宿主菌具有致病力, 如农杆菌等

39 Plasmids: 1. Basic features of plasmids 2. Size and copy number 3. Conjugation and compatibility 4. Plasmid classification 5. Plasmids in organisms other than bacteria 2um circle in yeast Saccharomyces cerevisiae

40 Bacteriophages: 1. Basic features of bacteriophages 2. Lysogenic phages 3. Viruses as cloning vehicles for other organisms

41 Structure Figure2.5 The two main types of phage structure.

42 Infection process Figure2.6 The general pattern of infection of a bacterial cell by a bacteriophage

43 Bacteriophages: 1. Basic features of bacteriophages(lytic Lytic) 2. Lysogenic phages (two cases:λ, M13) 3. Viruses as cloning vehicles for other organisms Prophage( 原噬菌体 ): 存在静止期或休眠期 M13( 丝状噬菌体 )

44 Figure2.8 The lysogenic infection cycle of bacteriophageλ

45 Figure2.8 The infection cycle of bacteriophage M13

46 Gene organization in the λdna molecule Control gene expression Control coat protein Control integration Figure2.9 The λ genetic map showing the positions of the important genes and the functions of the gene clusters.

47 . Figure2.10 The linear and circular forms of λ DNA.

48 Figure2.11 The M13 infection cycle showing the different types of DNA replication the occur.

49 Bacteriophages: 1. Basic features of bacteriophages(lytic Lytic) 2. Lysogenetic phages 3. Viruses as cloning vehicles for other organisms Simian virus 40(SV40): 猿猴病毒 40 Adenoviruses: 腺病毒 Baculoviruses: 杆状病毒

50 The end of chapter 2

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untitled 01 1-1 1-2 1-3 21 21 1-1 (biotechnology) Karl Ereky 1917 2 CHAPTER 1 1-2 19 1. 2. 3. DNA 1-1 3 1-1 1844 Herr Carl Nageli 1866 Gregor Mendel 1870 Theodor Schwann 1889 Altman 1905 Nettie Stevens 1913 Alfred