2. Molecular Biology Xu Liyan Chapter 14 gene recombination and gene engineering

3. section 1 DNA Recombination section 2 Recombinant DNA technology section 3 Relationship between Recombinant DNA technology and Medicine

4. section 1 DNA Recombination 1.1 Homologous Recombination 1.2 Gene Transfer and Recombine in Bacteria   Conjugation   Transformation   Transduction 1.3 Site-specific Recombination 1.4 Transpositional Recombination

5. 1.1 Homologous Recombination The covalence connection between different DNA moleculars is called DNA recombination or gene recombination The gene recombination includes two types as follows  homologous recombination  site-specific recombination  transpositional recombination

6.  Homologous Recombination The recombination between homologous sequence is known as homologous recombination

7. 5’ 3’ 5’ Rec BCD Rec A Rec BCD 5’ 3’ 5’ Ligase Holliday Mechanism of Homologous Recombination

10. Transformation  There is foreign DNA.  The phenotype of organisms is changed.  The c hanged Phenotype is passed down stably

12. Transduction  When virus is released from infected one cell and go to infect other cell, the DNA fragment transfer from one cell to other cell. This is called the transduction.

13. bacteria 1 phage integration some factor bacteria 2

14. bacteria 1 phage integration some factor bacteria 2  DNA fragment transformation between two bacterium  phage is carrier

15. 1.3 site-specific recombination The integration catalyzed by integrase between two site-specific DNA fragments is known as site-specific recombination.

16. 1.4 Transpositional recombination the displacement of some gene in the genome by insertion sequence or transposons

17. 1.4.1 insertion sequence and its mediated gene transposition  The length of typical insertion sequence is about 750~1500bp.  Typical insertion sequence includes two 9 ~ 41bp inverted repeat sequence and a transposase.  A 4 ~12bp positive repeat sequence always link to flanking of inverted repeat sequence.  Gene transposition by insertion sequence:  conservative transposition  duplication transposition

18. target transposase insertion sequences target 3’ 5’ transposase 5’ 3’ transposase 3’ 5’ transposase polymerase I ligase transposase duplication transposition12 3’

19. 1.4.2 structure of transposons  The transposon is a dispersive and repeat sequence in the genome.  The transposon can transfer from one region to other region of the genome.

20.  The structure of transposon is similar to one of insertion sequence.  The both insertion sequence and transposon contain transposase gene and flanking inverted repeat sequences, but transposon also contain a few other genes.  The insertion sequence is the most simple transposon in fact.

21. IS 10L amp-R generepressor gene transposase insertion sequence transposase gene Tn3 tet-R geneIS 10L Tn10

22. section 2 DNA recombination technology 2.1 the basic concept related with DNA recombination technology recombination technology 2.2 the basic principle of DNA recombination technology recombination technology

23. 2.1 the concept related with DNA recombination technology 2.1.1 DNA cloning 2.1.2 tool enzyme 2.1.3 target gene 2.1.4 gene vector

24. 2.1.1 DNA cloning  It is a process of DNA molecular amplification.  Usually, the first a target DNA fragment is inserted to a vector and a recombinant (replicon) is constructed.  The second the recombinant is transformed into host cell and screen out the cell containing the recombinant.  The last that cell is amplified, namely a mass of target DNA molecule is gained.

25. 2.1.2 tool enzyme  restriction endonuclease  DNA ligase  DNA polymerase I  reverse transcriptase  polynucleotide kinase  end-transferase  alkaline phosphatase

26. structural character of cutting site recognized by restriction enzyme restriction endonuclease recognized sequence and cut Bam H I Eco R I GAATTC CTTAAG GGATCC CCTAGG 5’ Pvu I Sst I GAGCTC CTCGAG CGATCG GCTAGC 5’ Alu I Sma I AGCT TCGA CCCGGG GGGCCC 5’

27. 2.1.3 target gene The interested gene is the target gene

28. source of the target gene * It is from genomic DNA directly, this is prokaryotic gene only generally. * It is from artificial synthesis, this is simple polypeptide gene generally. * It is from mRNA. * It is from genomic library or cDNA library. * Polymerase Chain Reaction (PCR).

29. synthesize cDNA from mRNA AAA…AAA 5’ 3’ mRNA AAA…AAA 3’ 5’ AAA…AAA TTT...TTT 5’3’ 5’ 3’ primer: oligo dT reverse transcriptase TTT...TTT 5’ 3’ basic hydrolysis TTT...TTT 5’ 3’ ? TTT...TTT 5’ 3’ AAA…AAA DNA polymerase I TTT...TTT 5’ 3’ AAA…AAA 3’ 5’ S1 nuclease

30. genomic library genomic DNA fragment 50-200kb extraction restrictively cut gene fragments recombination recombinant transformation genomic library target gene cDNA library recombination recombinant transformation cDNA libraryextraction mRNA cDNA double strands

31. 5'5' 3'3' extension 3'3'5'5' PCR Process 5' 3'3' 5'5' 3'3' 3'3'5'5' denaturation annealing 3'3'5'5' 5'5' 3'3' 5'5' 3'3' 3'3'5'5' 5'5' 3'3' 5'5' 5'5' 5'5' 3'3' 3'3'5'5' denaturation Next cycle 5'5' 3'3' 5'5' 3'3'

32. 2.1.4 gene vector  The gene vectors are DNA molecules, which structure is reconstructed.  They can carry target DNA fragment  The target gene or DNA fragment is amplified and expressed.

33. vector * plasmid * cosmid * phage * M13 phage * insect virus DNA (autograph californica virus, ACNPV) * yeast artificial chromosome DNA * vaccinia virus DNA * simian virus 40 DNA 3-10kb 40kb 29-48.5kb 5.243kb 180kb 6.407kb 20kb 4-8kb 15kb 0.3-1.0kb 2.5kb 128kb 100kb 25kb * bovine papilloma virus DNA 8.0kb10kb 0.2-2.2Mb 0.3-1.2Mb * retrovirus DNA * fowlpox virus DNA * adenovirus DNA * herpes simplex virus DNA * cytomegalovirus DNA * Epstein-Barr virus DNA 240kb 170kb 6.407kb 233-238kb 8-10kb 24-36kb

34. Xmn I 3966 2034 Xmn I Pst I 3612 2067 Pvu II 1424 Ava I 650 Sal I 375 BamH Iplasmid pBR322 4.36kb 29 Hind IIIEcoR I 0 A origin A screening gene A single restriction site condition tet r amp r ori

35. plasmid pUC19 2.69kb Eco R I Sac I Kpn I Sma I Bam H I Xba I Hinc II Pst I Sph I Hind III amp r ori polylinker 52bp P lac lac I lac Z’

36. 2.2 the basic principle of DNA recombination technology recombination technology

37. the procedure of gene cloning separate target gene as well as vector 1 cut target gene and vector restrictedly 2 join target gene and vector 3 recombinant transformation 4 separate target gene as well as vector 1 cut target gene and vector restrictedly 2 ligate target gene and vector 3 recombinant screening 5 recombinant screening 5 recombinant transformation 4 recombinant screening 5 go a step further... target gene amplify 6

38. incomplete 1 2 3 1+3 incomplete digestion 1 2 3 Sma I complete 1+2+3 1 ------ 2, 2 2 ------ 5, 3 3 ------ 9, 4 4 ------ 14, 5 5 6 n n+n(n+1)/2 n+1 separate target gene

39. cut and ligate target gene and vector CGG C C GGC Hpa II CCGG GGCC CCGG GGCC Hpa II genome DNA CGG C C GGC Hpa II CCGG GGCC CCGG GGCC Ligase recombinant CCGG GGCC plasmid Hpa II

40. recombinant transformation vectors and recombinants cells competent cells

41. recombinant screening amp or tet etc + plasmidextraction digist with restriction enzeme 1 2 1 2 marker - + 1 2 - +

42. target gene amplification

45. SHEEC genomic DNA PCR amplification cloned into pGEM-T easy vector pT-X pGL3-promoter Recovered by Minielute Gel Extraction Kit Xho I+ Bgl II Dephosphorylation,Purification by Quick PCR Purification Kit X target fragments pGL3-promoter dephosphorylated vector Ligation by T4 Ligase Transformed into JM109 competent cells plated on LB plates containing ampicillin, cultured at 37 ℃ for 16 h pB-X cell clones Isolation recombinant plasmids by QIAprep Miniprep Kit Xho I+ Bgl II pB-X recombinants

46. The agarose gel electrophoresis of PCR products of NGAL gene 5’ flanking regulation sequences from SHEEC cells 200bp  M 1431 1137 945 657 416 152 1124 2000bp  1000bp 

47. The agarose gel electrophoresis of recons pGEM- 1431~152 after XhoI + BglII digesting 200bp  M1 1431 1137 945 657 416 152 1124 M2 1000bp  ← 947 bp ← 5.0 kb ← 2.0 kb

48. The agarose gel electrophoresis of recons pGLP- 1431 ~ 152 after XhoI + BglII digesting M 1431 1137 945 657 416 152 5000bp  1375 bp  564bp 

49. target gene expression prokaryotic expression system D

50. Expression analysis of four expression vectors in E.coli by SDS-PAGE

51. eukaryotic expression system 21kDa → ← 25kDa 1 2 3 4 5 6 7 8 9 10

53. section 3 the between DNA the relationship between DNA recombination technology and medicine recombination technology and medicine  discover and separate pathogenic gene  biopharmacy  DNA diagnosis  gene therapy  prevent transmissibility disease

54. Summary Homologous Recombination Site-specific Recombination Transposition   Conjugation   Transformation   Transduction DNA cloning: separate, cut, ligate, transform, screen, amplify, express

55. 选择题练习 基因重组与基因工程

56. 1. 基因工程的特点是 A 在分子水平上操作, 在分子水平上表达 B 在分子水平上操作, 在细胞水平上表达 C 在细胞水平上操作, 在分子水平上表达 D 在细胞水平上操作, 在细胞水平上表达 E 以上均可以

57. 2. 限制性核酸内切酶不具有哪项特点 ? A 仅存在于原核细胞中 B 用于重组 DNA 技术中的位 I 类酶 C 能识别双链 DNA 中特定的碱基顺序 D 具有一定的外切酶活性 E 辨认得核苷酸序列常具有回文结构

58. 3. 有关质粒的叙述, 下列哪项是错误的 ? A 小型环状双链 DNA 分子 B 可小到 2-3kb, 大到数百个 kb C 能在宿主细胞中独立自主地进行复制 D 常含有耐药基因 E 只有一种限制性核酸内切酶切口

59. 4. 下列哪项不是重组 DNA 的连接方式 ? A 粘性末端与粘性末端的连接 B 平端与平端的连接 C 粘性末端与平端的连接 D 人工接头连接 E 同聚物加尾连接

60. 5. DNA 克隆不包括下列哪项步骤 ? A. A. 选择一个适合的载体 B. B. 限制性核酸内切酶在特异位点裂解质粒 和目的基因 C. C. 用连接酶连接载体 DNA 与目的 DNA, 形 成重组体 D. D. 用载体的相应抗生素抗性筛选含重组体 的细菌 E. E. 重组体用融合法导入细胞

61. 6. 下列哪种酶是重组 DNA 技术中最重要的 ? A 反转录酶 B 碱性磷酸酶 C 末端转移酶 D DNA 聚合酶 I E DNA 连接酶

62. 7. 基因工程中通常使用的质粒存在于 A 细菌染色体 B 酵母染色体 C 细菌染色体外 D 酵母染色体外 E 以上均不是

63. 8. 在已知 DNA 序列情况下, 获取目的 DNA 最方便的方法是 A 人工化学合成 B 基因组文库法 C cDNA 文库法 D PCR 法 E 从染色体 DNA 直接提取

64. 9. 基因工程中使目的基因与载体拼接的酶是 A DNA 聚合酶 B RNA 聚合酶 C DNA 连接酶 D RNA 连接酶 E 限制性核酸内切酶

65. 10. 表达人类蛋白质的最理想的细胞体系是 A E.coli 表达体系 B 原核表达体系 C 酵母表达体系 D 昆虫表达体系 E 哺乳类细胞表达体系

66. 11. The 11. The nucleotide number which restriction enzyme recognize in DNA nucleotide sequence is A 4, 5 or 6 B 5, 6 or 7 C 6, 7 or 8 D 4, 6 or 8 E 4 - 8

67. 12. The technique used in identification of DNA is A northern blotting B southern blotting C Western blotting D affinity chromatography E ion exchange chromatography

68. 13. The way of gene recombination doesn’t include A transformation B transduction C transposition D change-over 转换 E integration

69. 14. The abbreviation of polymerase chain reaction is A PRC B PER C PDR D BCR E PCR

70. 15. 对于重组体的筛选, 属于非直接选择法的是 A 免疫化学法 B 原位杂交法 C southern 印迹 D 补救标志筛选 E 酶联免疫筛选

71. 16. 基因工程中, 目的基因的来源有 A 化学合成 B PCR 合成 C cDNA 文库 D 基因组文库 E 组织细胞中染色体 DNA 直接提取

72. 17. 质粒 DNA 等作为基因工程载体必须具备的条件是 A 能独立自主复制 B 易转化 C 易筛选 ( 质粒 DNA 含有抗药性基因等 ) D 具有合适的限制性核酸内切酶酶切位点 E 易提取获得

73. 18. 将表达载体导入真核细胞的转染方法有 A 磷酸钙转染 B DEAE 葡萄糖介导转染 C 电穿孔 D 脂质体转染 E 显微注射

74. 19. gene cloning also be called A DNA recombination B RNA recombination C DNA cloning D RNA cloning E protein replication

75. 20. The enzyme tools commonly used in gene cloning technique are A restriction enzyme B DNA polymerase I C DNA ligase D reverse transcriptase E terminal transferase