1. DNA Recombination and Recombinant DNA technology Chapter 21

2. DNA recombination: different DNA molecules break and link to form new DNA molecules. recombinant DNA technology: two or more than two DNA molecules are combined together to form a new DNA molecules.

3. Section 1 DNA Recombination and Gene Transfer in Nature

4. DNA recombination homologous recombination site-specific recombination transposition recombination conjugation transformation transduction

5.  The recombination happened between homologous sequences is called homologous recombination, also called general recombination. I. homologous recombination

6. Holliday model ：  2 highly arranged homologous DNA molecules ； patch recombinant splice recombinant  1 strand of 1 DNA molecules break, and linked with relative strand of the other DNA to form Holliday midbody.  Holliday midbody separate to form heterogeneous double strands

7. endonulease (recA) Branching (recA) endonulease (recBCD) DNA ligase 5´3´ 5´ 3´ 5´ 3´ 5´3´ 5´3´ 5´3´ 5´3´ 5´3´ 5´3´ 5´3´ 5´3´ 5´3´ 5´ 3´ 5´3´ 5´3´ 5´3´ 5´ 3´ 5´3´ 5´3´ Holliday midbody 5´3´ 5´3´ 5´3´ 5´3´

8. Holliday 5´3´ 5´3´ 5´3´ 5´3´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ 5´ 3´ endonuclease (ruvC) endonuclease (ruvC) DNA 连接酶 DNA 连接酶 patch recombinant splice recombinant

9. Plasmid DNA molecules are transferred from one cell to another while cell-cell or bacteria-bacteria contacting. II. conjugation Small circle double strand DNA molecules with self-replication function.  plasmid

10. conjugation transfer

11. ransformation Exogenous DNA was obtained by cells and express new heredity phenotype. III. transformation

12. eg ： DNA fragment is absorbed by another bacteria while bacteriolysis.

13. Virus released from the host cell will infect another cell again, the DNA transfer happened between the donor cell and receptor cell is called transduction. IV. transduction

14. λ-phage bacteriolysis

15. Section 2 Recombinant DNA Technology

16. Also called gene engineering, means to repair or recombine genes and let the living body generate new phenotype.

17. 1865, G.J.Mendel, the peas cross-breeding experiment. The history

18. In 1973, the first recombinant DNA molecule was constructed in Stanford university. Mosaic plasmid anti-streptomycin plasmid anti-tetracycline plasmid splicing E.Coli E.Coli anti-streptomycin and anti-tetracycline

19. 1997, cloned sheep “Dolly” in England

20. Some concepts about recombinant DNA technique:  clone: an aggregation of the same copies coming from one ancestor.  cloning: the process that can obtain the same copies. DNA clone:

21.  Technique level ：  molecular clone (DNA clone)  cell clone  individual clone ( animal or plant)

22. Main process: to combine the purpose DNA fragment with vector to form a new recombinant DNA and to be replicated and amplified in receptor cell to obtain a large amount of copies of a gene.  molecular cloning, or DNA cloning or genetic engineering

23.  purposes ： ① to obtain a gene’s copy that we are interest in. ② to obtain the production of the gene---protein.

24. I. Common used tool enzymes in recombinant technology  r restrictive endonuclease  DNA polymerase Ⅰ  r reverse transcription enzyme  T 4 DNA ligase  a alkaline phosphatase  T Taq DNA polymerase

25. restriction endonuclease restriction endonuclease, RE is a kind of nucleic endonuclease, which can recognize specific internal sequence and split phosphate dieser bond. GGATCC CCTAGG G CCTAG GATCC G+ Bam H Ⅰ  concept ：

26. type Ⅰ、 type Ⅱ、 type Ⅲ  classification ：  nomenclature ： Hind Ⅲ category series plant sequnce Haemophilus influenzae d plant The third kinds of enzyme

27. Some restricted endonuclease recognize different palindrome sequence, but generate same sticky edge, the enzyme is called isocaudarner, the same end is called compatible end. Bam H Ⅰ Bg l Ⅱ GGATCC CCTAGG AGATCT TCTAGA G CCTAG GATCC G+ + A TCTAG GATCT A  isocaudarner

28. type Ⅱ —— palindrome Cut edge ： flat edge 、 sticky edge GGATCC CCTAGG

29. Bam H Ⅰ GTC CAG G CCTAG GATCC G+ GGATCC CCTAGG Hind Ⅱ GTCGAC CAGCTG GAC CTG+ Flat edge Sticky edge

30. Restricted endonucleases coming from different resource ， but recognize the same palindrome sequence. GGATCC CCTAGG G CCTAG GATCC G+ Bam H Ⅰ GGATCC CCTAGG G CCTAG GATCC G+ Bst Ⅰ  ： isoschizomers

31. Name palindrome BamH Ⅰ 5 ’… G ▼ GATCC...3’ Bgl Ⅱ 5 ’… A ▼ GATCT...3’ EcoR Ⅰ 5 ’… G ▼ AATTC...3’ Hind Ⅲ 5 ’… A ▼ AGCTT...3’ Hpa Ⅱ 5 ’… C ▼ CGG...3’ Mbo Ⅰ 5 ’… ▼ GATC...3’ Nde Ⅰ 5 ’… GA ▼ TATG...3’ Apa Ⅰ 5 ’… GGGCC ▼ C...3’ Hae Ⅱ 5 ’… PuGCGC ▼ Py...3’ Kpn Ⅰ 5 ’… GGTAC ▼ C...3’ Pst Ⅰ 5 ’… CTGCA ▼ G...3’ Sph Ⅰ 5 ’… GCATG ▼ C...3’ Alu Ⅰ 5 ’… AG ▼ CT...3’ EcoR Ⅴ 5 ’… GAT ▼ ATC...3’ Hae Ⅲ 5 ’… GG ▼ CC...3’ Pvu Ⅱ 5 ’… CAG ▼ CTG...3’ Sma Ⅰ 5 ’… CCC ▼ GGG...3’ restricted endonuclease

32. II. Common used vectors in recombinant technology  concept of vector: Some DNA molecules which can carry purpose gene we want to study and amplify the gene or express the protein encoded by the gene.  classification of vectors: cloning vector expression vector

33.  cloning vector The vectors designed for the insertion and amplification of exogenous DNA sequence.  expression vector The vectors designed for the expression of the gene inserted in.

34. 1. Clone vectors  At least 1 replication origin  be able to self replicate in host cell and the exogenous gene sequence can be amplified at the same time.  At least 1 selection marker to distinguish the vectors containing the exogenous gene eg: antibiotics gene, β-lactase gene(lac Z)  MCS （ multiple cloning sites ） a specific sequence in vector containing multiple RE sequences. Basic feature of clone vectors:

35. （ 1 ） plasmid concept: small circle double strand DNA molecules which can replicate themselves. Common used clone vectors

36. pUC18 质粒载体图谱

37.  λ phage DNA editing system λgt (suitable for cDNA cloning ） EMBL （ suitable for genomic DNA cloning ） （ 2 ） phage  M13 phage DNA editing system M13mp series pUC series

38.  cosmid  yeast artificial chromosome, YAC  bacterial artificial chromosome, BAC  animal virus (adenovirus, retroviral vectors) （ 3 ） other clone vectors

39.  Concept : Means those vectors that can express the exogenous gene in the host cells.  Classification according to the host ：  eukaryotes expression vectors  prokaryotes expression vectors 2. expression vectors

40. 1. prokaryotes expression vectors R ：调节序列； P ：启动子； SD ： SD 序列； TT ：转录终止序列 Transcription direction Coding sequence

41. 2.eukaryotes expression vectors Ori Pro ：原核复制起始序列； P ：启动子； MCS ：多克隆位点； TT ：转录终止序列； ori euk ：真核复制起始序列。 selection marker For eukaryotes selection marker For eukaryotes integration sequence Integration sequence

42. section 3 basic priciple and operation of DNA recombination

43.  basic principle obtaining of purpose gene vector selection and construction the connection of exogenous gene and vector recombinant DNA transferring to cells the screening of recombinant DNA the expression of recombinant DNA Isolationselectionconnectiontransformation screening and identification

44. DNA cloning recombinant DNA transferring to host cell replication cultured in solid culture media screening ofbacterial monoclone containing recombinant DNA bacteria

45. I. isolation——the isolation and obtain of purpose gene 1. chemical synthesis 2. genomic or cDNA library 3. PCR 4. other methods

46. 1.chemical synthesis primary structure possible DNA sequence prediction!

47. www.ensembl.org

48. ENSEMBL www.ensembl.org

58. EXON 1 of HOXD13

59. genomic DNA genomic fragment clone vector recombinant DNA bacteria containing recombinant DNA RE bacteria 2. genomic DNA library

60. RE sequnce RE internal sequence cutting cos L R cos cos L left arm R cos right arm genomic DNA RE reconbination ligase infect to E.coli by recombinant phage ~20 Kb DNA cos LR cos~20 Kb exogenous DNA genomic library genomic library established by random cutting method phage package

61. mRNA cDNA double strand recombinant DNA cDNA library reverse transcription vector bacteria  cDNA library reverse transcription A A T T T T AAAA SI nuclease DNA polymerase Ⅰ alkline hydrolysis T T T T replication

62. 3. PCR ATGTGA F ATG TGA R

63. II.rebuild of vector  purpose ： ① to obtain a ppurpose gene fragment ② to obtain protein encoded by the gene

64. vectorDNA insertionhost cell plasmid<10kbbacteria, yeast λ-phage~20kbbacteria cosmid~50 kbbacteria BAC~400kbbacteria YAC~3 Mbyeast different clone capacity and suitable host

65. EcoRISalIATGTAGBamHIXbaISmaI

66. BamHI 1 μl EcoRI 1 μl 10* H buffer 1 μl Vector / gene fragment up to 1 μg ddH2O fill up to 20 μl total volume 20 μl purification of DNA

67. Vector BamHI EcoRI

68. III. connection of vector and gene fragment （ 1 ） single sticky ends （ 2 ） different sticky ends （ 3 ） sticky ends generated by other methods 1. connection of sticky ends

69. Bam H Ⅰ GGATCC CCTAGG T4 DNA ligase 15ºC GATCC G CCTAG + Bam H Ⅰ cutting purpose gene Bam H Ⅰ cutting vector recombinant DNA self connection of vector self connection of purpose gene single sticky ends

70. different sticky ends Eco R Ⅰ Bg l Ⅱ + EcoR Ⅰ + Bg l Ⅱ T4 DNA ligase 15ºC recombinant DNA

71. artificial linker sticky ends of gene fragment generated by PCR exogenous DNA EcoR I FRFR

72. Flat ends generated by the cutting of RE flat ends generated by the sticky end filling Suitable for ： 2. connection of flat ends G CCTAG GGATC CCTAG DNA polymerase I

73. Purpose DNA vector RE T4 DNA ligase 15ºC Recombinant DNA Self connection Of vector Self connection Of fragment

74. 3. Connection of sticky and flat ends Sma Ⅰ Bg l Ⅱ CCCGGG GGGCCC CCCGGG GGGCCC + Sma Ⅰ + Bg l Ⅱ sma Ⅰ + Bg l Ⅱ GGG CCC GGG CCC T4 DNA ligase 15ºC recombinant DNA CCCGGG GGGCCC

75.  The features of host cells ：  Safe host  Lack of RE and recombinant E  Competent cell  The methods of transferring ：  Transformation  Transfection  infection Ⅳ. The transferring of recombinant DNA

76. 1. By the genetic marker on the vector (1)Antibiotics marker (2)Specific sequence Ⅴ. The identification of recombinant DNA RE digestion PCR nucleic hybridization DNA sequencing

77. DNA recombination technology ： summary isolation Isolation of purpose gene selection Selection of vector connection Connection of fragment and vector transformation Recombinant DNA transferring to host cell Identification Screening and identification of recombinant DNA

78. The establishment of expression system ：  the construction of expression vector  the establishment of host cell system  the isolation and purification of expression product Ⅵ. The expression of recombinant DNA

79. An expression vector is similar to cloning vectors, but with a major difference: the expression vector must contain a promoter so that proteins can be expressed.

80. Expression vector

82. Section 4 Application of Recombinant DNA Technology in Medicine

83. biological pharmacy  The protein product or polypeptide product by using gene engineering has become the most important industry nowadays.  First gene engineering product –artificial synthesized insulin from Eli Lilly company in US.

84. recombinant DNA product productfunction Tissue plasminogen activatoranticoagulation Blood coagulation factor VIIIcoagulation granule-macrophage colony stimulating factor Leukocyte growth stimulation Growth factorCell growth and differenciation Growth hormoneNanism treatment insulinDM reatment Interferin (  1b,  2a,  2b,  ) Anti-tumor or anti-virus Monoclonal antibody Specific detection of diagosis experiment, tumor target therapy Hepatitis B immunologyPrevention of hepatitis B

85. Other applications of recombinant DNA technology in medicine  the explore and indentify of pathogenic gene  transgenic engineering and gene targeting  accelerate the HGP  gene diagnosis and gene therapy