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Molecular Biology Xu Liyan

Molecular Biology Xu Liyan. Chapter 14 gene recombination and gene engineering. section 1 DNA Recombination section 2 Recombinant DNA technology section 3 Relationship between Recombinant DNA technology and Medicine. section 1 DNA Recombination. 1.1 Homologous Recombination

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Molecular Biology Xu Liyan

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

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

  3. section 1 DNA Recombination • 1.1 HomologousRecombination • 1.2 Gene Transfer and Recombine in Bacteria • Conjugation • Transformation • Transduction • 1.3 Site-specific Recombination • 1.4 Transpositional Recombination

  4. 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

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

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

  7. Holliday 5’ 3’ 3’ 3’ 5’ 5’ 3’ 5’ 5’ 5’ 3’ 3’ 3’ 5’ 5’ 3’ 5’ 5’ 3’ 3’ Ruv C Ruv C 3’ 3’ 5’ 5’ 3’ Ligase Ligase 5’ 5’ 3’ 3’ 3’ 5’ 5’ 3’ 5’ 5’ 5’ 3’ 3’ 5’ 3’ 5’ 5’ 3’ 3’ 3’ 5’ 5’ 3’

  8. 1.2 Gene Transfer and Recombine in Bacteria Conjugation F- F+ 1 2 F factor 3 n 5 4

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

  10. transformation of diplococcus pneunonine A well-known experiment Avery, 1943 DNA Diplococcus pneumoniae without capsule is untoxicity to human. Diplococcus pneumoniae with capsule is toxicity to human

  11. 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.

  12. bacteria 1 phage integration some factor bacteria 2

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

  14. 1.3site-specific recombination The integration catalyzed by integrase between two site-specific DNA fragments is known as site-specific recombination.

  15. 1.4 Transpositional recombination the displacement of some gene in the genome by insertion sequence or transposons

  16. 1.4.1 insertion sequence and its mediated gene transposition  Thelength of typical insertion sequence is about 750~1500bp.  Typical insertion sequence includes two 9 ~ 41bp inverted repeat sequence and a transposase.  A4 ~12bp positive repeat sequence always link to flanking of inverted repeat sequence.  Gene transposition by insertion sequence:  conservative transposition  duplication transposition

  17. duplication transposition insertion sequences target transposase transposase 5’ transposase 3’ 3’ transposase 3’ 5’ target transposase 5’ 3’ 3’ transposase 3’ 5’ polymerase I ligase 1 2 transposase transposase transposase transposase transposase

  18. 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.

  19.  The structure oftransposon is similar to one of insertion sequence.  The bothinsertion 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.

  20. insertion sequence transposase Tn3 transposase gene repressor gene amp-R gene Tn10 IS 10L tet-R gene IS 10L

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

  22. 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

  23. 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.

  24. 2.1.2 tool enzyme  restriction endonuclease DNA ligase  DNA polymerase I  reverse transcriptase  polynucleotide kinase  end-transferase  alkaline phosphatase

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

  26. 2.1.3 target gene The interested gene is the target gene

  27. 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).

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

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

  30. 5' 3' 3' 3' 3' 3' 5' 5' 5' 5' denaturation denaturation 5' 5' 3' 3' annealing 3' 5' 3' 5' 5' 5' 5' 5' 3' 3' 5' Next cycle 5' 3' 3' 5' 3' PCR Process extension

  31. 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.

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

  33. plasmid EcoR I 0 29 Hind III Xmn I 3966 ampr 375 BamH I tetr Pst I 3612 650 Sal I pBR322 4.36kb condition 1424 Ava I A origin Ascreening gene 2034 Xmn I ori 2067 Pvu II A single restriction site

  34. Eco R I Sac I Kpn I Sma I Bam H I Xba I Hinc II Pst I Sph I Hind III plasmid lac Z’ polylinker 52bp Plac ampr pUC19 2.69kb lac I ori

  35. 2.2 the basic principle of DNA recombination technology

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

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

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

  39. recombinant transformation vectors and recombinants competent cells

  40. recombinant screening plasmid extraction amp or tet etc 1 + 2 digist with restriction enzeme - - + + 1 1 2 2 marker marker

  41. target gene amplification

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

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

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

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

  46. target gene expression prokaryotic expression system D

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

  48. 1 2 3 4 5 6 7 8 9 10 ← 25kDa 21kDa → eukaryotic expression system

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