1 DNA,RNA, Recombinant DNA Technology. 2 Metabolic pathways expanded. - презентация

1 1 DNA,RNA, Recombinant DNA Technology

2 2 Metabolic pathways expanded

3 3 Model organisms: Cellular biology, biochemistry... molecular biology

4 4

5 5 Developmental biology......

6 6 Fly mutation eyeless The fly and you are not much different.

7 7 Jaenisch, R.Nat. Genet :

8 8 The first science: technology drives research drives technology dri...

9 9

10 10 Nucleic Acids – DNA and RNA

11 11

12 12 DNA + RNA RNA DNA

13 13 DNA structure -> sequence

14 14

15 15 Polymerase reaction: 5-> 3

16 16 Page 93 The central dogma

17 17

18 18 Gene expression. Page 93

19 19

20 20 What is a gene?

21 21

22 22

23 23

24 24 Page 95 Eukaryotes – Intron-Exon concept

25 25

26 26

27 27

28 28 Recombinant DNA Technology

29 29 Definitions Recombinant DNA, a DNA construct created by fusing different fragments of DNA Genetic Engineering, the deliberate alteration of DNA through the creation of recombinant DNA Genetically Modified Organism, a living entity modified through genetic engineering Transgenic, a genetically modified organism containing DNA from another source

30 30 Recombinant DNA Technology Clones -> Cells or organisms with identical DNA

31 31 Restrictionendonucleases 5-> 3 3 <- 5

32 32

33 33

34 34

35 35 Gel Electrophoresis

36 36 Gel Electrophoresis

37 37 Gel Electrophoresis

38 38 X-Ray structure of a complex of ethidium bromid with DNA. Page 1125

39 39 Construction of a restriction map. Page 104

40 40 Restriction map for the 5243-bp circular DNA of SV40. Page 104

41 41

42 42

43 43 Construction of a recombinant DNA molecule through the use of synthetic oligonucleotide adaptors Page 109

44 44

45 45 Plasmid Cloning Vectors

46 46 Plasmid Cloning Vectors

47 47 Insertional inactivation Gene in cloning site: LacZ -> pUC18 (lacZ complements the host defect in lacZ) -> pUC18 into host organism -> active lacZ (β-galactosidase) from plasmid-> cleavage of X-gal (blue colonies) -> gene cloned into polylinker -> lacZ gene disrupted -> no cleavage of X-gal (white colonies)

48 48 positive negative Blue/White Selection

49 49 Insertional inactivation Gene in cloning site: Resistance marker -> pBR322 (cloning sites within antibiotica resistence marker) -> plasmid into host -> resistance against 2 antibiotica -> gene cloned within one resistance marker -> gene for antibiotica resistance marker disrupted -> sensitive against one antibioticum

50 50

51 51 Transformation and Selection

52 52 Horizontal gene transfer - Transformation -> uptake of naked DNA (chemical transformation, electroporation) - Conjugation -> DNA transfer by cell – cell contact - Transduction -> DNA transfer by bacteriopage infection Other methods of Gene transfer -> used with fungi, animal and plant cells: - Microinjection - protoplasts

53 53 Electron micrograph of bacteriophage λ. Page 107 Electron micrograph of the filamentous bacteriophage M13. Bacteriophages

54 54 Bacteriophage T2 injecting its DNA into an E. coli Page 84

55 55 Life Cycle of Bacteriophage

56 56

57 57

58 58 Page 107 Replication of bacteriophage upon infection of a cell

59 59

60 60

61 61 Molecular genetics and bacteriophage

62 62

63 63 Cloning of foreign DNA in λ phages. Page 110

64 64 What is a gene library ?

65 65 Creation of Libraries

66 66 Creation of Libraries

67 67 Sizes of Some DNA Molecules. Page 92

68 68

69 69 Cosmid = Cos - Plasmid

70 70

71 71

72 72

73 73 Fragmentation of genomic DNA

74 74

75 75 cDNA synthesis

76 76 DNA Library Clones -> genetically identical

77 77 Genomic phage library

78 78 Evaluation of library

79 79 Evaluation of library

80 80 Ordered library Microarrays

81 81 Ordered library Chromosome Walking -> also used in Human Genome Project

82 82 Different ways to clone a gene

83 83 Bacterial host engineering Escherichia coli (E. coli) is a type of bacteria normally found in the intestines of people and animals. Although most strains of E. coli are harmless, some can cause illness or even death. The most serious form is E. coli 0157:H7. E. coli leads to about 73,000 cases of infection and 61 deaths each year in the United States.

84 84 Genetic and physical maps of the E. coli chromosome Fig. 8.14

85 85 E.Coli K12 strain has been used for further engineering The K12 strain was first isolated in 1921 from the stool of a malaria patient and it has been maintained in laboratory stocks as a pure strain for the last 75 years. Most strain in molecular biology are recA- endA- hsdR- Every strain comes with description of its genotype: DH5alpha (recA-; hsdR-; LacIq; uvrA-; mcrA-……) Asilomar Conference on Recombinant DNA (February 1975) NIH Recombinant Advisory Committee (RAC) (1973)

86 86 Additional changes in K12 E.coli for ease of the laboratory practice 1. Bacterial restriction modification systems have been removed. (To prevent its interferention with the replication of foreign DNA in bacteria). hsdR/hsdM/hsdS (EcoK) restriction system mcrA/mcrB/mrr complex Degrades DNA not methylated at the sequence 5'-AAC-(N)5-GTGC-3' hsdM recognises unmethylated DNA hsdM is also involved in methylation of DNA hsdR encodes an endonulease hsdS encodes DNA sequence specific protein hsdR- or hsdS- mutants facilitate propagation of any foreign DNA E.coli DNA is methylated by dcm, dam and hsdM -mcrA-/mcrB- strains are good for cloning eukaryotic DNA mcrA/mcrB/mrr cleaves DNA methylated by other systems

87 87 Additional changes in K12 E.coli for ease of the laboratory practice 2. DNA recombination systems are modified to prevent rearrangements (RecA-) (to prevent deletions and rearrangements) recA is a core recombination protein recA- strains allow cloning of repetitive sequences recA-/recB-/recC- are enhanced strains with very low recombination efficiency uvrC/umuC are involved in DNA repair uvrC-/umuC- are good for cloning of inverted repeats 3. Endonuclease activity has been mutated (EndA-) (to increase plasmid yields and improve the quality of DNA – no nicks)

88 88 Transformation of plasmid DNA in competent E. coli cells Competent (here) = able to uptake DNA

89 89 Transformation of plasmid DNA to competent E. coli cells -- Electroporation and electroporation-competent cells -- Heat shock transformation and chemically competent cells Recovery in rich growing media Electroporation Chemical transformation treating E. coli CaCl2 will batter the membranes and essentially make the bacteria very unhappy. CaCl2 is gaping holes in the membrane BRIEF HEAT SHOCK

90 90 Calcium/phosphate (heat shock) method

91 91 Conjugation Lederberg Monod F- to F+ 100 minutes 4000 genes