Презентация на тему "Recombinant DNA Technology"

Содержание

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  DNA,RNA, Recombinant DNA Technology

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  2 “Metabolic pathways” expanded

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

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  5 Developmental biology......

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

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  7 Jaenisch, R.Nat. Genet. 2001 27: 327-331

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  8 The ‘first’ science: “technology drives research drives technology dri...

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  10 Nucleic Acids – DNA and RNA

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  12 DNA + RNA DNA + RNA DNA + RNA RNA DNA

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  13 DNA structure -> sequence

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  15 Polymerase reaction: 5’-> 3’

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  16 Page 93 The central dogma

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  Gene expression.

  18 Page 93

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  20 What is a gene?

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  24 Page 95 Eukaryotes – Intron-Exon concept

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  28 Recombinant DNA Technology

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  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

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

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  31 Restrictionendonucleases 5’-> 3’ 3’

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  35 Gel Electrophoresis

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  36 Gel Electrophoresis

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  37 Gel Electrophoresis

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

  38 Page 1125

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  Construction of a restriction map.

  39 Page 104

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

  40 Page 104

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

  43 Page 109

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  45 Plasmid Cloning Vectors

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  46 Plasmid Cloning Vectors

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  Insertional inactivation

  47 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)

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  48 http://www.bio.davidson.edu/Courses/genomics/method/reporters.html positive negative Blue/White Selection

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  Insertional inactivation

  49 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

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  51 Transformation and Selection

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  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

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  Electron micrograph of bacteriophage λ.

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

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  Bacteriophage T2 injecting its DNA into an E. coli

  54 Page 84

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  55 Life Cycle of Bacteriophage

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  58 Page 107 Replication of bacteriophage upon infection of a cell

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  61 Molecular genetics and bacteriophage

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  Cloning of foreign DNA in λ phages.

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  64 What is a gene library ?

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  Creation of Libraries

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  Sizes of Some DNA Molecules.

  67 Page 92

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  69 Cosmid = Cos - Plasmid

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  Fragmentation of genomic DNA

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  75 cDNA synthesis

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  76 DNA Library Clones -> genetically identical

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  77 Genomic phage library

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  78 Evaluation of library

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  79 Evaluation of library

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  80 Ordered library Microarrays

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

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  82 Different ways to clone a gene

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  Bacterial host engineering

  83 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.

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

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  E.Coli K12 strain has been used for further engineering

  85 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)

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  Additional changes in K12 E.coli for ease of the laboratory practice

  86 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 hsdMis also involved in methylation of DNA hsdRencodes an endonulease hsdSencodes DNA sequence specific protein hsdR- or hsdS- mutants facilitate propagation of any foreign DNA E.coli DNA is methylated by dcm, damandhsdM mcrA-/mcrB- strains are good for cloning eukaryotic DNA mcrA/mcrB/mrr cleaves DNA methylated by other systems

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  87 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/umuCare 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)

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

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  Transformation of plasmid DNA to competent E. coli cells

  89 -- 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

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  Calcium/phosphate (heat shock) method

  90 www.bch.msu.edu/bchug/web/

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