DNA recombinant technology

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DNA recombinant technology

  1. 1. Recombinant DNA Technology
  2. 2. Required features 1. Origin of replication 2. Selectable marker 3. Screenable marker for recombinant molecules 4. Cloning sites
  3. 3. AAGATGCGAATTCGTACA DNA methylase AAGATGCGAATTCGTACA DNA methylase * * AAGATGCGAATTCGTACA AAGATGCGAATTCGTACA Restriction endonuclease Restriction endonuclease * * AAGATGCGAATTCGTACA AAGATGCG AATTCGTACA
  4. 4. 5’ ATGCGAATTCCGGTT 3’ 3’ TACGCTTAAGGCCTT 5’ EcoR1 Sticky-end cutter 5’-ATGCG-3’ 5’-AATTCCGGTT-3’ 3’-TACGCTTAA-5’ 3’-GGCCTT-5’ 5’ ATGCGATATCCGGTT 3’ 3’ TACGCTATAGGCCTT 5’ Blunt-end cutter EcoRV 5’-ATGCGAT-3’ 3’-TACGCTA-5’ 5’-ATCCGGTT-3’ 3’-TAGGCCTT-5’
  5. 5. T4 DNA Ligase
  6. 6.  All of the previous steps were performed in vitro.  We have generated a very small amount of a recombinant plasmid  Need to amplify in bacteria to get enough to work with.
  7. 7.  Transformation – process to mobilize DNA into bacterial host  Select for transformed bacteria on specific antibiotic that corresponds to the antibiotic resistance gene present on the plasmid
  8. 8. 0.1 to 1% of cellular protein 10 to 70% of cellular protein
  9. 9. cDNA
  10. 10. •Requires that you know something about the gene sequence •Can get sequence information form purified protein
  11. 11. Product name Protein type Application Company Adagen (Adenosine deaminase ) An enzyme Severe combined immunodeficiency disease (SCID) Enzon Genotropin (Recombinant growth hormone) A hormone Growth hormone deficiency (GHD) in children Pharmacia & Upjohn Humalog (Recombinant human insulin) A hormone Diabetes Eli Lilly Nabi-HB (AntiHepatitis B) An antibody Hepatitis-B Nabi Novo Seven (Recombinant coagulation factor VIIa) A modified factor Hemophillia patients with inhibitors Novo Nordisk Ontak (Diphtheria toxin-interleukin-2) A fusion protein Cutaneous T-cell lymphoma (CTCL) Ligand Pharmaceuticals Roferon-A (Recombinant interferon alfa-2a) A modifier Hairy cell leukemia or AIDS-related Kaposi's sarcoma Hoffmann-La Roche
  12. 12.  Can introduce gene into animals and plants  These modified organism are powerful research tools to study the effect of a specific gene product on metabolism, development etc….  Has also been used to develop improved agricultural products
  13. 13. Genetically Engineered Salmon Is Bigger Better?
  14. 14. http://www.agwest.sk.ca/sabic_index_tp.shtml

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