Unit 5.6 Recombinant DNA Technology

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  • Recombinant DNA Technology
    • Method
      • 1) Isolation of gene and vector
      • 2) Insertion of gene into vector
      • 3)Transformation of vector into cells
      • 4) Growth
      • 5) Selection of cells with genes
    • Reverse Transcriptase
      • 1) Extract mRNA
      • 2) Add reverse transcriptase and cDNA will be made
      • 3) Add DNA polymerase to make double stranded DNA
      • 4) Insert DNA into vector
    • Insulin Production
      • 1) Insulin gene is located using a gene probe and isolated 
      • 2) Same restriction enzymes used to cut DNA of the plasmid and the insulin gene. 
        • Forms complementary sticky ends
      • 3) sticky ends anneal and DNA ligase joins the DNA pieces
      • 4)The plasmid is transformed into E.coli bacteria
      • 5)E.coli grown in the presence of an antibiotic.
        • marker gene (antibiotic resistance) in the plasmid.
          • so cells that have taken in the plasmid are resistant to antibiotics
      • 6) E.coli cloned and insulin extracted
    • Adv
      • Complex proteins produced in large quantities
      • Don’t need to extract proteins from mammalian organs
    • Disadv
      • expensive for large scale production
      • Difficult to identify/isolate correct gene
      • More than 1 gene may be needed for complex proteins
      • Disruption of normal genes during insertion
      • Transfer of antibiotic resistant genes into E.coli in human gut


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