DNA Technology

DNA Technology

1. Reverse transcription

   • mRNA isolated from donor cell
     • e.g. insulin mRNA from beta-cells of human pancreas
   • Retroviral reverse transcriptase produces complementary strand of DNA (cDNA)
   • cDNA strand separated from mRNA strand by a helicase
   • DNA polymerase catalyses formation of complementary strand
     • Double-stranded cDNA produced coding for human insulin

2. Restriction endonucleases

   • Cleave DNA at specific palindromic recognition sites (may be 4,6,8 bp long)
   • Produce sticky ends
   • Gene can be inserted into a vector (bacterial plasmid) cleaved by the same restriction enzyme

3. Transformation

   • Insertion of recombinant plasmid into host cell (bacteria)
   • Bacteria & plasmid incubated with calcium chloride in cold conditions
   • Heat shock applied
   • Cell membrane becomes permeable to DNA

4. Selection

   • To find out which bacteria have taken up the recombinant plasmid
   • Tested for molecular markers
     • Replica plating
       • Plasmids contain antibiotic resistance genes, e.g. ampicillin, tetracycline
         • In hybridised plasmid one gene (say tetracycline resistance) will be inactivated
       • Place colonies of bacteria on plates of two antibiotics
       • Select those which grow on Amp but not Tet
     • Beta-galactosidase
       • Turns lactose (blue) to glucose and galactose (white)
       • Select those which remain blue with lactose.
     • GFP
       • Select the bacteria which do not fluoresce!
       • Green Fluorescence Protein - gene interrupted by insertion of foreign DNA

5. Growth

   • Cloning of those bacteria which contain the gene
     • En masse production of insulin!