Invivo Cloning

• Invivo Cloning


  Uses restriction endonucleases for 'sticky' ends, and modified to ensure transcription

  • Promoter region added at the start as a binding site for RNA polymerase
  • Terminator region added at the end to detach RNA polymerase and stop transcription
• Vectors


  Carriers of DNA fragments to the host cell

  • Plasmids are the most common vectors as circular DNA is seperated
• DNA Insertion in Vectors

  Uses the same restriction endonuclease

  • Creates the same sticky ends
  • Complementary binding
  • DNA ligase joins the ends together via a condensation reaction
• Transformation of Vector


  Needs to be inserted into the host cell, cell membrane of host needs to be more permeable

  • Host cells are mixed with Ca2+ and heat shocked
• Issues of Invivo Cloning

  • Plasmid may not be in the cell
  • Sticky ends may of rejoined on the plasmid
  • Sticky ends may of rejoined on the DNA
• Marker Genes


  Identification of successful bacteria

  • Antibiotic resistance
  • Fluorescent proteins
  • Enzymes
• Antibiotic Resistant Marker Genes

  Using 2 resistant genes

  • DNA fragment disrupts tetracycline gene, no longer resistant
  • Grow the bacteria on agar
  • Transfer bacterial colonies onto a plate with ampicillin using velvent blocks
  • Detect which colonies die, transfer to a plate with tetracycline antibiotic
  • Colonies which die but appear on ampicillin plate contain the DNA fragments
• Fluorescent Markers


  Jellyfish gene GFP inserted into the plasmid with DNA inserted between, those that dont glow have the DNA fragment
• Enzyme Markers


  Lactase can turn certain substances blue from colourless

  • Gene inserted with DNA fragment between, grown on colourless substance agar plate
  • Colourless area contains DNA