• Procedure that allows DNA fragments or genes to be copied
  1. Use a restriction enzyme to cut up the foreign DNA that contains a gene to be copied. The restriction enzyme produces multiple fragments of foreign DNA with sticky ends
  2. Use the same restriction enzyme to cut up the DNA of a cloning vector. This produces the same strictly ends in both foreign DNA and cloning vector: DNA molecule that can carry foreign DNA into a host cell and be replicated there                   
  • Plasmid is a common cloning vector bcuz can be introduced into bacteria for transformation
  • Using a plasmid that has one restriction site for restriction enzyme can help with identification of the copied gene
    • ampR gene: gives bacterial resistance against antibiotic ampicillin
    • GFP gene: makes bacteria fluorescence green
    • lacZ gene: codes for enzyme that breaks down lactose
      • Also breaks down artificially made X-gal
      • The one restriction site for the restriction enzyme occurs in within the lacZ gene
  1. Mix the cut foreign DNA with cut plasmids. This allows base-pairing at the sticky end
  2. Apply DNA ligase to stabilize attachments and close up the backbone. Forms recombinant plasmids (some plasmids will not pair)
  3. Mix plasmids with bacteria to allow transformation. Some of the bacteria will absorb the plasmids (transformation)
  • Not all bacteria will be competent enough to take up trait and express traits associated with gene
    • Competent: change in structure and permeability of cell membrane
  1. Grow the transformed bacteria in the presence of ampicillin and X- gal.
  • Only bacteria that have absorbed a plasmid (transformed bacteria) will grow in presence of ampicillin bcuz contain the resistant genel; they will also be white bcuz lack functioning lacZ gene (foreign DNA was inserted within lacZ gene of the plasmid, making gene dysfunctional )
  • Purposes of Gene Cloning: to make many copies/amplify particular gene and to produce a protein product from it
  • Can isolate gene and give product to different organism

Using Restriction Enzymes to Make a Recombinant Plasmid

  • Recombinant DNA tech uses Restriction enzymes (restriction endonucleases) to cut DNA
    • Restriction enzymes obtained from bacteria that manufacture these enzymes to combat invading viruses
      • DNA of a bacterial cell is protected from the cell’s own restriction enzymes by the addition of methyl groups (—CH3) within the sequences recognized by the enzymes
    • Restriction enzymes cut the sugar phosphate backbones of the two DNA strands in a staggered manner at short, specific nucleotide sequences (restriction sites) → yielding a set of double-stranded restriction fragments with single-stranded sticky ends
    • Sticky ends can form hydrogen-bonded base pairs with complementary sticky ends of a DNA molecules cut with the same restriction enzyme