Gene Cloning and Gene Transfer

Gene Cloning and Gene Transfer

1. Reverse Transcriptase

   • Found in retrovirus (HIV)
   • template mRNA is used to form cDNA
     • Using free nucleotides
     • cDNA (Complementary DNA)
   • Used to 'reverse engineer' genes from a protein
   • Issues
     • Copy errors
     • No Introns
   • Single stranded
     • Can be converted into double stranded DNA
       • via DNA polyermase

2. Gene Isolation

   • Must know loci of gene
   • Restriction endonuclease cuts DNA
     • At a recognition site.
       • Usually palindromic sequence
     • Produces blunt ends and sticky ends (useful)

3. In Vivo

   • Insertion
     • 1. Plasmid cut with same restriction enzyme
       • Restriction Endonuclease
     • 2. Plasmid only cut once
     • 3. Hydrogen bonds form between sticky ends
     • 4. DNA Ligase forms phosphodiester bonds
   • Transfer
     • 1. Plasmid and bacteria mixed
       • With ice-cold calcium chloride
       • Heat shock so bacteria take on plasmid.
     • 2. Options
       • No Plasmid
         • Most common
       • Self-ligated gene
       • Self-ligated plasmid
       • Recombinant Plasmid
     • 3. Marker Genes
       • Used for identification
       • Antibiotic resistance
       • Enzymes
       • Fluroescence
     • 4. Replica plating
       • Identify dead bacterium with recombinant Plasmid

4. In vitro - PCR

   • Requires
     • Template DNA
     • DNA Polyermase
     • DNA Nucleotides
     • Primers (Oglionucleotides
     • Thermocycler
   • Primers stop re-joining of strands and allow extending to begin.
   • Polyermase is thermostable
   • Stages
     • 1. Denaturing stage
       • 94-95c
     • 2. Annealing Stage
       • 50-55c
     • 3. Extending Stage
       • 72c