SU6(2)(1)

• Gene cloning


  Amplifying a specific piece of DNA via a bacterial cell

  
• Cloning vector


  Stable, replicating DNA molecule to which a foreign DNA fragment can be attached for introduction into a cell

  
• Components of gene cloning


  • Manipulation of DNA
  • DNA needs to be introduced into a bacterial cell – where it will replicate together with the cell

  
• Characteristics of an effective cloning vector


  • Origin of replication – ensures the vector is replicated within the cell
  • Selectable markers – means genes which allow any cells containing the vector to be selected or identified
  • One or more unique restriction sites into which a DNA fragment can be inserted

  
• Plasmids


  Small circular DNA molecules in bacteria that meet the requirements of an effective cloning vector

  
• puC19 plasmid


  A typical cloning vector that contains a cluster of unique restriction enzyme sites in a multiple cloning site, an origin of replication, and selectable markers (ampicillin resistance and lacZ gene)

  
• Bacterial transformation and selection


  1. Plasmids are transformed into bacterial host cells

  2. Plasmids can replicate, and selectable markers are used to screen for bacterial cells containing plasmids

  
• Selectable markers


  Allow identification/selection of transformed bacteria that have taken up the plasmid

  
• Transformed bacteria containing a plasmid


  Can survive in the presence of the antibiotic that the plasmid confers resistance to

  
• Step A: Cloning process

  • Assume you have a plasmid, 5kb in size, with a single BamHI restriction site, an ampicillin resistance gene and an origin of replication
  • You also have some DNA purified from e.g. mouse cells
  • You digest both DNA samples with BamHI
  • This cleaves the plasmid once, and cuts the mouse DNA into multiple fragments of different size
  • If you mix the 2 samples, some of the mouse DNA fragments will anneal with the plasmid, and can be joined by DNA ligase

  
• This results in a recombinant plasmid

  
• You can also generate some original non-recombinant plasmids, and have many unligated DNA fragments remaining

  
• Bacterial transformation and selection

  1. If you now add the plasmid to bacterial cells, some cells can take up the plasmid via the process of transformation

  2. In transformed bacteria, the plasmid can replicate to multiple copies as it contains an ori, and the antibiotic resistance gene on the plasmid can be expressed

  3. If you now grow these bacteria in the presence of the antibiotic, the normal bacteria will be killed, but the plasmid-containing bacteria will survive as they are able to resist the antibiotic

  
• This is the "power of the selectable marker"
• Result of successful gene cloning experiment – a bacterium containing a recombinant plasmid

  
• One can grow liquid culture of your transformed bacteria, and from there purify the plasmid DNA