gene cloning

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  • Created by: vezting
  • Created on: 24-02-16 09:06
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  • gene cloning
    • genetic engineering
      • is the manipulation of an organisms DNA. when they have DNA altered they are said to be transformed and their DNA is recombinant
      • 1: desired DNA fragment isolated using rest. enzymes
      • 2: insert DNA into vector. Eg plasmid or bacteria phages.
      • 3: vector DNA is isolated using same rest. enzymes DNA ligase used to stick DNA fragment and vector together
    • sticking DNA and vector together
      • 1: as vector and DNA have been cut using same rest. enzymes their stick ends are complimentary to each other
      • 2: mixed together with DNA ligase, forming sugar phosphate backbone
      • 3: new combination of bases- recombinant DNA
    • transforming the cells
      • heat shock- ice cold calcium chloride solution, cell walls permeable, mixture heated 42 degrees, 1-2 mins.
      • electroporation - bacteria given an electric shock, makes it temporarily permeable, allowing plasmid in
      • benefits of taking up plasmid- genes that code for resistance are within, contain genes that help microorganism invade host, genes allow micro.to use nutrients they wouldn't normally
    • in vivo vs PCR (in vitro)
      • in vivo +
        • cheaper as bacteria grows on cheap materials
        • large fragments can be cloned
          • useful if you don't know the exact sequence location of the gene you want to clone
        • less technically difficult as PCR needs optimum conditions
      • in vivo -
        • DNA fragment has to be isolated and this takes a long time
        • a lot of space and equipment needed
      • in vitro +
        • only replicates DNA of interest no isolation needed
        • fast process, millions of copies in a few hours
        • safer as not dealing with living cells
        • uses less space so saves money
      • in vitro -
        • primer and chemicals expensive
        • more mutations as DNA polymerase isn't good at 'proof reading'
        • can only replicate small DNA fragment

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