Summary - Gene Cloning

  • Gene cloning is the process in which a gene of interest is located and copied (cloned) out of all the DNA extracted from an organism.
  • The basic steps in gene cloning are:
    1. DNA extracted from an organism known to have the gene of interest is cut into gene-size pieces with restriction enzymes.
    2. Bacterial plasmids are cut with the same restriction enzyme.
    3. The gene-sized DNA and cut plasmids are combined into one test tube. Often, a plasmid and gene-size piece of DNA will anneal together forming a recombinant plasmid(recombinant DNA).
    4. The recombinant plasmids are transferred into bacteria using electroporation or heat shock.
    5. The bacteria is plated out and allowed to grow into colonies. All the colonies on all the plates are called a gene library.
    6. The gene library is screened to identify the colony containing the gene of interest by looking for one of three things:
      1. the DNA sequence of the gene of interest or a very similar gene
      2. the protein encoded by the gene of interest
      3. a DNA marker whose location has been mapped close to the gene of interest
  • Antibiotic resistance genes are naturally present in the bacterial plasmids used in gene cloning. The presence of the antibiotic resistance trait allows genetic engineers to screen out any bacteria that have not been transformed (are not recombinant and could not contain the gene of interest). This is why some transgenic plants have antibiotic resistance and is a source of controversy for those opposed to genetic engineering.
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