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Biology LibreTexts

9.12: Making Recombinant DNAs

Molecular biologists often create recombinant DNAs by joining together DNA fragments from different sources. One reason for making recombinant DNA molecules is to enable the production of a specific protein that is of interest. For example, it is possible to engineer a recombinant DNA molecule containing the gene for human growth hormone and introduce it into an organism like a bacterium or yeast, which could make massive quantities of the human growth hormone protein very cheaply. To do this, one needs to set up the proper conditions for the protein to be made in the target cells. For bacteria, this typically involves the use of plasmids. Plasmids are circular, autonomously replicating DNAs found commonly in bacterial cells. Plasmids used in recombinant DNA methods

  1. replicate in high numbers in the host cell;
  2. carry markers that allow researchers to identify cells carrying them (antibiotic resistance, for example) and
  3. contain sequences (such as a promoter and Shine Dalgarno sequence) necessary for expression of the desired protein in the target cell. A plasmid that has all of these features is referred to as an expression vector (see an example in the figure at left).

Plasmids may be extracted from the host, and any gene of interest may be inserted into them, before returning them to the host cell. Making such recombinant plasmids is a relatively simple process. It involves

  1. cutting the gene of interest with a restriction enzyme (endonucleases which cut at specific DNA sequences);
  2. cutting the expression plasmid DNA with restriction enzyme, to generate ends that are compatible with the ends of the gene of interest;
  3. joining the gene of interest to the plasmid DNA using DNA ligase;
  4. introducing the recombinant plasmit into a bacterial cell; and
  5. growing cells that contain the plasmid. The bacterial cells bearing the recombinant plasmid may then be induced to express the inserted gene and produce large quantities of the protein encoded by it.

Figure 9.12.1: An expression vector

Contributors

Dr. Kevin Ahern and Dr. Indira Rajagopal (Oregon State University)