Recombinant DNA Technology - Microbiology and Immunology

Recombinant DNA Technology

In recombinant DNA technology, first, the DNA responsible for a particular phenotype is identified and isolated. Once purified, the gene or genes are fused with other pieces of DNA to form recombinant DNA molecules. These are prop-agated (gene cloning) by insertion into an organism that need not even be in the same kingdom as the original gene donor.

Cloning Vectors and Hosts

A good recombinant vector has two indispensable qualities: it must be capable of carrying a significant piece of the donor DNA and it must be readily accepted by the cloning host.

◗ Cloning vectors

Cloned vectors include: (a) plasmids, (b) bacteriophages, and (c) hybrid vectors.

    Plasmids are excellent vectors because they are small, wellcharacterized, easy to manipulate, and they can be trans-ferred into appropriate host cells through transformation.coli plasmid carries genetic markers for resistance toantibiotics, although it is restricted by the relatively small amount of foreign DNA it can accept.

      Bacteriophages are also good vectors because they havenatural ability to inject DNA into bacterial hosts through transduction. The Charon2 phage is a modified phage vector that lacks a large part of its genome; hence it can carry a fairly large segment of foreign DNA.

      Hybrid vectors have been developed by splicing two dif-ferent vectors together. A cosmid is an example of a hybrid vector that combines a plasmid and a phage and is capable of carrying relatively large genomic sequences. A hybrid coli–yeast vector can be inserted in both bacterial andyeast cloning hosts.

◗ Cloning hosts

E. coli is the traditional cloning host that is still used in themajority of experiments. This is because this bacterium was the original recombinant host and the protocols using it are well established, relatively easy, and reliable. Hundreds of specialized cloning vectors have been developed for it. The main disadvan-tage with E. coli is its lack of versatility in correctly expressing eukaryotic genes.

The yeast Saccharomyces cerevisiae is another alternative host used for certain industrial processes and research. This host being eukaryotic already possesses mechanisms for processing and modifying eukaryotic gene products. Certain techniques may also employ different bacteria (Bacillus subtilis), animal cell culture, and even live animals and plants to serve as cloning hosts.

Biological Products of Recombinant DNA Technology

Recombinant DNA technology is used by pharmaceutical companies to manufacture medicines that cannot be manu-factured by any other means. Diseases, such as diabetes and dwarfism, caused by lack of an essential hormone are now being treated by replacing the genes of missing hormone. Porcine and bovine insulin were once the only forms avail-able to treat diabetes, even though such animal products used to cause allergic reactions in certain sensitive individuals. In contrast, dwarfism that cannot be treated with animal growth hormones was treated only with human growth hormone (HGH) obtained from the pituitaries of cadavers. At one time, not enough HGH was available to treat thousands of children in need. However, now the scenario is changed by advent of recombinant HGH. Recombinant technology has changed the outcome of these and many other conditions by enabling large-scale manufacture of lifesaving hormones and enzymes of human origin.