Techniques : Recombinant DNA

The Details:Techniques:
Recombinant DNA

Timeline: 1972
Timeline: 1973
Discovery of DNA
rDNA Animation
Polymerase Chain Reaction

Scientist Profiles
Paul Berg
Kary B. Mullis

Web Link:
Speaking the Language of Recombinant DNA

Were it not for recombinant DNA (rDNA) technology, also referred to as gene splicing, cloning would merely be an interesting, but useless side path of molecular biology. The opposite is also true: without cloning, recombinant DNA would continue to be an expensive and time-consuming process. The reason for this is simple. Recombinant DNA is difficult and has a low success rate. When it is achieved, there is no guaranteed way to pass the genetically altered organism's newly obtained trait on to subsequent generations. However, a organism can be cloned, thus producing thousands of organisms with the desired trait.

The first task of the gene splicing process is to isolate the gene that is to be inserted into the DNA. For this example, a human protein will be inserted into another animal’s DNA, just as in the case of Polly. This can be done through the lengthy process of searching through the entire genome. In the case of humans, the genome is being mapped by the Human Genome Project. However this is costly and has not yet been completed. Another way is to isolate the RNA which is producing the protein from the specific area of the body. This RNA can then be converted into DNA using reverse transcriptase, an enzyme used by retroviruses to convert their RNA genes into DNA.

Recombinant DNA is made possible by two important enzymes. Restriction enzymes and DNA ligase are the two principal tools, first used by Paul Berg in 1972, employed to alter DNA. Restriction enzymes are used to "cut" DNA at a specific location. These are used if the DNA must be removed from the entire strand of DNA (as in the first method for gene isolation above) and also to open the section of DNA into which the isolated gene will be inserted. DNA ligase is used to "glue" two sections of DNA together.

However, the isolated gene cannot be directly inserted into the target DNA. A cloning vector must be used. In the Cohen and Boyer experiment, a plasmid was used. A plasmid is a circular section of DNA which can be inserted into a cell. The cell then takes on the characteristics of this DNA. This technique is easy, however it does not work on larger animals. The other cloning vector is a virus. The isolated gene is inserted into the DNA of a virus and "glued" using DNA ligase. The virus then injects the gene into the cell’s main DNA. The cell then begins producing the desired protein. Cloning is looked upon as a much easier, cost effective, and reliable way of mass producing genetically altered larger organisms.

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