Recombinant DNA

Recombinant DNA refers to a collection of techniques for creating (and analyzing) DNA molecules that contain DNA from two unrelated organisms. One of the DNA molecules is typically a bacterial or viral DNA that is capable of accepting another DNA molecule; this is called a vector DNA. The other DNA molecule is from an organism of interest, which could be anything from a bacterium to a whale, or a human. Combining these two DNA molecules allows for the replication of many copies of a specific DNA. These copies of DNA can be studied in detail, used to produce valuable proteins, or used for gene therapy or other applications.

The development of recombinant DNA tools and techniques in the early 1970s led to much concern about developing genetically modified organisms with unanticipated and potentially dangerous properties. This concern led to a proposal for a voluntary moratorium on recombinant DNA research in 1974, and to a meeting in 1975 at the Asilomar Conference Center in California. Participants at the Asilomar Conference agreed to a set of safety standards for recombinant DNA work, including the use of disabled bacteria that were unable to survive outside the laboratory. This conference helped satisfy the public about the safety of recombinant DNA research, and led to a rapid expansion of the use of these powerful new technologies.

Overview of Recombination Techniques

The basic technique of recombinant DNA involves digesting a vector DNA with a restriction enzyme, which is a molecular scissors that cuts DNA at specific sites. A DNA molecule from the organism of interest is also digested, in a separate tube, with the same restriction enzyme. The two DNAs are then mixed together and joined, this time using an enzyme called DNA ligase, to make an intact, double-stranded DNA molecule. This construct is then put into Escherichia coli cells, where the resulting DNA is copied billions of times. This novel DNA molecule is then isolated from the E. coli cells and analyzed to make sure that the correct construct was produced. This DNA can then be sequenced, used to generate protein from E. coli or another host, or for many other purposes.

There are many variations on this basic method of producing recombinant DNA molecules. For example, sometimes researchers are interested in isolating a whole collection of DNAs from an organism. In this case, they digest the whole genome with restriction enzyme, join many DNA fragments into many different vector molecules, and then transform those molecules into E. coli. The different E. coli cells that contain different DNA molecules are then pooled, resulting in a "library" of E. coli cells that contain, collectively, all of the genes present in the original organism.

Another variation is to make a library of all expressed genes (genes that are used to make proteins) from an organism or tissue. In this case, RNA is isolated. The isolated RNA is converted to DNA using the enzyme called reverse transcriptase. The resulting DNA copy, commonly abbreviated as cDNA, is then joined to vector molecules and put into E. coli. This collection of recombinant cDNAs (a cDNA library) allows researchers to study the expressed genes in an organism, independent from nonexpressed DNA.

Applications

Recombinant DNA technology has been used for many purposes. The Human Genome Project has relied on recombinant DNA technology to generate libraries of genomic DNA molecules. Proteins for the treatment or diagnosis of disease have been produced using recombinant DNA techniques. In recent years, a number of crops have been modified using these methods as well.

As of 2001, over eighty products that are currently used for treatment of disease or for vaccination had been produced using recombinant DNA techniques. The first was human insulin, which was produced in 1978. Other protein therapies that have been produced using recombinant DNA technology include hepatitis B vaccine, human growth hormone, clotting factors for treating hemophilia, and many other drugs. At least 350 additional recombinant-based drugs are currently being tested for safety and efficacy. In addition, a number of diagnostic tests for diseases, including tests for hepatitis and AIDS, have been produced with recombinant DNA technology.

Gene therapy is another area of applied genetics that requires recombinant DNA techniques. In this case, the recombinant DNA molecules themselves are used for therapy. Gene therapy is being developed or attempted for a number of inherited human diseases.

Recombinant DNA technology has also been used to produce genetically modified foods. These include tomatoes that can be vine-ripened before shipping and rice with improved nutritional qualities. Genetically modified foods have generated controversy, and there is an ongoing debate in some communities about the benefits and risks of developing crops using recombinant DNA technology.

Since the mid-1970s, recombinant DNA techniques have been widely applied in research laboratories and in pharmaceutical and agricultural companies. It is likely that this relatively new area of genetics will continue to play an increasingly important part in biological research into the foreseeable future.

Patrick G. Guilfoile

Bibliography

Cooper, Geoffrey. The Cell: A Molecular Approach. Washington, DC: ASM Press, 1997.

Glick, Bernard, and Jack Pasternak. Molecular Biotechnology: Principles and Applications of Recombinant DNA, 2nd ed. Washington, DC: ASM Press, 1998.

Kreuzer, Helen, and Adrianne Massey. Recombinant DNA and Biotechnology, 2nd ed. Washington, DC: ASM Press, 2000.

Lodish, Harvey, et al. Molecular Cell Biology, 4th ed. New York: W. H. Freeman, 2000.

Old, R. W., and S. B. Primrose. Principles of Gene Manipulation, 5th ed. London: Blackwell Scientific Publications, 1994.

Internet Resource

"Approved Biotechnology Drugs." Biotechnology Industry Organization. <http://www.bio.org/aboutbio/guide2.html>.