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Recombinant DNA refers to a collection of techniques for creating (and analyzing) DNAmolecules 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 iscalled 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 indetail, 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 tomuch concern about developing genetically modified organisms with unanticipated and

 potentially dangerous properties. This concern led to a proposal for a voluntary moratorium onrecombinant 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 arestriction 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 samerestriction 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 constructisthen 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 thecorrect 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 DNAmolecules. 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. Thiscollection of recombinant cDNAs (a cDNA library) allows researchers to study the expressed

genes in an organism, independent from nonexpressed DNA.

Applications

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Recombinant DNA technology has been used for many purposes. The Human GenomeProject has relied on recombinant DNA technology to generate libraries of genomic DNA

molecules. Proteins for the treatment or diagnosis of disease have been produced usingrecombinant 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 recombinantDNA technology include hepatitis B vaccine, human growth hormone, clotting factors for 

treating hemophilia, and many other drugs. At least 350 additional recombinant-based drugs arecurrently 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 DNAtechnology.

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. Genetherapy 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 anongoing 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 researchlaboratories 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 intothe foreseeable future.

References:

Cooper, Geoffrey. T he 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: BlackwellScientific Publications, 1994.