Recombinant DNA and Biotechnology

Biotechnology is an industrial process that uses the scientific research on DNA for practical benefits. Biotechnology is synonymous with genetic engineering because the genes of an organism are changed during the process and the DNA of the organism is recombined. Recombinant DNA and biotechnology can be used to form proteins not normally produced in a cell. In addition, bacteria that carry recombinant DNA can be released into the environment to increase the fertility of the soil, serve as an insecticide, or relieve pollution.

Tools of biotechnology. The basic process of recombinant DNA technology revolves around the activity of DNA in the synthesis of protein. By intervening in this process, scientists can change the nature of the DNA and of the gene make-up of an organism. By inserting genes into the genome of an organism, the scientist can induce the organism to produce a protein it does not normally produce.

The technology of recombinant DNA has been made possible in part by extensive research on microorganisms during the last century. One important microorganism in recombinant DNA research is Escherichia coli (E. coli). The biochemistry and genetics ofE. coli are well known, and its DNA has been isolated and made to accept new genes. The DNA can then be forced into fresh cells of E. coli, and the bacteria will begin to produce the proteins specified by the foreign genes. Such altered bacteria are said to have been transformed.

Interest in recombinant DNA and biotechnology heightened considerably in the 1960s and 1970s with the discovery of restriction enzymes. These enzymes catalyze the opening of a DNA molecule at a “restricted” point, regardless of the DNA's source. Moreover, certain restriction enzymes leave dangling ends of DNA molecules at the point where the DNA is open. (The most commonly used restriction enzyme is namedEcoRl.) Foreign DNA can then be combined with the carrier DNA at this point. An enzyme called DNA ligase is used to form a permanent link between the dangling ends of the DNA molecules at the point of union (Figure 1 ). 

Figure 1

The production of a a recombined bacterium using a gene from a foreign donor and the synthesis of protein encoded by the recombinant DNA molecule.

The genes used in DNA technology are commonly obtained from host cells or organisms called gene libraries. A gene library is a collection of cells identified as harboring a specific gene. For example, E. coli cells can be stored with the genes for human insulin in their chromosomes.

Pharmaceutical products. Gene defects in humans can lead to deficiencies in proteins such as insulin, human growth hormone, and Factor VIII. These protein deficiencies may lead to problems such as diabetes, dwarfism, and impaired blood clotting, respectively. Missing proteins can now be replaced by proteins manufactured through biotechnology. For insulin production, two protein chains are encoded by separate genes in plasmids inserted into bacteria. The protein chains are then chemically joined to form the final insulin product. Human growth hormone is also produced within bacteria, but special techniques are used because the bacteria do not usually produce human proteins. Therapeutic proteins produced by biotechnology include a clot-dissolving protein calledtissue plasminogen activator (TPA) and interferon. This antiviral protein is produced within E. coli cells. Interferon is currently used against certain types of cancers and for certain skin conditions.

Vaccines represent another application of recombinant DNA technology. For instance, the hepatitis B vaccine now in use is composed of viral protein manufactured by yeast cells, which have been recombined with viral genes. The vaccine is safe because it contains no viral particles. Experimental vaccines against AIDS are being produced in the same way.

Diagnostic testing. Recombinant DNA and biotechnology have opened a new era of diagnostic testing and have made detecting many genetic diseases possible. The basic tool of DNA analyses is a fragment of DNA called the DNA probe. A DNA probe is a relatively small, single-stranded fragment of DNA that recognizes and binds to a complementary section of DNA in a complex mixture of DNA molecules. The probe mingles with the mixture of DNA and unites with the target DNA much like a left hand unites with the right. Once the probe unites with its target, it emits a signal such as radioactivity to indicate that a reaction has occurred.

To work effectively, a sufficiently large amount of target DNA must be available. To increase the amount of available DNA, a process called the polymerase chain reaction (PCR) is used. In a highly automated machine, the target DNA is combined with enzymes, nucleotides, and a primer DNA. In geometric fashion, the enzymes synthesize copies of the target DNA, so that in a few hours billions of molecules of DNA exist where only a few were before.

Using DNA probes and PCR, scientists are now able to detect the DNA associated with HIV (and AIDS), Lyme disease, and genetic diseases such as cystic fibrosis, muscular dystrophy, Huntington's disease, and fragile X syndrome.

Gene therapy. Gene therapy is a recombinant DNA process in which cells are taken from the patient, altered by adding genes, and replaced in the patient, where the genes provide the genetic codes for proteins the patient is lacking.

In the early 1990s, gene therapy was used to correct a deficiency of the enzymeadenosine deaminase (ADA). Blood cells called lymphocytes were removed from the bone marrow of two children; then genes for ADA production were inserted into the cells using viruses as vectors. Finally, the cells were reinfused to the bodies of the two children. Once established in the bodies, the gene-altered cells began synthesizing the enzyme ADA and alleviated the deficiency.

Gene therapy has also been performed with patients with melanoma (a virulent skin cancer). In this case, lymphocytes that normally attack tumors are isolated in the patients and treated with genes for an anticancer protein called tumor necrosis factor.The genealtered lymphocytes are then reinfused to the patients, where they produce the new protein which helps destroy cancer cells. Approximately 2000 single-gene defects are believed to exist, and patients with these defects may be candidates for gene therapy.

DNA fingerprinting. The use of DNA probes and the development of retrieval techniques have made it possible to match DNA molecules to one another for identification purposes. This process has been used in a forensic procedure called DNA fingerprinting.

The use of DNA fingerprinting depends upon the presence of repeating base sequences that exist in the human genome. The repeating sequences are called restriction fragment length polymorphisms (RFLPs). As the pattern of RFLPs is unique for every individual, it can be used as a molecular fingerprint. To perform DNA fingerprinting, DNA is obtained from an individual's blood cells, hair fibers, skin fragments, or other tissue. The DNA is extracted from the cells and digested with enzymes. The resulting fragments are separated by a process called electrophoresis. These separated DNA fragments are tested for characteristic RFLPs using DNA probes. A statistical evaluation enables the forensic pathologist to compare a suspect's DNA with the DNA recovered at a crime scene and to assert with a degree of certainty (usually 99 percent) that the suspect was at the crime scene.

DNA and agriculture. Although plants are more difficult to work with than bacteria, gene insertions can be made into single plant cells, and the cells can then be cultivated to form a mature plant. The major method for inserting genes is through the plasmids of a bacterium called Agrobacterium tumefaciens. This bacterium invades plant cells, and its plasmids insert into plant chromosomes carrying the genes for tumor induction. Scientists remove the tumor-inducing genes and obtain a plasmid that unites with the plant cell without causing any harm.

Recombinant DNA and biotechnology have been used to increase the efficiency of plant growth by increasing the efficiency of the plant's ability to fix nitrogen. Scientists have obtained the genes for nitrogen fixation from bacteria and have incorporated those genes into plant cells. By obtaining nitrogen directly from the atmosphere, the plants can synthesize their own proteins without intervention of bacteria as normally needed.

DNA technology has also been used to increase plant resistance to disease. The genes for an insecticide have been obtained from the bacterium Bacillus thuringiensis and inserted into plants to allow them to resist caterpillars and other pests. In addition, plants have been reengineered to produce the capsid protein that encloses viruses. These proteins lend resistance to the plants against viral disease.

The human genome. One of the most ambitious scientific endeavors of the twentieth century was the effort to sequence the nitrogenous bases in the human genome. Begun in 1990 and completed in 2003, the effort encompassed 13 years of work at a cost of approximately $3 billion. Knowing the content of the human genome is helping researchers devise new diagnostics and treatments for genetic diseases and will also be of value to developmental biologists, evolutionary biologists, and comparative biologists.

In addition to learning the genome of humans, the project has also studied numerous bacteria. By 1995, the genomes of two bacteria had been completely deciphered (Haemophilus influenzae and Mycoplasma genitalium), and by 1996, the genome of the yeast Saccharomyces cerevisiae was known. The Human Genome Project is one of colossal magnitude that will have an impact on many branches of science for decades to come. The project remains the crowning achievement of DNA research in the twentieth century and the bedrock for research in the twenty-first.

Figure 1

Figure 1