Recombinant DNA

Biotechnology is an industrial process that uses the scientific research on DNA for practical means. Biotechnology is synonymous with genetic engineering because the genes of an organism are changed during the process. Because the genes are changed, the DNA of the organism is said to be recombined. The result of the process is recombinant DNA. Recombinant DNA and biotechnology can be used to form proteins not normally produced in a cell, to produce drugs or vaccines, or to promote human health. In addition, bacteria that carry recombinant DNA can be released into the environment under carefully controlled conditions to increase the fertility of the soil, serve as an insecticide, or relieve pollution.

Biotechnology and recombinant DNA can also be used in forensic medicine to "fingerprint" individuals and identify DNA at a crime scene. In addition, transgenic plants and animals are being created. Humans can also have the genes in their cells modified to produce proteins that relieve health-related deficiencies. Finally, at the time of publication, scientists at Celera Genomics and the National Human Genome Research Institute have sequenced a substantial portion of the human genome in an effort to identify genes linked to human disease. The sequenced fragments of DNA are currently not organized into contiguous reading frames. Although a substantial portion of the genome has been sequenced, it may require several more years for all the pieces to come together to form a complete and accurate genetic map of the human genome.

Tools of Biotechnology

The basic process of recombinant DNA technology revolves around DNA activity in the synthesis of protein. During this synthesis, DNA provides the genetic code for the placement of amino acids in proteins. By intervening in this process, scientists can change the nature of the DNA, thereby changing the nature of the protein expressed by that DNA. 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 half-century. One important microorganism in recombinant DNA research is Escherichia coli, commonly referred to as E. coli. The biochemistry and genetics of E. coli are well known, and its DNA has been isolated and made to accept new genes. The DNA can then be forced into fresh E. coli cells and the bacteria will begin to produce the proteins specified by the foreign genes. Such altered bacteria are said to have been transformed.

Knowledge about viruses has also aided the development of DNA technology. Viruses are fragments of nucleic acid surrounded by a protein coat. In some cases, viruses attack cells and replicate within the cells, thereby destroying them. In other cases, the viruses enter cells, and their nucleic acid joins with the nucleic acid in the cell nucleus. By attaching DNA to viruses, scientists use viruses to transport foreign DNA into cells and to connect it with the nucleic acid of the cells.

Another common method for inserting DNA into cells is to use plasmids, which are small loops of DNA in the cytoplasm of bacterial cells. Working with a plasmid is much easier than working with a chromosome, so plasmids are often the carriers, or vectors, of DNA. Plasmids can be isolated, recombined with foreign DNA, then inserted into cells where they multiply as the cells multiply.

Interest in recombinant DNA and biotechnology heightened considerably during 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 source of the DNA. The video below shows that a human DNA molecule is opened at a certain site by the restriction enzyme EcoRl (upper left), and the desired DNA fragment is isolated (lower left). Plasmid DNA is treated with the same enzyme and opened. The DNA fragment is spliced into the plasmid to produce the recombinant DNA molecule.

Certain restriction enzymes leave dangling ends of DNA molecules at the point where the DNA is opened. Foreign DNA can therefore be combined with the carrier DNA at this point. An enzyme called DNA ligase forges a permanent link between the dangling ends of the DNA molecules at the point of union.

Recombinant DNA technology is sophisticated and expensive. Genes must be isolated, vectors must be identified, and gene control must be maintained. Stability of the vector within a host cell is important, and the scientist must be certain that nonpathogenic bacteria are used. Cells from mammals can be used to synthesize proteins, but cultivating these cells is difficult. In addition, the proper gene signals must be identified, RNA molecules must be bound to ribosomes, and the presence of introns must be considered. Collecting the gene product and exporting it from the cell are other considerations.

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 that may result in problems, such as diabetes, dwarfism, and impaired blood clotting, respectively. Proteins for these chemicals 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, such as the following, can also be produced by biotechnology: 

    --      Tissue plasminogen activator (TPA), a clot-dissolving protein, can now be produced in recombined mammalian cells. 

    --       Interferon, an antiviral protein produced in E. coli cells, is currently used to fight certain types of cancers and for certain skin diseases.

An antisense molecule is a molecule of RNA that reacts with and neutralizes the mRNA molecule used in protein synthesis. In doing so, the antisense molecule prevents the synthesis of a protein involved in a specific disease. For example, an antisense molecule can prohibit human host cells from producing key portions of the human immunodeficiency virus (HIV) when infection has occurred.

Vaccines represent another application of recombinant DNA technology. The hepatitis B vaccine now in use is composed of viral proteins manufactured by yeast cells and 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. One vaccine uses vaccinia (cowpox) virus as a vector. The virus has been combined with genes from several viruses, and it is hoped that injections of the vaccine will stimulate resistance to multiple diseases. Vaccines can also be produced by eliminating certain disease-inducing genes from a pathogen, leading to a harmless organism that will stimulate an immune response.

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 on a larger DNA molecule. The probe mingles within the mixture of DNA molecules and unites with the target DNA much like a left hand unites with a right. After the probe unites with its target, it emits a signal like that from a radioactive isotope 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). This has yielded a direct test for AIDS that is preferred over the AIDS antibody test. Lyme disease and genetic diseases such as cystic fibrosis, muscular dystrophy, Huntington's disease, and fragile X syndrome can be identified by DNA probes. (Cystic fibrosis is a respiratory disease in which mucus clogs the respiratory passageways and makes breathing difficult; muscular dystrophy is a disorder of the nervous system in which destruction of nerve fibers leads to erratic muscular activity; Huntington's disease is a disease of the nervous system accompanied by erratic movements and nervous degeneration; and fragile X syndrome is a disease of the X chromosome accompanied by a form of mental retardation.)

Segments of DNA called restriction fragment length polymorphisms (RFLPs) are the objectives of the tests with gene probes. RFLPs are apparently useless bits of DNA located near genes associated with the diseases. By locating RFLPs, the biotechnologist can locate the disease gene. DNA probes also detect microorganisms in the environment and identify viral and bacterial pathogens.

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. The genes then provide the genetic codes for proteins the patient is lacking. Nonreproductive cells are used in gene therapy, so there is no carryover of inserted genes to the next generation.

In the early 1990s, gene therapy was used to correct a deficiency of the enzyme adenosine 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 in the bodies of the children. Once established in the bodies, the gene-altered cells began synthesizing the enzyme ADA. Thus, the deficiency was removed and the disease resolved.

Approximately 2,000 single-gene defects are believed to exist, and patients with these defects may be candidates for gene therapy. A multilayered review system now exists to ensure the safety of gene therapy proposals. Many aspects must be considered before approval is granted for gene therapy experiments.

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.

DNA fingerprinting depends on 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 each 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 then extracted from the cells and digested with enzymes. The resulting fragments are separated by a process called electrophoresis. Electrophoresis is a process in which electrical charges separate DNA fragments according to size. The separated DNA fragments are then detected with DNA probes and used to develop a fingerprint. A statistical evaluation enables the forensic pathologist to compare a suspect's DNA with the DNA recovered at a crime scene and to state with a high degree of certainty (usually 99 percent) that the suspect was at the crime scene.

Searching for DNA

The ability to retrieve DNA from ancient materials and museum specimens has given archaeologists and anthropologists hopes of a glimpse at ancient life. Biochemists have successfully obtained DNA from extinct animals and plants. Evolutionary biologists have used the DNA to draw lineage patterns from the data. This often gives a better understanding of relationships between species. DNA isolated from ancient humans has been used to trace the movements of populations, such as the Anglo-Saxons, as well as to determine whether males were favored over females in certain societies.

Studies have also been performed on human origins by using the DNA found in the mitochondria. All of an offspring's mitochondrial DNA is derived from its mother. Because this DNA represents an unbroken line of genetic information, an analysis of mutation sites in the mitochondrial DNA can conceivably lead one back to the first human female.

DNA and Agriculture

Although plants are more difficult to work with than bacteria, gene insertions can be made into single plant cells. Then the cells can be cultivated to form a mature plant. The major method for inserting genes is through the plasmids of the 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. The plant cells can then perform a process that normally takes place only in bacteria.

DNA technology has also been used to increase plant resistance to disease by reengineering the plant to produce viral proteins. Also, the genes for an insecticide obtained from a bacterium have been inserted into plants to allow the plants to resist caterpillars and other pests.

One of the first agricultural products of biotechnology was the rot-resistant tomato. This plant was altered by adding a gene that produces an antisense molecule. The antisense molecule inhibits the tomato from producing the enzyme that encourages rotting. Without this enzyme, the tomato can ripen longer on the vine.

Transgenic Animals

Transgenic animals are animals in which one or more genes have been introduced into its nonreproductive cells. The first transgenic animal was produced in 1983 when genes for human growth hormone were introduced into mice.

Transgenic animals can be used to produce valuable products. For example, a transgenic pig has been produced with the ability to synthesize human hemoglobin for use as a blood substitute. Also, a transgenic cow has been bred with the ability to produce human lactoferrin, an iron-building milk protein and a potential antibacterial agent. A transgenic sheep can synthesize a protein that helps emphysema patients breathe easily, and a transgenic goat has been made to produce a protein needed by cystic fibrosis patients.

The Human Genome

In 1990, researchers at Celera Genomics and at the National Human Genome Research Institute began an ambitious endeavor to sequence the entire human genome. In 2000, researchers revealed to the general public that a substantial portion of this work had been completed. This "rough draft" of the human genome is currently in fragments much like pieces of a jigsaw puzzle. Current efforts are underway to match the different pieces to complete the puzzle. Recently, Celera Genomics revealed their startling estimation of the number of human genes to be 30,000. This estimation, based on the sequence data, is substantially below previous predictions. The sequence data has led to the estimation that less than 5% of the human genome actually encodes functional proteins. Once the jigsaw puzzle is completed, the data will undoubtedly help researchers devise new diagnostics and treatments for genetic diseases.

In addition to sequencing the human genome, researchers have sequenced the genomes of Drosophila melanogaster (fruit fly), Arabidopsis thaliana (plant), Saccharomyces cerevisiae (budding yeast), and C. elegans (worm). In addition, mouse, rat, and zebrafish genomes have been sequenced. Not only eukaryotic organisms are useful to the research community. The genome of Plasmodium (the organism that causes malaria) has also been sequenced. The goals of these sequencing projects are to prepare gene linkage maps and physical maps. A gene linkage map is a map that pinpoints the location of genes based on their connection to certain marker gene sequences. A physical map, in comparison, gives the actual number of bases between genes on a chromosome; therefore, it locates the gene of interest more precisely.

Ultimately, scientists hope to learn the actual names and sequences of all 3 billion nitrogenous bases in the human genome. Automation and computerization are essential tools in the sequencing, and the development of the specific technology is underway.