DNA TECHNOLOGY

Genetic engineering, the direct manipulation of genetic material for practical purposes, has begun an industrial revolution in biotechnology. This use of living organisms to manufacture desirable products dates back centuries, but advances in DNA technology, have resulted in hundreds of new products. Genes from different organisms and species are now routinely combined to form recombinant DNA and inserted into living cells in which these genes are replicated and may be expressed and their products studied or harvested. DNA technology has led to major advances in all fields of biology and in our knowledge of the humane genome.

DNA Cloning

Techniques for gene cloning are used to prepare multiple identical copies of pieces of DNA.

· DNA technology makes it possible to clone genes for basic research and commercial applications

One of the approaches used to clone pieces of DNA makes use of the plasmids of bacterial cells. Recombinant DNA may be made by inserting foreign DNA into plasmids. These plasmids are put back into bacterial cells where they will replicate as the bacteria

reproduce. Such cloned DNA provides multiple copies of the gene and may also be used to produce protein coded for by the foreign DNA.

· Restriction enzymes are used to make recombinant DNA

Restriction enzymes protect bacteria from the DNA of viruses or other bacteria by cutting up foreign DNA in a process called restriction. Most restriction enzymes recognize short nucleotide sequences and cut at specific points within them. The cell protects its own DNA from restriction by methylating nucleotide bases within its own recognition sequences.

A recognition sequence, or restriction site, is usually a symmetrical sequence of four to eight nucleotides running in opposite directions on the two strands. The restriction enzyme usually cuts phosphodiester bonds in a staggered way, between the same adjacent nucleotides on both strands, leaving sticky ends of short single-stranded sequences on both sides of the resulting restriction fragment.

DNA from different sources can be combined in the laboratory when the DNA is cut by the same restriction enzyme and the complementary bases on the sticky ends of the restriction fragments pair by hydrogen bonding. DNA ligase is used to seal the strands together.

· Genes can be cloned in recombinant DNA vectors

Cloning vectors are DNA molecules that can move foreign DNA into a cell and replicate there. Recombinant plasmids are easily returned to bacterial cells where they will replicate cloned DNA. The ease with which DNA can be isolated from and returned to bacterial cells make bacteria the most common host for gene cloning.

The plasmid method of gene cloning involves treating antibiotic-resistant plasmids with a restriction enzyme that cuts the DNA ring at a single restriction site and disrupts a gene whose activity is easily determined, such as lacZ, the gene for b-galactosidase. The clipped plasmids are mixed with DNA containing the gene of interest, which has also been treated with the same restriction enzyme and has complementary sticky ends. The sticky ends form hydrogen bonds with each other, and DNA ligase seals the recombinant molecules.

The plasmids are introduced by transformation into bacterial cells that are lacZ- and thus unable to produce b-galactosidase. Bacteria are plated onto a medium containing ampicillin and X-gal, a compound that is cleaved by b-galactosidase and yields a blue product. Colonies that are able to grow on the medium (because they contain a plasmid with the amp^R gene) and are not blue (because of the foreign DNA inserted in the middle of the b-galactosidase gene) are carrying a recombinant plasmid.

The colonies are tested to find the ones that contain the gene of interest. If the gene is expressed, the presence of the protein product can be determined by its activity or structure (using antibodies).

The gene itself can be detected by nucleic acid hybridization, using a nucleic acid probe, which has complementary sequences to segments of the gene and can hybridize with the DNA of the gene after denaturation of the cell's DNA by heat or chemicals produces single-stranded DNA. The probe is located by its radioactively labeled molecules or fluorescent tag. Once the desired clone is identified, it can be grown in culture and the gene of interest isolated in large quantities.

Differences between prokaryotic and eukaryotic mechanisms for gene expression can be overcome by using an expression vector, a vector that has a prokaryotic promoter just upstream from the eukaryotic gene insertion site.

Creating artificial genes eliminates the problem caused by large introns that can make bacterial cells, which lack RNA-processing enzymes, unable to express eukaryotic genes. Using reverse transcriptase from retroviruses, mRNA which has no introns--is used as a template to produce complementary DNA, or cDNA.

Yeast cells are eukaryotic hosts used to clone eukaryotic genes; they are easy to grow and have plasmids that serve as vectors. Artificial chromosomes have also been constructed that carry foreign DNA and contain an origin for DNA replication, a centromere, and two telomeres. These vectors behave normally in mitosis and are able to clone large pieces of DNA. Plant and animal cells in culture can serve as hosts and may be necessary when a protein must be modified following translation.

Yeasts and other eukaryotic cells can take up DNA that may become incorporated into a chromosome through recombination. More efficient means for introducing DNA into eukaryotic cells include electroporation, in which an electric pulse briefly opens holes in the plasma membrane through which DNA. can enter, injection into cells using microscopically thin needles, or firing into plant cells on microscopic metal particles using a gene gun.

· Cloned genes are stored in DNA libraries

The genes used for cloning can be isolated from an organism by cutting its DNA into thousands of pieces with restriction enzymes and inserting them into plasmids. The collection of the thousands of clones of bacteria containing recombinant plasmids derived from this shotgun approach is called a genomic library.

Bacteriophages are also used as vectors for creating genomic libraries. DNA fragments are spliced into phage DNA, which is packaged into capsids and used to infect bacteria. The production of new phage clones the foreign DNA.

A partial genomic library can be produced from the mRNA molecules found in a cell. Such a cDNA library contains only the genes that are expressed (transcribed) within the cell.

· The polymerase chain reaction (PCR) clones DNA entirely in vitro

A technique developed in 1985, known as polymerase chain reaction (PCR), has revolutionized molecular biology by being able to produce billions of copies of a section of DNA in vitro in only a few hours. A solution of DNA containing the region of interest is heated to separate the strands, then cooled and incubated with the four nucleotides, a special heat-resistant type of DNA polymerase, and specially synthesized primers that bind upstream from the target sequence. DNA polymerase adds nucleotides to the 3' ends of the primer. The solution is heated again and the process repeated. The desired DNA segment does not need to be purified from the starting material and very small samples can be used.

Analysis of Cloned DNA

Many of the methods for analyzing and comparing DNA make use of the technique called gel electrophoresis that separates nucleic acids and proteins on the basis of their size and electrical charge. Due to the negative charge of their phosphate groups, restriction fragments of DNA migrate through the electric field produced in a thin slab of gel toward the positive electrode, Fragments move at a rate inversely proportional to their size, producing band patterns in the gel of fragments of decreasing size.

· Restriction fragment analysis detects DNA differences that affect restriction sites

Cutting a long DNA molecule with a particular restriction enzyme and separating the resulting restriction fragments by gel electrophoresis produces a characteristic pattern of bands. Pure samples of such bands can be recovered from the gel and retain their biological activity.

Two DNA samples, such as cloned alleles of a gene, will produce different patterns of bands when differences in their DNA sequences add or delete restriction sites. The addition of nucleic acid hybridization allows two or more unpurified samples of DNA (such as the entire genome) to be compared for the presence and band locations of a particular DNA sequence. In a technique known as Southern hybridization or Southern blotting, DNA is treated with a restriction enzyme; the fragments are separated on a gel and then transferred to nitrocellulose or nylon paper by blotting; labeled probes of single-stranded DNA are added and hybridize with complementary DNA sequences; and the restriction fragment bands of interest are identified by autoradiography.

Northern blotting is used to hybridize mRNA with probes to determine whether a particular gene is being transcribed and how much mRNA is present.

Samples of noncoding DNA treated with restriction enzymes also produce different band patterns due to differences in nucleotide sequences in restriction sites. Such restriction fragment length polymorphisms (RFLPs) can serve as genetic markers on a chromosome. A particular RFLP marker often occurs in several variants in a population. RFLPs are detected by Southern blotting, and the entire genome can be used as the DNA starting material.

· Entire genomes can be mapped at the DNA level

Locating Genes by In Situ Hybridization DNA probes can map genes on eukaryotic chromosomes with an in situ hybridization technique. Labeled DNA probes base-pair with denatured DNA of intact chromosomes on a microscope slide, and autoradiography and chromosome staining show the location of the gene.

Geneticists are attempting to map entire genomes by determining the location of all of an organism's genes and noncoding DNA. The current international effort to map the human genome is called the Human Genome Project and includes three stages: genetic (linkage) mapping, physical mapping, and DNA sequencing. The project also includes the mapping of important research organisms, such as E. coli, yeast, the nematode C. ele-gans, Drosophila, and mouse.

The first stage in mapping a large genome is to develop a linkage map of several thousand genetic markers, either genes or other identifiable sequences such as RFLPs or short repetitive sequences , (micro-satellites). Testing for genetic linkage to these known

markers enables researchers to place other genes or markers.

In physical mapping, the actual distances between markers are determined. The DNA of each chromosome is cut into restriction fragments, which are then ordered along the chromosome. First large fragments are cut, cloned, and ordered. Then those fragments are cut and their fragments ordered. Finally, fragments are cut, cloned, and ordered that are small enough to be sequenced.

The ordering of fragments is facilitated by the technique of chromosome walking. Two sets of overlapping fragments (cut by two different restriction enzymes) are prepared. A probe made from the 3' end of a known sequence is used to identify the fragment in the other set that overlaps that sequence. A 3' probe from that fragment is used to search the first library for the next overlapping fragment, continuing to "walk" down the chromosome.

Clones of DNA fragments are ultimately used to determine the nucleotide sequence of an entire genome. A sequencing machine is used that combines DNA labeling, DNA synthesis with special chain-terminating nucleotides, and high-resolution gel electrophoresis.

In the Sanger method of DNA sequencing, samples of a single-stranded restriction fragment are incubated with modified nucleotides (di-deoxyribonucleotides) that randomly block further synthesis when they are incorporated into a growing DNA strand. The sets of radioactively or fluorescently tagged strands of varying lengths are separated by gel electrophoresis, and the nucleotide sequence is read from the sequence of bands.

Faster sequencing techniques will have to be developed to complete the human genome by 2005. The genomes of E. coli, some other bacteria, several archaea, and yeast have been completed.

Computer software is used to scan DNA sequences for the nucleotide triplets that code for start and stop codons, thus identifying a list of putative genes and their sequences. These sequences are then compared with sequences of known genes of that species and other species to look for similarities that might indicate the function of the new gene. Many of the putative genes identified so far have never been encountered before, but many others have been found in even distantly related organisms.

In order to study patterns of gene expression, researchers isolate the mRNA made in different cells, create a cDNA library using reverse transcriptase, and then use the cDNA as probes to explore other DNA collections. Using this cDNA in DNA microarray assays, scientists can test all the genes expressed in a tissue for hybridization with thousands of short, single-stranded DNA fragments from different genes arrayed on a microscope slide. The intensity with which the hybridized spots fluoresce indicates the relative amount of mRNA that was in the tissue. Gene expression in different tissues and at different stages of development can be determined. DNA microarray assays are used to compare gene expression in cancerous and noncancerous tissues.

The function of unknown genes can be studied using in vitro mutagenesis, in which changes are made to a cloned gene, the gene is returned to the cell, and changes in physiology or developmental patterns that result from the altered gene product are monitored.

Practical Applications of DNA Technology

· DNA technology is reshaping medicine and the pharmaceutical industry

DNA technology and the Human Genome Project are identifying genes responsible for genetic diseases, hopefully leading to new ways to diagnose, treat, and even prevent those disorders. Finding the genes that are turned on or off in particular diseases may also lead to preventions or therapies.

PCR and labeled DNA probes are being used to identify difficult pathogens and to diagnose infectious diseases.

DNA technology has led to the diagnosis of hundreds of human genetic disorders. Genes have been cloned for a number of genetic diseases, making it possible to probe for the gene in DNA from individuals who are being tested. Even if the gene has not yet been cloned, a disease gene may be diagnosed when closely linked with an RFLP marker. Comparisons within a family must be used to determine which variant of the RFLP marker is linked to the abnormal allele.

Genetic engineering may provide the means for correcting genetic disorders in individuals by replacing or supplementing defective genes. New genes would be introduced into somatic cells of the affected tissue. For the correction to be permanent, the cells must be types that actively reproduce within the body, such as bone marrow cells.

Some of the technical problems involved with human gene therapy include how to get the proper control mechanisms to operate on the transplanted allele. The most difficult ethical question is whether germ cells should be treated to correct defects in future generations. Opponents fear that tampering with human genes will eventually lead to the practice of eugenics, the deliberate effort to control the genetic makeup of human populations.

Many pharmaceutical proteins are produced using DNA technology. Large quantities of proteins can be made by inserting a gene into vector DNA with a highly active promoter. Engineering host cells to secrete a protein as it is made simplifies its purification.

Insulin and human growth hormone were among the first hormones made by recombinant DNA techniques. Tissue plasminogen activator (TPA), another important pharmaceutical product, is a protein that helps dissolve blood clots and reduce the risk of subsequent heart attacks.

Two new approaches to fighting diseases that do not respond to traditional therapies are under development. Antisense nucleic acid is single-stranded DNA or RNA molecules that base-pair with and block the translation of mRNA. Interfering with viral mRNA or the transformation of cells to a cancerous state could prevent diseases. Surface-receptor blockers or mimics are drugs that interfere with the binding of viral particles to receptors or bind with the virus to keep it from infecting cells.

Recombinant DNA techniques have been used to make large amounts of protein molecules from the surfaces of pathogens, which can be used as vaccines if the protein subunit triggers an immune response. Genetic engineering methods can also directly modify the genome of a pathogen so as to attenuate it (make it nonpathogenic) so it can be used as a vaccine.

· DNA technology offers forensic, environmental, and agricultural applications

RFLP analysis can be used in criminal cases to compare the DNA fingerprint, or specific pattern of RFLP bands, of a victim, suspect, and crime sample. Variations in the number of tandem repeated base sequences (simple tandem repeats or STRs) found in satellite DNA are now commonly used in forensic DNA fingerprint analysis. Forensic tests require only five small regions of the genome that are known to be highly variable from one person to the next in order to provide a high statistical probability that matching DNA fingerprints come from the same individual.

Microorganisms that are able to extract heavy metals, such as copper, lead, and nickel, may become important in mining and cleaning up mining waste. Microbes are used in sewage treatment plants to degrade many organic compounds into nontoxic form. Engineering organisms to degrade chlorinated hydrocarbons and other toxic compounds is an area of active research. Environmental disasters such as oil spills and waste dumps are other areas for which detoxifying microbes are being developed.

Genetic engineering is providing means to improve the productivity of agricultural plants and animals. Vaccines, growth hormones, and antibodies are produced with recombinant DNA technology. Transgenic organisms containing genes from other species are being developed for potential agricultural use.

The ability to regenerate plants from single cells growing in tissue culture has made plant cells easier to genetically manipulate than animal cells. The bacterium Agrobacterium tumefaciens produces crown gall tumors in the plants it infects when its Ti plasmid (tumor inducing) integrates a segment of its DNA into the plant chromosomes. Using DNA technology, foreign genes are inserted into Ti plasmids whose disease-causing properties have been eliminated, and the recombinant plasmids are introduced into plant cells growing in culture. When these cells regenerate whole plants, the foreign gene is included in the plant genome.

Only dicotyledons are susceptible to infection by Agrobacterium. Techniques such as electroporation and DNA guns are being used to transfer foreign genes into important monocots such as corn and wheat.

Early results of genetic engineering in plants have been positive. Plant strains that carry a bacterial gene for resistance to herbicides have been developed, which will enable crops to be grown while weeds in their midst are killed. Crop plants are being engineered to be resistant to infectious pathogens and insect pests. Crop yields may be improved through the selective enlargement of plant parts or improvements to a plant's food value.

The production of bacteria with increased nitrogen-fixing potential and the engineering of plants that can fix nitrogen themselves are examples of a valuable potential use of DNA technology for improving plant productivity and reducing use of expensive and polluting fertilizers.

· DNA technology raises important safety and ethical questions

Several federal agencies set policy and regulate new developments in genetic engineering. Safety issues include concerns about potential harmful side effects of medical products. With genetically engineered agricultural products, there are potential dangers in the impact of such organisms on native species. Transgenic species may become "superweeds" or introduce some of their genes for resistance to herbicides, insect pests, and natural diseases into wild plants. Researchers are trying to develop mechanisms to prevent the escape of engineered plant genes.

Ethical questions about human genetic information include who should have access to information about a person's genome and how that information should be used. Potential ethical, environmental, and health issues must be considered in the development of these powerful genetic techniques and remarkable products of biotechnology.