PCR had an immediate impact on cloning technology. It could produce large quantities of DNA that could be readily cloned and subsequently used to study the functions and behavior of genes in living systems.DNA cloning involves four basic steps. Scientists first isolate the source and vector DNA and free them from contaminants. They then use restriction enzymes to cut these two DNAs, creating ends that can connect the source DNA with the vector. Next they bond the source's DNA to the vector's with a DNA ligase enzyme that repairs the cuts and creates a single length of DNA. Finally the DNA is transformed into a host cell — a bacterium or another organism.
PCR-mediated cloning is a family of methods rather than a single technique. TA cloning, for example, uses Taq polymerase, an enzyme known as Tth DNA polymerase, or one of a group of other polymerases that preferentially add the base adenine (A) to particular ends of PCR products. Such products can be cloned into a vector containing complementary overhangs of the base thymidine (T). Blunt-end cloning uses DNA polymerases that possess proofreading activity, such as Pwo DNA polymerase. These actively remove mispaired nucleotides from the ends of double-stranded DNA and generate blunt-end PCR products. Researchers have also been able to amplify long lengths of DNA using mixtures of several different DNA polymerases. When the DNA fragments become longer than 10,000 bases, the conventional vectors do not work well as carriers of the target DNA. Instead scientists use hybrid vectors that contain drug resistance marker genes to allow for positive selection of the DNA fragment of interest. They are especially well suited for cloning large mammalian genes or multigene fragments.
What cloning method should a research team use? That depends on several factors, including the type of DNA polymerase, the length of the PCR product, and the purpose of the cloning experiment. Whatever approach they choose, researchers must test the bacterial host cells for the presence of the source DNA in their cytoplasm once they have the chance to divide. If the procedure has successfully transformed the vector into the host cell, the cell will test positive for the vector via a selectable marker.
In the early days of cloning, very few scientists had the skill and understanding to perform these fairly sophisticated techniques. Recently, however, the invention of pretested kits has given most life scientists simple access to this technique. CLONTECH Laboratories, Epicentre Technologies, Promega, Stratagene, and several other firms offer cloning kits and tools, along with effective technical support.
New cloning methods continue to emerge. "There will be a push to get away from traditional cloning methods toward other types of enzymes to do the cloning," says Carsten Carstens of Stratagene. "A major development will be the use of site-specific recombinant technology. We're about to release technology for using linear vectors in bacterial cells. That will be a lot more efficient in making libraries." Adds Henry Ji, Stratagene's director of new product development: "Responding to market need we have put together a program to clone antigens into expression vectors." Eppendorf Scientific has just introduced a method of cloning through electrofusion. "The general applications include monoclonal antibodies, different tumor cells, and ornamental plants," says Sharon Durbin, the company's product manager for electrofusion products.
A VARIETY OF APPLICATIONS
The Human Genome Project and the commercial sequencing effort led by Celera Genomics have made significant progress in determining the DNA sequences of humans. The teams completed working drafts last year. Several labs have started to proof those drafts and to determine some of the missing sequence data. DNA sequencing uncovers important variations in the nucleotide bases, or polymorphisms, that make up our genes. These single nucleotide polymorphisms (SNPs) are associated with an increased risk of developing diseases such as cancer and heart disease. Without PCR and cloning to generate enough DNA and permit examination of the functions of the genes that contain it, life science teams would not have made this kind of progress.
PCR technology promises advances in human genetics. For example, Thilly and colleagues have founded a company, Peoples Genetics, that aims to discover the inherited mutations that occur in minuscule proportions in the population (see accompanying story, "Genetics for the People"). They believe that this is essential to discovering disease-causing mutations in human populations.
Fields of science beyond traditional molecular biology laboratories have benefited from PCR. It has become a well-recognized tool in forensic science. Police labs routinely use it to identify blood and other forms of evidence (see accompanying story, "Science in a 'Dirty, Grungy World'"). In Manchester, UK, the Forensic Science Service uses Extract-N-Amp, a kit from Sigma-Aldrich, to differentiate between marijuana and other plants.
PCR has also become a useful tool in some unexpected scientific disciplines. For example, archaeologists have found it effective to determine relationships between ancient civilizations and to study the evolutionary biology of different animal species. PCR can amplify very small samples of DNA from virtually any tissue, including examples thousands of years old. These molecular readings have become very important in validating (and sometimes disproving) scientific conclusions based on circumstantial evidence.
Cloning technology has also enabled the routine study of gene function. It is now a relatively simple process to isolate large DNA fragments that contain genes and then to express the genes in transgenic hosts. In addition, the RT-PCR technique permits scientists to determine the genes responsible for producing very low levels of messenger RNA that may play an important role in cellular metabolism and the disease process. Researchers have only scratched the surface of understanding the many processes of the living cell.
Ultimately, the tools of PCR and cloning are geared toward understanding, treating, and preventing the diseases that affect the quality of human life each day. Take rheumatoid arthritis, a disease that affects both young and old individuals. Researchers know that a cytokine called tumor necrosis factor (TNF) plays a major role in this disease. By causing immune cells to attack the body's own cells, it causes very painful inflammation. TNF binds to a specific membrane-bound receptor on the immune cells. Scientists have used recombinant DNA technology to clone an altered form of the receptor gene that codes for a soluble form of the receptor containing the TNF binding site. Injected into an arthritis patient, this modified protein binds to TNF and inhibits it from binding to the receptors on immune cells. That prevents the immune cells from initiating the signal cascade that causes inflammation.
Research teams are investigating many other disease processes in the hope of finding the key element or elements in the cell's signal transduction pathways that can present a point of attack on a disease. Several pharmaceutical companies are studying signal transduction pathways to develop drugs which can affect these key elements and prevent or treat disease.
Fewer than 20 years have passed since Kary Mullis took the celebrated drive that led to his discovery of PCR. Mullis may have left the field, but manufacturers have taken it up, making a series of refinements in the reagents and instruments used for PCR and cloning. Using these products, curious researchers will continue to identify fresh applications as they ponder what seems to be the unlimited potential of PCR and DNA cloning.
