PCR Primed To Spur Chain Of Applications

What would you do if your research interests revolved around obtaining DNA from a bacterium preserved for millions of years in the gut of a bee stuck in amber, matching up a murderer to crime- scene blood half a century old, or cloning genes from a 1,000- year-old mummy? Most scientists would first consider PCR--the polymerase chain reaction--as a technique for approaching problems such as these. With PCR, minute quantities of nucleic acids can be amplified millions of times into sufficient quantities for use in molecular cloning, sequencing, DNA fingerprinting, and diagnostics, among an increasing number of applications. PCR technology, owned and licensed through Hoffmann-La Roche Inc., a Swiss-based pharmaceutical company with United States headquarters in Nutley, N.J., is considered by many to be the most important development in the field of molecular biology since the discovery of restriction enzymes--the molecular "scissors" that selectively cut DNA--two decades ago. Hoffmann-La Roche, which includes Roche Diagnostic Systems Inc., purchased the patents and licensing rights from Cetus Corp. of Emeryville, Calif., in 1992. ISOLATION AID: Gentra Systems' Purescript kit allows researchers to isolate RNA from cells or tissues to be used for PCR. "PCR is probably the most revolutionary thing that has happened to molecular biology in the last 20 years," declares molecular biologist Elliott Dawson, president of BioVentures Inc., a Murfreesboro, Tenn.-based company whose products include kits for rapidly isolating DNA or RNA from cells in preparation for PCR. "It would be very hard to overestimate its usefulness." "There are so many areas of molecular biology that have been impacted by PCR, notably in areas of basic research, where PCR is really moving things forward," agrees molecular biologist Nancy Casna, president of Therion Corp., a Troy, N.Y., laboratory that performs DNA-based tests to determine animal identity. "The tedious things that we all used to have to do--like cutting out bands from gels, cloning, and subcloning--can be replaced by PCR." Simply leafing through an issue of an interdisciplinary scientific journal punctuates how broadly PCR can be applied. While many times PCR is used as a research tool for tasks as basic as cloning, nearly as often it's applied to scientific problems reminiscent of Jurassic Park. Montana State University paleontologists Mary Schweitzer and Jack Horner relied on PCR to pluck molecules of DNA from the core of 65-million-year-old bones believed to have once belonged to a Tyrannosaurus rex. Separately, molecular geneticist Raul Cano and his colleagues at California Polytechnic State University in San Luis Obispo have amplified DNA sequences believed to be of dinosaur origin from bones fossilized in amber. (Both research projects were detailed in V. Morell, Science, 261:160-1, 1993.) Also of interest to Jurassic Park fans is the potential of retrieving dinosaur DNA from the stomach contents of prehistoric blood-sucking insects, whose last meal may have been a T. rex. "PCR is the only tool that could allow us to extract dinosaur DNA from the gut of insects preserved in amber, and then get enough of that DNA to clone or sequence," notes Will Gergits, vice president of Therion Corp. Cloning dinosaur genes is only one of numerous applications made possible by PCR. This powerful technique can also be applied when the goal may be something as simple as subcloning a gene, or as complex as sequencing the human genome. Whereas once molecular biologists required huge quantities of painstakingly purified DNA as a starting point for their procedures, with PCR this step is, in theory, obsolete. "For PCR, the starting material can be anything that contains DNA or RNA, such as bacterial cells in culture, whole blood or tissues, even aged blood stains," says Dawson of BioVentures. "The beauty of PCR is that it allows you to work with infinitely small quantities of DNA, taken from samples such as animal carcasses or old blood stains, where the DNA might be degraded," Gergits points out. Degraded DNA left over from cells at an old crime scene, for instance, or dissected from the preserved tissues of ancient mummies can serve as a template for amplification. "Theoretically, you can start with a single molecule of DNA and make copies of it, just as you would make photocopies of an important document," he explains. The end product of PCR is enough DNA to clone, sequence, mutate, or subject to forensic or genetic analysis. The amplification process is actually a series of enzyme- catalyzed reactions carried out by a bacterial DNA polymerase, the P in PCR. Specific primers--generally short oligonucleotides of known or random sequence--bind to target DNA denatured by heat into single strands. The primers, which now flank the target sequence, provide the polymerase with a starting point for replication. The enzyme moves along the single strands of DNA, making complementary copies of the DNA segment targeted by the primers. As the chain reaction continues, double strands of DNA are again denatured and extended, in continuing cycles of heating and cooling that ultimately create billions of copies of the original molecule. The earliest versions of the technology, developed by Nobel laureate Kary Mullis in 1985 while he was with Cetus Corp., relied on a polymerase isolated from E. coli to carry out the repetitive rounds of DNA replication. Other polymerases isolated from heat-loving bacteria living in natural hot springs soon replaced the E. coli enzyme, because they could withstand the successive cycles of heating and cooling required in PCR. Currently, heat-stable polymerases, such as Taq polymerase--from the thermophilic bacterium Thermus aquaticus--are available from companies such as PCR pioneers Roche and Perkin-Elmer Corp. of Norwalk, Conn., along with several other biotech firms, including Fisher Scientific of Pittsburgh; Invitrogen Corp. of San Diego; Promega Corp. of Madison, Wis.; and Boehringer Mannheim Corp. of Indianapolis. They also supply other biology and biotechnology kits, including restriction enzymes. Several companies produce Taq DNA polymerase and offer the enzyme to customers as a single product or as part of specialized PCR- related kits for cloning or sequencing. Recently, however, Hoffmann-La Roche took a firm stand against companies that promote their own products, such as Taq, for use with Roche's patented PCR technology and scientists who use PCR without paying licensing fees. In late May, Roche filed an amended suit in U.S. District Court in San Francisco naming more than 40 American universities and government laboratories as infringing on Roche's PCR patents. Among the institutions cited were the University of California's Lawrence Berkeley Laboratories, Stanford University School of Medicine, the Massachusetts Institute of Technology, Harvard University, the National Cancer Institute, and the National Institute of Allergy and Infectious Diseases, as well as a long list of individual scientists. The scientists and institutions were named as part of an ongoing federal suit filed by Roche against Promega Corp. in New Jersey in 1992. Roche alleged that the Wisconsin firm infringed on PCR patents by promoting its own Taq polymerase for use in PCR- related applications. In a statement, William Linton, president and CEO of Promega Corp., said, "If U.S. courts uphold Roche's claims of infringement and demand for licensing fees from those engaged in basic research, the costs of such research will skyrocket at a time of diminishing budgets, threatening scientific progress as well as the survival of critical economic development tool for American business and industry." Roche, however, which spent $350 million to acquire the patents from Cetus Corp. in 1992, claims it is merely protecting its investment in this highly lucrative technology. Legal considerations aside, PCR is a technology that is rapidly becoming a standard in molecular biology research. Although specialized instrumentation is not an absolute requirement for PCR, scientists should consider that rapid temperature changes are necessary for the repeated rounds of DNA strand separation and primer extension to occur. Unless a researcher wants to shuttle tubes back and forth between hot and cold water baths once every minute for a couple of hours, "a thermal cycling instrument is a necessary piece of equipment," says Cheryl Helton, technical services scientist with Boehringer Mannheim Corp. Thermal cyclers, such as the microprocessor- controlled device produced by Perkin Elmer Corp., are designed to perform the rapid temperature changes and incubations automatically. Using a heat-stable polymerase and a thermal cycler, researchers can set up their reactions, place the microfuge tubes into a preprogrammed instrument, and just walk away. The thermal cycler raises and lowers the temperature of the heating block as many times as desired. In a category with the standard plastic microcentrifuge tubes and micropipettor tips that are commonly used in molecular biology laboratories are a number of products modified specifically for PCR. Micropipettor tips, for example, such as ART Barrier pipette tips from San Diego-based Molecular BioProducts Inc., come with a special barrier material that blocks the passage of liquids and aerosols to the micropipettor when samples are sucked into the disposable tip. This eliminates the potential for cross- contamination of DNA specimens. "Many very useful technological developments in molec-ular biology have been directly driven by PCR," comments Therion Corp.'s Casna. Although barrier tips were created expressly for PCR, products such as this have been found to have other uses, such as preventing radioactive contamination of laboratory instruments and personnel. Since temperature is critically important for both denaturing the DNA and the annealing and extension steps, it is important that the reactants in the tubes reach the desired temperature as quickly as possible. Therefore, PCR "cocktails" are usually made up in thin-walled microcentrifuge tubes that allow the hot or cold temperatures to pass through to the mixture quickly. A newer idea is to set up the reaction in glass microcapillary tubes that are placed into an air thermal cycling device, the Air Thermo- Cycler from Idaho Technology Inc. in Idaho Falls, Idaho. The reactions in this so-called rapid cycling technique can be conveniently mixed up in the wells of a microtiter plate and then loaded by capillary action into tubes that are placed into the air thermal cycler. Temperature changes are transmitted quickly through the glass walls of the microcapillaries, allowing for rapid cycling. To prevent evaporation during the heating and cooling cycles, a layer of oil is often added to form a barrier between the PCRreactants and the air. In response to increased demands for PCR-related products during the early 1990s, developers at Perkin-Elmer created a thermal cycler with a heated lid. "The heated lid on the thermal cycling instrument replaces the need for oil in PCRreactions," says Murray Anderson, product manager for PCR instrumentation at Perkin-Elmer. "With this device, investigators can perform oil-free PCR. There is no need to add oil before PCR or get rid of it after the reaction is complete." Using microcentrifuge tubes with wax is another way researchers can perform oil-free PCR. These products offer an additional advantage during hot-start PCR, a variation on the standard theme in which some of the reaction components are kept separate until the annealing temperature is reached. A convenient way to perform hot-start PCRis to set up the reactions in tubes containing beads of wax. The wax serves as a physical barrier to prevent key components from reacting until the proper temperature is reached. It also eliminates the need for overlays of mineral oil to protect against evaporation. Many researchers use hot-start PCR to keep the nucleotides and the primers away from the template DNA and Taq polymerase until the optimal temperature for priming is reached. Hot-start PCR therefore improves the specificity of priming and reduces the number of mispriming events, as well as the formation of "primer dimers," which form when primers anneal to themselves instead of the target DNA. "Hot-start PCR avoids the gradual increase in temperature that occurs in the thermal cycler," explains Richard Oberste, an associate professor in the department of diagnostic medicine/pathobiology at Kansas State University's College of Veterinary Medicine. "Theoretically, it increases the efficiency of the initial rounds of amplification." Wax beads used for hot-start PCR, such as the HotWax beads produced by Invitrogen Corp., are simply dropped into PCR reaction tubes along with the reaction mix. The HotWax beads release magnesium when they melt, which allows the Taq polymerase to become active. HotStart tubes from Molecular BioProducts have a wax bead pre-adhered to an inner wall to eliminate the bead transfer steps. An additional advantage to wax beads is that they can be used for storage of the PCR product, for example, or to store pre-mixed cocktails, which can be popped out of the freezer and into the thermal cycler on a mo-ment's notice. When it comes to PCR, acronyms are in abundance. Following on the heels of standard PCR came techniques such as RT (reverse transcriptase) PCR; RAPD (rapid amplified polymorphic DNA) PCR, a DNA fingerprinting technique using primers of random sequence; LT (long-template) PCR; Q (quantitative) PCR; and AMPFLP (amplified fragment-length polymorphism), another fingerprinting method detecting genetic variations. These outgrowths have come along regularly since the discovery of the basic technology in 1985. Several companies--such as Qiagen Inc. of Chatworth, Calif.; Boehringer Mannheim; Invitrogen; Life Sciences Inc. of St. Petersburg, Fla.; Minneapolis-based Gentra Systems Inc.; and Perkin-Elmer--sell individual products and kits relating to PCR in its various guises. Although the starting material does not have to be highly purified, in many PCR applications the first steps involve isolating template DNA from a cellular source. Kits and reagents for isolating DNA and RNA are produced by a number of molecular biology supply companies, such as Boehringer Mannheim; Pharmacia Biotech Inc., Piscataway, N.J.; Promega; Fisher Scientific; and Qiagen Inc. Qiagen carries several products, including QIAquick, for speedy isolations of nucleic acids in gels; QIAmp, for the rapid purification of genomic DNA and viral nucleic acids; and RNeasy kits that facilitate the extraction of cellular RNA. Gentra Systems' Puregene and Purescript kits allow researchers to isolate either DNA (Puregene) or RNA (Purescript) from cells or tissues to be used in PCR or RT PCR. RT PCR utilizes the viral enzyme reverse transcriptase to make DNA copies of RNA isolated from cells. The AMV Reverse Transcriptase First Strand cDNA synthesis kit from Life Sciences Inc. produces a first strand of cDNA (complementary DNA) from an mRNA transcript. The synthesized cDNA can then be amplified by PCR for the purpose of analyzing genes that are actively expressed. "Virtually every cell in the body has the same DNA," says Dawson of BioVentures. "To look at the patterns of expression of the genes contained within that DNA, we need to look at the RNA. This can be conveniently accomplished with RT PCR." One of the latest techniques is LT, or long-template, PCR. Until last year, the length of DNA fragments that could be amplified by PCR remained relatively small--in the range of 3,000 to 5,000 base pairs long. Wayne Barnes of Washington University School of Medicine in St. Louis changed all that when he and others at Roche Molecular Systems in Alameda, Calif., and Perkin-Elmer discovered that adding two polymerases to the PCR mixture--one that synthesizes and one that "proofreads" the newly synthesized DNA and replaces mismatched bases--increased the size of the amplified products at least tenfold. LT PCR kits are already commercially available. "With LT PCR, a researcher can generate long PCR products without mistakes," explains Helton of Boehringer Mannheim. "The Expand kit combines Taq polymerase with a proofreading polymerase that allows Taq to synthesize long pieces of amplified DNA that are correct." "The fidelity and processivity [accuracy and speed] of Taq polymerase deteriorates after approximately 5 kb," says Dawson of BioVentures, whose company markets kits for rapidly isolating DNA or RNA from cells in preparation for PCR. As Taq copies pieces of DNA longer than about 3,000 base pairs, the polymerase begins to make errors and may "stall," ultimately falling off the DNA. "By adding supplemental enzymes, you can overcome the stall points of the enzyme and increase the length of the amplification product. There are instances in which entire mitochondrial genomes have been amplified by LT PCR." Usually a researcher amplifies a sequence of DNA with a specific purpose in mind. Perhaps the amplicon, as PCR products are called, is an intriguing gene found only in one or a few copies in a genome. Following PCR, enough of the gene's DNA will exist for cloning, after which the genetic segment can be sequenced, mutated, or expressed for further studies. Detecting the amplified DNA can be accomplished in a number of ways using a variety of tools, a list that includes antibody-based detection through enzyme-linked immunosorbent assay (ELISA). PCR products can be separated on gels and visually analyzed to determine the size of the amplified segment or the quantity of the DNA. This also requires transillumination equipment-- available from companies such as Spectronics Inc. in Brookline, Mass.--along with photography or videodocumentation equipment. Molecular Dynamics Inc. in Sunnyvale, Calif., markets a line of gel-scanning instruments that can be used for imaging PCR products on gels that involve different measuring techniques, notably fluorescence. The FluorImager system and reagents from Vistra Fluorescence, an alliance of Molecular Dynamics and Arlington Heights, Ill.-based Amersham Life Science Inc., let investigators tag their PCR products with fluorescence that can be measured directly in the fluorescence-imaging instrument. "The fluorescence-imaging instrument allows you to detect fluorescent signals on gels or membranes, in microtiter plates, and on TLC [thin-layer chromatography] plates," says Dick Rubin, applications manager for Molecular Dynamics. Many times, investigators sequence PCR products to screen for mutations, for identification, or to search for homologies with other sequences in protein data bases. Researchers can sequence the product in their own labs, or send it to a company such as Sequetech in Mountain View, Calif., which sequences PCR products and other types of templates on a contract basis. "We offer sequencing surfaces for all major template types, although our specialty is PCR products," says Michael Chin, Sequetech director of research and development. PCR products can also be analyzed for size or nucleotide composition by capillary electrophoresis if the laboratory budget allows. For sequencing or to identify PCR fragments, computer- controlled capillary electrophoresis systems available from Perkin-Elmer/Applied Biosystems, Beckman Instruments Inc. of Fullerton., Calif., and other companies provide resolution that reportedly exceeds that of standard gel electrophoresis, especially for small pieces of DNA that might be present in a sample in limited amounts. Capillary electrophoresis also gives a quicker result: Whereas it may take an hour or more to electrophorese and visualize PCR DNA on a gel, capillary electrophoresis takes just minutes. With all of its applications, PCR seems to have an exceptionally bright future. The current trends seems to be taking PCR deeper into the fields of diagnostics, to detect pathogenic organisms and to look for genetic disease. And in forensics, where PCR- based tests for genetic variability at the polymorphic D1S80 and DQa genetic loci have gained perhaps its greatest notoriety, through the murder trial of O.J. Simpson and other cases. "Through [Perkin-Elmer's] strategic alliance with Roche, we are working to expand PCR even further into applied fields, particularly in the areas of environmental food testing, agriculture, and diagnostics," says Anderson. Richard Oberste of Kansas State University, who is developing PCR-based tests to detect viral or bacterial DNA in animals and in the food supply, believes: "In some ways, PCR in diagnostics has lagged behind the advances in other areas of biology, but it is a powerful genetic tool that should be pressed forward." Agrees Casna of Therion Corp.: "The applications are endless." Holly Ahern is a science writer and an assistant professor of biology at Adirondack Community College in Queensbury, N.Y. (The Scientist, Vol:9, #13, pg.16, June 26, 1995) (Copyright, The Scientist, Inc.)

PCR Primed To Spur Chain Of Applications

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