Innovations Expand Lab Power, Uses Of PCR Technique

The gene amplification technique invented by genetics researcher Kary Mullis on a moonlit drive through the northern California hills a decade ago--the polymerase chain reaction (PCR)-- continues to revolutionize the life sciences. Uses in molecular biology research and in diagnostic tests are proliferating, and PCR is even bringing a new molecular approach to such fields as paleontology and epidemiology. The following companies are among those supplying PCR-related products for the research laboratory. J.T. Baker Inc. 222 Red School La. Phillipsburg, N.J. 08865 (800) 582-2537 Fax: (908) 859-9318 Product: MDE heteroduplex starter kit, $250 Bio-Rad Laboratories Inc. 3300 Regatta Blvd. Richmond, Calif. 94804 (800) 4BIO-RAD Fax: (415) 232-4257 Product: InstaGene DNA Purification Matrix, $30 National Biosciences Inc. 3650 Annapolis La. Plymouth, Minn. 55447 (800) 747-4362 Fax: (800) 369-5118 Product: Oligo ver. 4.1 for PC, 4.0 for Mac; call for prices Perkin-Elmer Corp. 761 Main Ave. Norwalk, Conn. 06859-0310 (800) 762-4000 Fax: (203) 761-5424 Product: QPCR System 5000, $30,000 Stratagene Corp. 11099 N. Torrey Pines Rd. La Jolla, Calif. 92037 (800) 424-5444 Fax: (619) 535-0034 Products: A variety of poly- merases and ligases from organisms other than those patented by Hoffmann-La Roche Driving the ever-expanding applications are advances in PCR technology itself and in the tools that support it. Key to the most recent PCR-based discoveries is the development of more sensitive detection and quantitation methods, as well as the discovery of enzymes that not only polymerize nucleic acids but also can proofread (correcting misincorporated bases as new DNA strands are built) and reverse transcribe RNA to DNA. Other tools help before and after PCR by setting experiment parameters and analyzing PCR products. With these new tools, PCR has recently shown, among many other things, that: Florida dentist David Acer most likely infected at least five of his patients with HIV (C.-Y. Ou, et al., Science, 256:1165, 1992). Northern Italian endangered bear populations are more genetically like Romanian bear populations than American bears, information needed to successfully introduce members of other bear populations to bolster the breeding base (P. Taberlet, J. Bouvet, Nature, 358:197, 1992). Seed pods found in the truck of a suspect in an Arizona murder case came from a specific Palo Verde tree near the body (C.K. Yoon, Science, 260:894, 1993). HIV lurks in lymph nodes during latent infection (G. Pantaleo, et al., Nature, 362:355, 1993; and J. Embretson, et al., Nature, 362:359, 1993). Two gene pieces can be sequenced from a 120- to 135- million- year-old weevil preserved in Lebanese amber (R.J. Cano, et al., Nature, 363:536, 1993). PCR borrows nature's way of replicating DNA. A researcher supplies pieces (primers) of DNA that bracket a DNA sequence of interest, a hefty supply of DNA nucleotides, a heat-tolerant polymerase, and various reagents. A thermal cycler is used to control temperature, shifting up to open the double helices, down to bind the primers, and up a bit to build the new strands, then up again to cycle anew. The cell's DNA replication machinery rapidly mass produces the desired DNA sequence, at first exponentially, then leveling off. >From this simple scheme has grown an entire industry. "[PCR] is a technology that has ramifications in a hugely broad spectrum of applications, and where a variety of improvements are being made, and can still be made," says Phillips L. Kuhl, president of Cambridge Healthtech Institute in Massachusetts, a company that sponsors and organizes technical conferences in biomedical areas. "PCR: Applications and Alternative Technologies" was the subject of the institute's second conference, held in Boston last month. The PCR conference is expected to be an annual event. PCR supply needs are met by Perkin-Elmer Corp. of Norwalk, Conn., which has an exclusive arrangement to market temperature cyclers, DNA sequencers, devices to detect and quantitate PCR products, reagents, and kits. The kits combine reagents for a specific function other than human diagnostics, such as forensics or environmental testing. For example, the EnviroAmp Legionella kit detects in water samples the microbe responsible for Legionnaire's disease. The rights to PCR research and development, as well as human diagnostic tests, are held by Roche Molecular Systems Inc., Newport Beach, Calif., an arm of Hoffmann-La Roche in Nutley, N.J., which purchased the PCR patents from Cetus Corp. of Emeryville, Calif., in late 1991. The first PCR-based human diagnostic test, for chlamydia, received FDA clearance for marketing in mid-June. Cambridge Healthtech Institute estimates that the potential worldwide market for human DNA-based diagnostics, most of which would use PCR to amplify nucleic acids in patient samples, could top $1 billion within the next four years. Using PCR requires a license. "There are different terms for for- profit and not-for-profit use, and two different rates depending upon whether Perkin-Elmer products are used," says Ellen Daniell, director of licensing at Roche. "Rates are as low as 9 percent of net service revenues [on a particular test] for a nonprofit organization. There is no per-test minimum." PCR was rather difficult to carry out as conceived back in 1983, because the temperature-sensitive DNA polymerase first used had to be replenished after every heat cycle. The application of thermostable polymerase revolutionized the technology by making automation possible. The search for additional DNA replication enzymes continues, taking scientists to hot springs where bacteria harboring the enzymes thrive, such as those in Yellowstone National Park. Newly discovered enzymes continue to ease PCR. Stratagene Corp. in La Jolla, Calif., for example, researches, develops, and markets thermostable enzymes other than those offered by Perkin- Elmer. The company offers a variety of polymerases and ligases. Perkin-Elmer's new UlTma DNA polymerase proofreads. And rTth DNA polymerase, also introduced by Perkin-Elmer this year, reverse transcribes RNA to DNA, then amplifies the DNA. This polymerase is particularly valuable in studying gene expression because it can amplify rare gene messages. It is synthesized in recombinant E. coli bacteria engineered to produce a modified polymerase from the thermophilic bacterium Thermus ther-mophilus. "It is a significant advance to be able to use a single enzyme to do both reactions," says Thomas J. White, vice president of research and development at Roche Molecular Systems. In some PCR experiments, researchers want to go beyond detecting DNA to quantitating it. To aid them, Perkin-Elmer has just introduced the automated QPCR System 5000. Unlike standard methods that distinguish PCR products by size, QPCR uses a probe- based system. "The probe has an electrochemical label incorporated, which is tris 2,2-bipyridine ruthenium chelate. We sell the label, so users can synthesize their own probes," says Elena Katz, senior staff scientist at Perkin-Elmer, who worked on developing the technique. She adds that, when bound, "the probe goes to an excited state. When it decays to the ground state, it emits light at 620 nanometers. The chemiluminescence reaction is controlled by applying a voltage in an electrochemical flow cell. We have developed an instrument to exploit this phenomenon." Quantitating a PCR product with the system takes less than three minutes. A different approach--not brand-new, but finding new uses in AIDS research--from quantitating PCR products is competitive PCR, in which an internal control is run along with the experimental amplification (M. Becker-Andre, K. Hahlbrock, Nucleic Acids Research, 17:9437, 1989; G. Gilliand et al., Proceedings of the National Academy of Sciences, 87:2725, 1990). Two sets of primers are used that correspond to the same target DNA sequence. The control primers incorporate a distinguishing feature, such as an intron or a restriction enzyme cutting site, that enables it and its amplified products to be separated from the non-control primers and products. The control primers lead to production of so-called competitive DNA because they bind to the same site as the experimental primers. Quantitation is possible because the amount of competitor DNA is known. Known amounts of competitor DNA are added in increments to a series of PCR reaction tubes, each holding the same amount of sample DNA. After amplification, the tube with equimolar amounts of competitor and experimental DNA is identified. By inference, it contains the same amount of experimental PCR product as the known amount of control product in that tube. Competitive PCR is being developed at Clontech Laboratories in Palo Alto, Calif., and the Palo Alto Institute for Molecular Medicine (P.D. Siebert, J.W. Larrick, Nature, 359:557, 1992). A variety of products target pre- and post-PCR needs (R. Lewis, The Scientist, Dec. 9, 1991, page 23). Bio-Rad Laboratories Inc. in Hercules, Calif., for example, offers InstaGene DNA Purification Matrix to prepare samples. Plymouth, Minn.-based National Biosciences Inc.'s Oligo software helps researchers set parameters and select primers for PCR experiments. An important niche in the PCR market, especially as the technology enters the clinical front, is for products that prevent contamination of a PCR run with product from a previous run. This type of glitch, called "product carryover," can lead to a false positive result. Perkin-Elmer markets a method developed at Life Technologies Inc., Gaithersburg, Md., that incorporates modified thymine (deoxyuridine triphosphate) into primers, then uses an enzyme, uracil N-glycosylase, to cleave at the replacement site, preventing reamplification of the old products containing the modified base. Many tools help analyze PCR products. Applied Biosystems Inc., Foster City, Calif., for example, specializes in DNA sequencing instrumentation that is often used on PCR products. The company will probably become even more tied into PCR now that it has been acquired recently by Perkin-Elmer. But sequencing is just one way to analyze a PCR product-- shortcuts can also provide valuable information about a gene. When PCR is used to amplify part of a gene responsible for a disorder, for example, several techniques can detect how the disease-causing gene variant (allele) differs from the normal (wild type) form of the gene. This can be done in about 20 different ways, says Peter Yesjar, technical specialist at J.T. Baker Inc. of Phillipsburg, N.J. "One conventional way to detect a mutation, for example, is to sequence the whole strand and identify one nucleotide and compare it to a standard," he says. J.T. Baker's Mutation Detection Enhancement (MDE) ultra-high- resolution electrophoresis gels are used in simpler procedures to detect genetic variants--single-strand conformational polymorphism analysis (SSCP) on single DNA strands and heteroduplex analysis on double stranded DNA. They are based on the fact that a mutation in a gene alters the conformation (three-dimensional shape) that either single- or double-stranded DNA assumes after its strands are separated and allowed to reanneal. MDE uses a proprietary modified polyacrylamide to visualize mutant alleles that migrate on a gel to a different position than do their wild type counterparts. PCR continues its trademark role of ferreting out needles in biological haystacks. The technology was crucial in revealing HIV's hiding places in the quiescent period before AIDS symptoms appear. A similar application with enormous commercial potential is fetal cell sorting. In development at Genzyme Corp. in Framingham, Mass., fetal cell sorting homes in on fetal nucleated red blood cells that escape into the maternal circulation at a rate of one in a million. This provides a far less invasive target for prenatal diagnosis than the current techniques of amniocentesis and chorionic villus sampling. As few as 15 fetal cells can provide enough DNA for PCR to proceed. The first experiments targeted Y chromosome-specific sequences, because these obviously cannot come from maternal contamination. But fetal cell sorting using PCR can be adapted to search for any gene variant, as long as primers are available. Another rare-cell situation involves cancer. In pre-symptomatic stages, PCR can spot the few cells with altered proto-oncogenes or tumor suppressor genes. After treatment, such as a bone marrow transplant, chemotherapy, or surgery, PCR of affected tissues can amplify residual disease or the first signs of a recurrence. PCR is, and will continue to be, pivotal in the largest-scale sequencing project, the Human Genome Project, as well as other genome research. Says White, "PCR is one of the main technologies used in all phases of the genome project. It is used for all sequence-tagged sites; identification of different markers; characterizing mutations for disease associations; physical mapping and putting together contigs [a method of approaching a gene by aligning overlapping sequences]; looking at variations in different populations; and, of course, in sequencing." Kary Mullis received a $10,000 bonus from Cetus Corp. for inventing PCR in 1983. Last year he received another $10,000, along with the John Scott Award from the city of Philadelphia, to honor his invention. This spring, he was awarded the $450,000 Japan Prize for that work--a dramatic signifier of the impact his technique has had on the life sciences.

Innovations Expand Lab Power, Uses Of PCR Technique

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