Convenient, Performance-Boosting Products Put PCR On The Fast Track

PCR On The Fast Track The polymerase chain reaction (PCR) revolutionized researchers' ability to manipulate DNA and RNA. With PCR-an amplification procedure that mimics DNA replication-molecular biologists no longer have to painstakingly purify large quantities of DNA for genetic experiments. The growing number of plastics and laboratory disposables, combined with the advent of reagent kits designed expressly for PCR, are making it easier for scientists to apply the technology in new areas. PCR minimizes considerations of initial quantities of DNA or RNA. "The beauty of PCR is that it allows us to work with infinitely small quantities of DNA," states Will Gergits, vice president of Therion Corp. in Troy, N.Y. DNA EXTRACTION SYSTEM: Biotecx Laboratories' EZout System removes DNA fragments from the agarose gel matrix. Therion uses tests such as DNA fingerprinting to determine the genetic identity of animals. "Any material containing DNA-such as [internal organs] left behind in the dirt by poachers, an old carcass, or perhaps blood cells or semen left behind at a crime scene-provides the starting material for PCR," Gergits says. The technique can be used in applications as simple as subcloning a gene or as complicated as sequencing a genome. Using PCR, scientists have obtained intact genetic sequences from sources as unusual as the bones of dinosaurs or mummified human remains. PCR technology was developed in 1985 by Nobel laureate Kary Mullis at Cetus Corp., a biotech company in Emeryville, Calif. Mullis's vision was to repeatedly separate the two polynucleotide strands in DNA and allow each to serve as a template for new strand synthesis. Mullis's earliest attempts to amplify genetic material relied on a replicative enzyme, DNA polymerase, derived from the laboratory bacterium E. coli. However, amplifying DNA by PCR requires repeated temperature increases to denature the double DNA strands. This also has the unfortunate effect of inactivating the E. coli enzyme in halting the PCR reaction. A polymerase isolated from thermophilic bacteria, Thermus aquaticus, which lives naturally at high temperatures, remains active during PCR's rapid rounds of temperature cycling. Because of its importance in PCR, the thermostable polymerase from T. aquaticus, called Taq polymerase, became Science magazine's first "Molecule of the Year" in 1989. The patent for Taq polymerase now is owned and licensed through Hoffmann-La Roche Inc. The Swiss-based pharmaceutical company, with United States headquarters in Nutley, N.J., bought the patent rights from the now-defunct Cetus Corp. in 1990 for more than $300 million. It now is asserting its exclusive rights to both the PCR process and Taq polymerase. In 1992, Hoffmann-La Roche filed a lawsuit against Promega Corp., a biotech company in Madison, Wis. Promega holds a license to manufacture and sell the enzyme, but only for non-PCR applications such as mutagenesis or genetic sequencing. Promega offers Taq as a single product and in cloning and mutagenesis kits. The suit alleges that Promega infringed on PCR patents by promoting its Taq polymerase for PCR, thus avoiding royalty payments to Hoffmann-La Roche. Last year, Hoffmann-La Roche amended the lawsuit to include more than 40 American universities and government laboratories, along with a long list of individual scientists. Promega Corp. filed a countersuit alleging that Hoffmann-La Roche's patent on Taq polymerase should be invalidated based on "inequitable conduct," a term synonymous with fraud. Promega submitted reports and experimental data from experts that it believes indicates that Cetus scientists were not the first to "discover" Taq polymerase, but instead were working with an enzyme that had been purified from T. aquaticus by Russian researchers nearly a decade before Mullis's PCR breakthrough. In March, pre-trial hearings were held in U.S. District Court of Northern California in San Francisco to narrow the issues that will be presented to a jury. The hearings are expected to move the trial forward. Legal battles aside, Taq remains the most popular of the polymerases used for PCR. Perkin-Elmer Corp. of Norwalk, Conn., markets AmpliTaq, the Taq polymerase manufactured by Roche Molecular Systems Inc., a division of Hoffmann-La Roche in Branchburg, N.J. Perkin-Elmer also carries kits designed to optimize PCR performance in several applications. One such application is a technique referred to as RT (reverse transcriptase)-PCR, in which the amplification target resides in RNA, not DNA. The first step of RT-PCR is the synthesis of a DNA strand complementary to a nucleotide sequence in RNA, known as cDNA. This so-called first strand is synthesized by reverse transcriptase, an enzyme produced by viruses with RNA-based genomes. The first strand serves as a template for synthesis of a second DNA strand by the Taq polymerase. "Once a double-stranded DNA molecule is formed from RNA, PCR proceeds in the same way that it would starting from a DNA-containing sample," explains Cheryl Helton, technical services scientist at Boehringer Mannheim Corp. of Indianapolis. "All of the cells in the body contain essentially the same genetic material," comments molecular biologist Elliot Dawson, president of BioVentures Inc., a Murfreesboro, Tenn., company that supplies reagents and kits for quick isolation of DNA or RNA used in PCR. "To study the expression of particular genes in various cells, we have to look at the RNA, which is the chemical message produced from genes and translated by ribosomes into proteins. With RT-PCR, we can amplify the message to determine which genes are being expressed." OPTIMIZED SYSTEMS: Perkin-Elmer's GeneAmp family of PCR kits for different applications Perkin-Elmer's family of GeneAmp RNA PCR kits consists of optimized systems for different RT-PCR applications. The GeneAmp EZ rTth RNA PCR Kit is a single-buffer, single-enzyme system that simplifies screening multiple samples for a particular RNA message. Tth polymerase is a thermostable enzyme purified from the bacterium Thermus thermophilus, another denizen of steamy depths. Tth polymerase is a thermostable enzyme that exhibits an intrinsic reverse transcriptase activity, which makes the enzyme quite efficient at first-strand synthesis. Another kit from Perkin-Elmer, the GeneAmp Thermostable rTth Reverse Transcriptase RNA PCR Kit, also facilitates first-strand cDNA synthesis. The thermostability of this enzyme permits researchers to set up reactions at higher temperatures. This overcomes the problem of secondary structure (folds and loops), which can form in the normally single-stranded RNA. Boehringer Mannheim markets both Taq polymerase and Tth polymerases as separate products. The company's Taq polymerase is sold under licensing arrangements with Hoffmann-La Roche and Perkin-Elmer that allow purchasers to use this enzyme in PCR applications. In addition to using Tth polymerase in first-strand synthesis reactions for RT-PCR, researchers can use this enzyme in thermal cycling DNA sequencing procedures. Boehringer Mannheim's PCR Core Kit and PCR Core Kit+ contain all of the necessary reagents (except primers and the template) required to amplify DNA by PCR. The PCR Core Kit+ additionally contains reagents for the UNG system, which consists of uracil DNA glycosylase (an enzyme that slices uracil residues out of DNA) and dUTP (a uracil-containing nucleotide). With this system, researchers amplify the target DNA in the presence of dUTP. In subsequent reactions, dUTP-labeled DNA is degraded by the glycosylase enzyme, thus eliminating the risk of re-amplifying any PCR products carried over from a previous reaction. Under a licensing arrangement with Hoffmann-La Roche and Perkin-Elmer, Life Technologies Inc. of Gaithersburg, Md., sells both native (derived directly from T. aquaticus) and recombinant forms of Taq polymerase for use in PCR. The recombinant enzyme is purified from the cloned T. aquaticus DNA polymerase gene expressed in E. coli, which is easier to grow in the laboratory. Life Technologies' PCR Reagent System is a kit that contains the reagents necessary for amplifying nucleic acid templates by PCR, including buffers, nucleotides, magnesium chloride (MgCl2) to provide the requisite cations, and Taq polymerase. To facilitate RT-PCR, the 5« RACE System from Life Technologies is designed for rapid amplification of cDNA ends (RACE) and a process termed anchored PCR, in which one end of a first strand cDNA is labeled with an "anchor" sequence. With the 5« RACE system, researchers can amplify rare RNA messages for which little sequence information is available. The kit includes reagents for synthesis of first-strand cDNA, purification of first-strand products, addition of the oligo-dC anchor, and preparation of the target cDNA for PCR amplification. Taq polymerase is not included with this system. The first step in PCR usually is releasing target nucleic acids from the confines of cells. The exquisite sensitivity of PCR amplifies DNA that matches the sequence of the primers, so time-consuming DNA and RNA purification steps often can be simplified. GeneReleaser from BioVentures Inc. is a universal cell lysis reagent that releases DNA or RNA from cells in a quick five-minute protocol (10 minutes if the researcher performs the lysis in a thermal cycling device). Cells from various sources-including whole blood or blood spots, cell cultures, plants, or paraffin-embedded tissues-can be lysed directly in the reaction tubes, to which PCR reagents are subsequently added. GeneReleaser kits include the reagent and other equipment such as microwave racks and reaction tubes. Also for genomic DNA isolations, DNAzol Reagent from Life Technologies is a guanidine-detergent cell lysing solution that frees nucleic acids from cells. The reagent and associated protocol permit investigators to isolate genomic DNA from cells in a few steps. Isolation of DNA from paraffin-embedded tissues, such as lung tissue or other biopsied material, can be achieved using Life Technologies' GlassMAX DNA Isolation Spin Cartridge system. With this system, the paraffin first is dissolved and then the DNA is separated on a spin cartridge containing a silica membrane. Qiagen Inc. of Chatsworth, Calif., offers several kits to help purify nucleic acids for PCR. With Qiagen's RNeasy Kits, researchers can perform small-scale purifications of plant or animal cell RNA. The RNeasy Kits incorporate a silica-based separation matrix in a spin column format with which RNA isolations can be achieved in 20 minutes. Genomic-tips, also from Qiagen, are specially designed plastic tips in which genomic DNA suitable for PCR applications is separated by a gravity-driven chromatography method. Two kits from Gentra Systems Inc. of Minneapolis are designed for purifying DNA or RNA from blood and other tissues prior to PCR. The Puregene Kit contains two reagents necessary for isolating genomic DNA from cells and tissues. Gentra Systems' Purescript Kit removes RNA from cells for use in RT-PCR. PURELY CONVENIENT: Gentra Systems Puregene kit for isolating genomic DNA and Purescript kit for removal of RNA from cells for use in RT-PCR. SPIN CONTROL: Princeton Separations' CentriSpin family of disposable spin columns to clean up PCR products. In addition to obtaining the template prior to PCR, it often is necessary to "clean up" the PCR products after an amplification procedure. "Most researchers use PCR as a tool and intend to perform further manipulations on the amplification products," comments Dennis Stocker, senior scientist with Princeton Separations Inc. The Adelphia, N.J.-based company supplies kits and consumables for molecular biology applications, including PCR. "In general, before an amplified DNA product can be used in additional applications such as sequencing or subcloning, a 'clean-up' step is required to purify the PCR products away from free nucleotides or excess primers left over from the reaction," Stocker explains. The Centri-Spin family of products from Princeton Separations are disposable spin columns that researchers can use to clean up PCR products. In particular, the company's Centri-Sep spin columns remove contaminants from amplified DNA fragments used during automated fluorescent sequencing. QIAquick PCR Purification Kits from Qiagen contain spin columns to quickly purify amplified DNA away from primers and unincorporated nucleotides. For researchers who identify PCR products according to their size on an agarose gel, the EZout System from Biotecx Laboratories Inc. in Houston removes DNA fragments from the gel matrix. One PCR application that has proved itself in the research arena is gene cloning. "If the genetic sequence of a gene is known, it is a relatively simple matter to design PCR primers and amplify the gene for cloning," says Jeffrey Porter, a research associate in the department of chemistry at Louisiana State University in Baton Rouge. While researchers can design their own systems for cloning genes, several kits designed to optimize gene cloning by PCR are commercially available. The CloneAmp Systems from Life Technologies are kits for rapidly cloning amplified DNA sequences. CloneAmp kits utilize the enzyme uracil DNA glycosylase to produce "sticky ends," which are single-stranded overhangs at either end of the target DNA. Kits are available with the plasmids pAMP1, pUC18, pUC19, and pAMP10. The cloning vectors contain sticky ends that are complementary to those of the enzyme treated amplification product. When the amplified DNA fragment is added to the cloning vector, the sticky ends anneal by forming complementary base pairs, thus joining the fragment and vector. Stratagene Cloning Systems in La Jolla, Calif., offers pCR-Script Cloning kits, which incorporate Stratagene's Pfu polymerase, a thermostable DNA polymerase isolated from Pyrococcus furiosus. The pCR-Script vectors are phagemids (a combination between a bacteriophage and a plasmid) with a cloning site specific for blunt-ended fragments. DNA sequences amplified with Pfu polymerase are well suited for cloning into the pCR-Script vectors because this enzyme lacks the activity that leaves sticky ends behind. Selected Vendors of Equipment and Supplies for PCR Biotecx Laboratories Inc. Bio Ventures Inc. Boehringer Mannheim Corp. Clontech Laboratories Inc. Gentra Systems Inc. Hoffmann-La Roche Inc. Invitrogen Corp. Life Technologies Inc. Molecular BioProducts Inc. Perkin-Elmer Corp. Pharmacia Biotech Princeton Separations Inc. Promega Corp. Qiagen Inc. Roche Molecular Systems Inc. Stratagene Cloning Systems. Stratagene's Seamless cloning kit permits researchers to clone DNA fragments that lack naturally occurring restriction sites. This system combines PCR technology with the activity of a restriction enzyme that cleaves DNA sequences distant from the recognition site. As opposed to methods in which restrictions sites are created or eliminated in time-consuming, multistep mutagenesis methods, the Seamless system allows researchers to clone these difficult DNA fragments in one day, according to the company. SOME LIKE IT HOT: Molecular BioProducts' HotStart tubes use a wax bead to keep reaction components separate until a critical temperature is reached. When it comes to PCR, items created expressly for increased speed and convenience abound. The ART Barrier tips, produced by San Diego-based Molecular BioProducts Inc., contain a special barrier to block liquids and aerosols from contaminating micropipettors to which the tips are attached. This decreases the possibility for cross-contamination caused by carryover of substances from previous samples. HotStart Storage and Reaction tubes, also from Molecular BioProducts, are plastic microtubes designed to hold PCR cocktails with a small wax bead attached to an inside wall. The wax serves as a physical barrier to keep reaction components separate until a critical reaction temperature is reached. Dubbed "hot-start PCR," this technique lessens the possibility of false priming events (when primers bind to regions other than the target sequence), or formation of "primer-dimers" (when primers stick to each other rather than to the template nucleic acid). INCLUDED: Clontech Laboratories' new Advantage PCR kit contains TaqStart Antibody. HotWax Beads, from San-Diego-based Invitrogen Corp., contain magnesium, a divalent cation that functions as a cofactor in the elongation stages of PCR. As the HotWax beads melt, they release magnesium into the reaction mixture, reportedly doubling the likelihood that primer binding and elongation will not occur until optimal temperatures are reached. A different approach to keeping reactants apart is using monoclonal antibodies to block the reactive site of the replicative enzyme until appropriate reaction temperatures are achieved. TaqStart Antibody and TthStart Antibody, products from Clontech Laboratories Inc., are included with the polymerase mixes in the Palo Alto, Calif.-based company's new Advantage PCR kits. During the initial set-up steps, the antibody is bound to the polymerase, preventing the enzyme's activity. When the temperature increases, the antibody dissociates from the enzyme, permitting its full activity. Pharmacia Biotech of Piscataway, N.J., has contributed to PCR convenience with the Ready-To-Go bead, a polymeric bead found in its Ready-To-Go kits for RT-PCR. Each bead is a pre-formulated, single-dose reaction. Rather than pipetting several different reagents and risk introducing contaminants into PCR reactions, a single Ready-To-Go bead is added to each sample. The bead adds all of the necessary reagents to begin first strand synthesis in RT-PCR. Current trends in technology are taking PCR to an even smaller scale with a higher level of automation for applications that include noncycling amplification approaches, quantitative PCR, and in situ hybridization. Concludes Porter: "The advances in PCR technology will open up new realms of scientific study, notably in the areas of genomics and clinical diagnosis, where PCR has yet to reach its full potential." Holly Ahern is a science writer and an assistant professor of biology at Adirondack Community College in Queensbury, N.Y.

Convenient, Performance-Boosting Products Put PCR On The Fast Track

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