A PCR Primer

PCR Enhancement Products Courtesy of RedasoftRedasoft's Visual Cloning 2000 includes primer design tools. Courtesy of Sigma-AldrichDirect loading of PCR products onto an agarose gel using Sigma-Aldrich's REDTaq. The art of PCR isn't hard to master. An abundance of products, ranging from relatively low-cost reagents to sophisticated optimization software, exists to meet most, if not all, PCR challenges. This profile looks at commonly used additives, PCR optimization kits and protocols, software tools for PCR enhancement, and hot-start polymerases. Other thermostable polymerases will be reviewed in the Sept. 3, 2001, issue of The Scientist. The key to an optimal PCR experiment is primer design. In general, primers should be between 18 and 24 nucleotides long and have a GC content between 40 and 60 percent. Ideally primers should contain a relatively GC-rich 5'-end and an AT-rich 3'-end (i.e., containing three A or T residues in the last five bases).1 Designing primer pairs with complementary 3'-ends should be avoided, as this leads to the formation of primer dimers, which depletes the effective primer concentration. A number of companies have developed computer software to aid primer design. Some of these programs are available for free on the Internet. For example, both the Weizmann Institute of Science Bioinformatics Unit Web site (bioinformatics. weizmann.ac.il/mb/bioguide/pcr/software.html) and LabVelocity (www.labvelocity.com) provide links to various primer design programs. Integrated DNA Technologies (www.idtdna.com) offers a Web-based Oligo Analyzer that is built on technology from DNAStar Inc. of Madison, Wis., whereas Ulm, Germany-based Interactiva offers DoPrimer™ software for the design of PCR and sequencing primers on its Web site (www.interactiva.de). Courtesy of PREMIER BiosoftScreen capture of PREMIER Biosoft's Primer Premier 5, showing all parameters considered in primer design. A number of commercially available software titles are on the market as well. These programs may be either stand-alone applications or exist as part of a larger software suite. Primer Premier 5, from PREMIER Biosoft of Palo Alto, Calif., can be used to design primers for PCR, sequencing, and hybridization. The newest version of the software offers features such as the ability to design primers across species and support for file formats found on commonly used databases such as GenBank and SWISS-PROT. Sci-Ed Software of Durham, N.C., sells Primer Designer 4, a primer design and analysis program available either as a stand-alone product or as part of the company's Clone Manager Suite. DNASTAR's PrimerSelect™ features a "virtual lab" that demonstrates the effect various design choices have on reading frames and restriction sites. Redasoft of Toronto, Canada, offers a primer design search tool as part of its Visual Cloning 2000 suite. The software features standard design tools and permits online ordering of custom-designed primers. Similarly, InforMax Inc. of Bethesda, Md., offers primer design as part of its Vector NTI® Suite of sequence analysis and data management software. Princeton, N.J.-based Techne Inc.'s PCR Help! software features primer and template design and analysis, as well as PCR protocol design and optimization. The software includes a "Gel Wizard" that analyzes PCR electrophoresis results and offers recommendations for improving amplification. The Expression software package from Genamics of Hamilton, New Zealand, is a sequence analysis application that includes primer design functions. The Genamics Web site (genamics.com/software/index.htm) offers a "SoftwareSeek" database listing molecular biology- related software tools from a variety of vendors and research institutions; the list includes primer design and other PCR-related software. Finally, iOligo, from Portsmouth, N.H.-based Caesar Software, can be used to design, analyze, and purchase oligonucleotides for PCR and sequencing reactions. The program accepts DNA, RNA, or reverse-translated proteins as input sequences. When All Else Fails, Try a Kit One common way to deal with a PCR-gone-wrong is to add a chemical additive to the reaction buffer. These reagents serve several functions, including disrupting secondary structure, decreasing melting temperature, stabilizing the polymerase, and enhancing enzyme processivity (the tendency of the polymerase to keep polymerizing). The list of these additives is growing, but one of the most popular is dimethyl sulfoxide (DMSO), which is added at a concentration of 2-10 percent. DMSO is believed to decrease secondary structure and lower the melting temperature of the primers but can decrease Taq polymerase activity at concentrations higher than 10 percent. Betaine (N, N, N-trimethylglycine), a component present in a number of commercially available PCR enhancement kits, has been shown to be useful for amplification of GC-rich templates.2 Nonionic detergents such as Triton X-100, Tween 20, or Nonidet P-40, and additives such as glycerol and gelatin, can help to stabilize Taq polymerase and are believed to decrease secondary structure.3 Other additives include bacteriophage T4 Gene 32 protein, which is believed to increase the yield of long PCR products, bovine serum albumin (BSA), and Escherichia coli single-strand binding protein (SSBP). A number of companies offer pre-made PCR buffers containing one or more of the above components. In fact, Taq polymerase is frequently supplied in a buffer that contains stabilizing agents such as nonionic detergents and glycerol (check product technical inserts). Roche Molecular Biochemicals of Indianapolis sells the Expand Long Template PCR System and the GC-RICH PCR System, both of which include a DMSO-containing buffer. The reaction buffer for the Advantage GC 2 Kit from Palo Alto, Calif.-based BD Biosciences-CLONTECH also contains DMSO. FailSafe™ PCR Enhancer from Epicentre of Madison, Wis., contains betaine. Some companies offer proprietary additive mixtures--for example, La Jolla, Calif.-based Stratagene's Taq Extender™ PCR Additive and PerfectMatch® PCR Enhancer; Valencia, Calif.-based QIAGEN's Q-Solution™; Lincoln Park, N.J.-based CPG Inc.'s MIGHTY™ Buffer; Westbury, N.Y.-based Brinkmann's TaqMaster™; CLONTECH's GC-Melt™; and Madison, Wis.-based Novagen's NovaTaq™ PCR Optimization Buffer. Many of these reagents can be found in a variety of PCR optimization and enhancement kits. These kits contain buffer sets, often with varied pH levels and MgCl2 concentrations, to help researchers experiment with different PCR conditions in an organized and efficient manner. Invitrogen Corp. of Carlsbad, Calif., offers the PCR Optimizer™ Kit. The kit contains 16 5X buffers containing 300 mM Tris-HCl and 75 mM ammonium sulfate, with varying pH and Mg2+ concentrations, as well as a stock solution of DMSO. The protocol suggests a three-stage, color-coded optimization scheme; the last stage, if needed, involves the addition of DMSO to each of the 16 buffers. Epicentre offers the MasterAmp™ PCR Optimization Kit, which contains 12 reaction buffers with dNTPs and varying concentrations of Mg2+, and MasterAmp PCR Enhancer, which contains betaine. Epicentre's optimization kits are provided with or without ammonium sulfate; the kit without ammonium sulfate is recommended for use with Tfl and Tth DNA polymerase and with proofreading DNA polymerases that have 3'-5' exonuclease activities. Fermentas of Hanover, Md., offers a PCR Optimization Kit that contains 16 10X buffers with varying pH and MgCl2 concentrations. The kit includes various PCR enhancement reagents such as glycerol, formamide, and DMSO solutions. Stratagene's Opti-Prime™ PCR Optimization Kit contains 12 buffers with varying pH and concentrations of MgCl2 and KCl; the PCR Optimization Kit II from Sigma-Aldrich of St. Louis is a similar product. Stratagene's Opti-Prime kit includes six buffer additives commonly used as PCR enhancers: formamide, DMSO, betaine, ammonium sulfate, glycerol, BSA, as well as Perfect Match PCR Enhancer. Sigma's kit contains the same additives, plus SSBP, which has been shown to enhance PCR specificity. Similarly, the PCR Optimization Kit from Roche Molecular Biochemicals contains a set of 16 buffers with varying pH and MgCl2 concentrations as well as reagents such as DMSO, gelatin, and glycerol. Maxim Biotech of South San Francisco, Calif., offers optimization kits for multiplex PCR. Maxim's Multiplex PCR Optimizer Kit provides six buffers and can be used to simultaneously amplify up to 18 DNA fragments from genomic DNA and cDNA. The company's EZ-GC Amp™ High-GC PCR/MPCR Optimizer Kit includes GC-Normalizer™ and DMSO to help amplify templates with high GC-content. Finally, Brinkmann offers the TripleMaster PCR System, a unique kit optimized for long-range, high fidelity, and GC-rich/difficult template PCR. Online PCR Help A variety of companies provide help to struggling researchers via their Web sites. For example, Qbiogene of Carlsbad, Calif., offers AmpliSURE™, an online optimization program based on modified Taguchi mathematical methods.4 The system includes a kit that generates comparative data for nine different reaction mixtures and allows the user, with the help of an online calculator (www.amplisure.com), to optimize the concentrations of dNTPs, Taq DNA polymerase, primers, and MgCl2, in one experiment. Users assign "scores" to the individual reactions based on agarose gel results, then plug these scores into the online calculator, which determines the optimal mixture of components. While Qbiogene does not sell custom PCR mixes, the company does sell a range of master mixes that customers can choose from based on their AmpliSURE results. According to Qbiogene, the AmpliSURE protocol is easy to use and helps ensure reproducibility of PCR experiments. For those on a tighter budget, Promega Corp. of Madison, Wis., has created the Amplification AssistantSM PCR Troubleshooting Program, an interactive help session that can be accessed via the Internet (www.promega.com/amplification/assistant). The program is designed to help researchers after a failed PCR attempt and requires the user to input reaction conditions, results, and component information. The Amplification Assistant provides custom advice based on the information supplied by the user and saves all the parameters entered during a particular session so that users can easily review or modify previous entries. Other online PCR resources can be found on the Web sites of some of the companies mentioned in this article. Roche, for example, provides a PCR Application Manual on its Web site, and Invitrogen offers a Web-based PCR/RT-PCR Applications Guide. Likewise, Brinkmann's Web site offers a PCR primer design guide. Feeling Hot, Hot, Hot Although the technique is not new, hot-start PCR continues to be a popular method for increasing the specificity of reactions by preventing mispriming and the creation of primer dimers. The method involves the separation of one reaction component (usually the DNA polymerase) from the rest of the reaction mixture until the temperature inside the reaction tubes reaches the annealing temperature. This procedure was originally achieved simply by adding Taq when the temperature reached 72ºC.5 To avoid the problems associated with this technique (e.g., the risk of losing reaction components when opening tubes at high temperatures, as well as the risk of introducing contaminants into the reaction tube), researchers developed some clever alternative strategies. These include the use of a wax barrier to separate the polymerase from the rest of the mixture,6 the use of wax beads containing PCR reagents,7 the use of an antibody against the polymerase that is inactivated at high temperatures,8 and the use of mutant polymerases that are activated at high temperatures.9 More recently, scientists have described techniques in which the addition of single-stranded DNA aptamers (short nucleic acid fragments that bind target molecules) or short double-stranded DNA fragments inhibits Taq polymerase at low temperatures.10,11 As the inhibition of Taq is dependent on the melting temperatures of the added fragments, these techniques allow greater control over the temperature at which functional enzyme is introduced into the reaction. Several companies offer hot-start reagents that rely on antibody-mediated inactivation of the polymerase. For instance, CLONTECH's TITANIUM™ Taq DNA polymerase includes TaqStart™ antibody for a built-in hot-start, as does the company's Advantage™ line of PCR kits. Invitrogen's Platinum® Taq DNA Polymerase is another antibody-based hot-start enzyme. A thermolabile inhibitor containing monoclonal antibodies to Taq is bound to this enzyme and is denatured during the initial PCR denaturation step, releasing active Taq polymerase. Stratagene offers Hotstart variants of their PfuTurbo®, Herculase™, and YieldAce™ DNA polymerases. These enzymes provide increased specificity and sensitivity and are antibody-deactivated until the initiation of thermal cycling. Other companies offer approaches based on the use of wax beads. Invitrogen's HotWax™ OptiStart™ Kit allows users to optimize their hot-start reactions by providing buffers with four different pH values, and HotWax beads containing three different Mg2+ concentrations, giving the user 12 possible reaction conditions to test. Stratagene's StrataSphere™ Magnesium Wax Beads are a similar product. Since Mg2+ is necessary for activation of Taq polymerase, placing magnesium ions in wax beads inactivates Taq until the beads melt. Promega's TaqBead™ Hot Start Polymerase isolates Taq polymerase in a wax bead until the tube temperature reaches 60ºC. Finally, Foster City, Calif.-based Applied Biosystems' AmpliWax® PCR Gem beads form a protective seal between PCR reaction components and the DNA template, polymerase, and buffer, until thermal cycling begins. Still other companies offer mutant or modified hot-start polymerases. Applied Biosystems' AmpliTaq Gold® DNA Polymerase is a chemically modified form of AmpliTaq® polymerase that is inactive until incubation at 95ºC for 1-10 minutes. The company also sells this enzyme in its GeneAmp™ Gold PCR Reagent Kit, which includes a multiplex control. QIAGEN's HotStarTaq™ DNA Polymerase is a modified form of Taq that is inactive at ambient temperatures and is activated by a 15-minute incubation at 95ºC. The company's HotStarTaq Master Mix Kit is a mixture of the enzyme, QIAGEN PCR Buffer, and dNTPs, for quick room-temperature setup of reactions. Recently, QIAGEN introduced ProofStart™, a hot-start proofreading enzyme that is chemically modified to inactivate polymerase and exonuclease activity, thus preventing degradation of primers. QIAGEN supplies Q-Solution as well as a specially formulated buffer with the enzyme. Stratagene's SureStart™ Taq DNA polymerase, a chemically inactivated version of Stratagene's Taq2000™ DNA polymerase, can be activated by incubation for 12 minutes at 92-95ºC or during the denaturation steps of thermal cycling; a similar product, FastStart Taq DNA Polymerase, is offered by Roche Molecular Biochemicals. Despite the abundance of PCR optimization products available to meet most researchers' needs, difficult templates still exist. Nevertheless, new protocols to combat these difficulties continue to be developed. For example, a group of researchers at the City University of New York recently demonstrated that the amplification of genomic DNA from mammalian cells can be enhanced after partial restriction digestion.12 Indeed, the art of PCR is not hard to master, and with the right tools, even the most difficult PCR won't be a disaster. Aileen Constans can be contacted at aconstans@the-scientist.com. References 1."PCR/RT-PCR Applications Guide," Invitrogen Corp., www.invitrogen.com/Content/Tech-Online/molecular_biology/manuals_pps/pcrtechguide.pdf. 2.S. Frackman et al., "Betaine and DMSO: Enhancing agents for PCR," Promega Notes, 65. 3.An excellent Web-based source of information and references related to common PCR additives can be found at the PCR additives page, taxonomy.zoology.gla.ac.uk/~rcruicks/additives.html. 4.B.D. Cobb, J.M. Clarkson, "A simple procedure for optimising the polymerase chain reaction (PCR) using modified Taguchi methods," Nucleic Acids Research, 22:3801-5, 1994. 5.M.A. Frohman et al. "Rapid production of full-length cDNAs from rate transcripts: Amplification using a single gene-specific oligonucleotide primer," Proceedings of the National Academy of Sciences, 85:8998-9002, 1988. 6.Q. Chou et al., "Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications," Nucleic Acids Research, 20: 1717-23, 1992. 7.S. Kaijalainen et al., "An alternative hot start technique for PCR in small volumes using beads of wax-embedded reaction components dried in trehalose," Nucleic Acids Research, 21:2959-60, 1998. 8.D.E. Kellog et al., "TaqStart antibody: 'Hot Start' PCR facilitated by a neutralizing monoclonal antibody directed against Taq DNA polymerase," BioTechniques, 16:1134-8, 1994. 9.C. Kebelmann-Betzing et al., "Advantages of a New Taq DNA polymerase in multiplex PCR and time-release PCR," BioTechniques, 24:154-8, 1998. 10.P. Kainz et al., "Specificity-enhanced hot-start PCR: Addition of double-stranded DNA fragments adapted to the annealing Temperature," BioTechniques, 28:278-82, Feb., 2000. 11.C. Dang, S.D. Jayasena, "DNA inhibitors of Taq DNA polymerase facilitate detection of low copy number targets by PCR," Journal of Molecular Biology, 264:268-78, 1996. 12.R. Vázquez, M.L. Steinberg, "Enhancement of PCRs by partial restriction digestion of genomic templates," BioTechniques, 26:91-95, 1999. Vendors of Commercially Available Primer Software Caesar Software FAX: (603) 436-7984 www.caesarsoftware.com DNASTAR Inc. (608) 258-7420 www.dnastar.com Genamics +64 (21) 923-440 www.genamics.com InforMax Inc. (800) 357-3114 www.informaxinc.com PREMIER Biosoft International (650) 856-2703 www.premierbiosoft.com Redasoft (603) 506-0437 www.redasoft.com Scientific and Educational Software (919) 471-6064 www.scied.com Techne Inc. (800) 225-9243 www.techneusa.com Editor's Note: To the best of our knowledge, this table is a comprehensive listing of suppliers.

A PCR Primer

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