High Fidelity PCR: Enhancing the Accuracy of DNA Amplification

Date: January 5, 1998 Chart 1, Chart 2 n the beginning there was Taq. Actually, there were others before Taq. There were precursory polymerases, such as that from E. coli, that lost their enzymatic activities at elevated temperatures. This shortfall made thermal cycling a time-consuming chore, with the necessity of adding new polymerase after each cycle. Then came the thermostable polymerases such as Taq DNA polymerase, which was isolated from the thermophilic, aerobic bacterium Thermus aquaticus. Taq is the mother of thermophilic DNA polymerases. Where would the molecular biology lab be without Taq? The laboratory would be substantially quieter and less cluttered without Taq churning away in thermal cyclers. The polymerase chain reaction (PCR)* has definitely kept scientists busy and altered their approach to scientific problems. The foothold PCR has gained in labs has prompted scientists to optimize and refine this technique. As such, this has put the enzymes themselves under scrutiny, leading to the discovery of new polymerases and mixtures of polymerases optimized for a variety of applications. Taq's enzymatic activity at high temperatures allows for primer extensions and sequencing of nucleic acid templates with complex secondary structures. Yet while Taq's thermostability is commendable, its error rate could use some improvement. Using Taq DNA polymerase, the misincorporation of bases during amplification could potentially be a trouble spot, especially if you are copying a specific gene that might ultimately be expressed. In the last 10 years, numerous studies have quantified the error rate of thermostable DNA polymerases (http://listeria.nwfsc.noaa.gov/protocols/taq-errors.html), and several enzymes have been found to copy DNA more accurately than Taq. While many enzymes outshine Taq in the area of fidelity, though, it is important to keep in mind that error rates are not totally attributable to the polymerase used. One study found fidelity of the DNA polymerase from Thermococcus litoralis was affected by changes in deoxynucleoside triphosphate (dNTP) concentration, units of enzyme used per reaction, and the ratio of MgSO4 to dNTPs present in the reaction (P. Mattila et al., Nucleic Acids Research, 19:4967-73, 1991). However, if fidelity is of concern in your laboratory, a number of thermostable polymerases and enzyme mixes on the market can help cut down on the error rate in your reactions. A high fidelity polymerase is an enzyme with "proofreading" ability. Polymerases with inherent 3¢ to 5¢ exonuclease activity are considered proofreaders, as this activity gives the polymerase the ability to correct any misincorporated nucleotides on the strand of DNA being synthesized. While high fidelity PCR can be carried out with a single high fidelity polymerase, several optimized polymerase mixtures have been developed. High fidelity polymerases alone should definitely increase fidelity rates but will probably not amplify long fragments that a 3'-exonuclease-free enzyme such as Taq can handle. Enzyme mixtures that combine a standard polymerase with a small amount of proofreading polymerase may provide a nice balance between fidelity and yield. A study published in 1994 illustrated that the use of a high level of exonuclease-free polymerase (Klentaq-1) with a very low level of a thermostable DNA polymerase exhibiting 3¢-exonuclease activity (Pfu, Vent, or Deep Vent) generated products with increased base-pair fidelity with a maximum yield of 35 kb DNA from 1 ng of lambda DNA template (W. Barnes, Proceedings of the National Academy of Sciences, 91:2216-20, 1994). In mixtures such as these, the proofreading enzyme is there to act as the "misincorporation police," correcting errors behind the nonproofing enzyme. Which is for you? Weighing your requirements for fidelity and yield should help in the decision-making process. Also, some of the kits contain polymerase(s), buffers, primers, and even template, so deciding exactly what you want will be important prior to purchasing. If you're still not certain exactly which enzyme or mix is right for you, contact the following companies or visit their web sites to get more information. Thermostable polymerases with inherent proofreading ability have been isolated from a number of thermophilic organisms. Scientists needing high fidelity DNA polymerases have several options available, so if there is a high fidelity polymerase on your shopping list, read on. From the thermophile Pyrococcus woesei, Pwo DNA polymerase from Boehringer Mannheim is supplied as the recombinant form, cloned in E. coli. Pwo polymerase has a half-life of more than 2 hours at 100°C. Pwo DNA polymerase generates blunt-ended PCR products, ideal for blunt-end ligation. The enzyme is particularly suited for cloning of PCR products, characterization of rare mutations in tissue, and characterization of a population of cells in culture, among other applications. VentRTM and Deep VentRTM DNA polymerases from New England BioLabs are for primer extension, thermal cycle sequencing, and high temperature dideoxy sequencing applications only. VentR is purified from strains of E. coli that carry the VentR DNA polymerase gene from the archaebacterium Thermococcus litoralis. The native organism, isolated from a submarine thermal vent, can grow at temperatures up to 98°C. The fidelity of VentR DNA polymerase is 5- to 15-fold higher than that observed for Taq DNA polymerase. Deep VentR DNA polymerase is isolated from Pyrococcus species GB-D, also a hyperthermophilic archaebacterium. Deep VentR is more stable than VentR and has a calculated half-life at 95°C of 23 hours. UlTma® DNA polymerase from PE Applied Biosystems is encoded by a modified form of a polymerase gene from the thermophile Thermotoga maritima. The gene has been cloned and expressed in E. coli. UlTma provides high yields of PCR products, produces blunt-ended PCR products suitable for cloning and gene expression, and has integral proofreading ability. Promega Corporation provides Tli DNA polymerase, isolated from the hyperthermophilic archaebacterium Thermococcus litoralis. The enzyme replicates DNA at 75°C and remains functional after incubation at 100°C. Promega recommends using Tli for applications other than sequencing. Stratagene markets Pfu DNA polymerase, derived from the hyperthermophilic archaebacterium Pyrococcus furiousus. Pfu is highly thermostable; the enzyme retains 95% activity after incubation at 98°C for one hour. A published average error rate per nucleotide of 1.3 x 10-6 is the lowest profiled of any other DNA polymerase (J. Cline et al., Nucleic Acids Research, 24:3546-51, 1996). The use of Pfu polymerase with PCR reactions also results in blunt-ended PCR products, ideal for blunt-ended cloning. Stratagene provides both native and recombinant versions of the polymerase. PfuTurboTM DNA polymerase, Stratagene's latest offering, is a blend of high fidelity of Pfu polymerase and a newly discovered PCR enhancing factor that delivers enhanced PCR yield, product size, and throughput performance. PfuTurbo DNA polymerase exhibits the same high fidelity as Pfu polymerase and can successfully amplify genomic targets up to 10 kb, and vector-based targets to 15 kb. Long and Accurate PCR (LA PCR) refers to a mixture of polymerases containing a minor proofreading enzyme. LA technology is the concept of mixing two thermostable enzymes together for longer and more accurate DNA extension, primarily for PCR. Invented by Wayne Barnes (U.S. Patent 5,436,149) and foreign counterparts, the patent for LA technology is now held by TaKaRa Shuzo (Japan). Several companies provide not only polymerase mixtures but also kits optimized for specific applications such as RT-PCR. Components of these mixtures and kits vary-something to remember when looking at price tags-but many contain all the necessary items for reactions, including buffers, primers, template, and dNTPs. The LG PCR Kit from Ana-Gen is comprised of an optimized polymerase mix, buffer, and dNTPs. The kit allows the amplification of standard and long distance templates (greater than 20 kb lambda DNA) with high efficiency and yield. The Ready-Mix eliminates the need for a hot start technique in most applications. The ExpandÔ High Fidelity PCR System from Boehringer Mannheim contains a mixture of Taq DNA Polymerase and Boehringer Mannheim's Pwo DNA polymerase. The kit is optimized for genomic DNA fragments less than 5 kb and offers an error rate of 8.5 x 10-6. Each lot of Expand High Fidelity is function tested in PCR; amplification is performed using human genomic DNA and specific primers for collagen and the tPA gene. Boehringer Mannheim's ExpandTM Long Template PCR System is optimized for the amplification of long fragments of DNA, including genomic DNA. This system includes three buffers that are optimized for the amplification of fragments from 0.5 to 12 kb, 12 to 15 kb, and larger than 15 kb, respectively. The ExpandTM 20 kbPLUS PCR System from Boehringer Mannheim is an optimized polymerase and buffer mixture specifically optimized for the amplification of extra-long pieces of DNA. The Expand 20 kbPLUS PCR System is capable of producing fragments up to 47 kb in length. The AdvantageTM cDNA Polymerase mixes and kits from CLONTECH are designed for long and accurate amplification of PCR products. AdvantageÔ Mix combines KlenTaq-1 polymerase with a minor amount of proofreading polymerase and a novel antibody called TaqStartTM that provides an automatic hotstart. Optimized mixes increase the performance and reliability of PCR under a variety of conditions. These mixes are also ideal for robust amplification of templates up to 10 kb with three times higher fidelity than Taq DNA polymerase. The AdvantageÔ Genomic Polymerase Mix is a special formulation of proofreading Tth DNA polymerase and TthStartTM antibody for automatic hot starts. Tth DNA polymerase is isolated from Thermus thermophilus HB8 and has a half-life of greater than 1 hour at 95°C. This mix is especially useful for PCR applications involving large targets and/or complex DNA mixtures, such as human genomic DNA. Also available from CLONTECH is the Advantage-HFTM PCR Kit, designed for high fidelity amplification of cDNA and genomic templates. Each Advantage-HFTM PCR Kit contains HF polymerase mix, reaction buffers, dNTP mix, and control template and primers. Sequence analysis of PCR products amplified with Advantage-HFTM Mix revealed a 20-fold improvement in fidelity over Taq DNA polymerase. Both genomic and cDNA high fidelity mixes are available. Distributed through Bio/Can Scientific in Canada and BioNexus Inc. and TetraLink International in the U.S. are DNAmp's AccuraseTM HF PCR Mix and CalypsoTM RT-PCR Mix. DNAmp Ltd. has implemented a hot-spring sampling program dedicated to discovering new thermophilic enzymes. Accurase is a blended, optimized mixture of two recombinant DNA polymerases. The nonproofing enzyme is from an Azorean hot-spring Thermus isolate, while the proofreading enzyme is from a deep sea Thermococcus isolate. Compared to Taq DNA polymerase, the combination of both polymerases allows amplification of longer length products coupled with improved product yield (PCR of up to 25kb lDNA and 12 kb human genomic DNA) and a three-fold increase in fidelity. Calypso is an optimized blend of the two DNA polymerases that make up Accurase and AMV reverse transcriptase (AMV RT). The combination of AMV RT and Accurase allows RT PCR of long products, improved product yield, a three-fold increase in fidelity, and increased sensitivity due to the efficiency of the three-enzyme system. Use of AMV RT and a DMSO-containing reaction buffer minimizes problems encountered with secondary structures in RNA. The eLONGaseTM Enzyme Mix from Life Technologies is an optimized mixture of Taq DNA polymerase and Pyrococcus species GB-D polymerase. A single, optimized buffer system allows amplification of simple and complex genomic targets. This mixture contains the enzyme mix and 5X buffer system and provides five times higher fidelity than Taq DNA polymerase. The mix is suitable for amplification of DNA fragments from 250 bp to 30 kb. The eLONGaseTM Amplification System includes the enzyme mix, buffer system, primers, control genomic DNA, distilled water, and silicone oil. This system is suitable for amplification of long cDNA templates in RT-PCR applications. PE Applied Biosystems' rTth DNA Polymerase, XL (Extra Long), was developed and formulated for use with the GeneAmpTM XL PCR Kit. This polymerase formulation, which combines enzymes from Thermus thermophilus and Thermococcus litoralis, is designed to amplify target DNA sequences from 5 to 40 kb using the GeneAmp PCR process. The GeneAmp XL PCR Kit creates optimal conditions for generation of extra-long PCR products from a wide range of templates with high reproducibility and specificity. The GeneAmp XL PCR Kit includes control reagents, buffer pack, and dNTPs. Also available from PE Applied Biosystems is the GeneAmp XL RNA PCR Kit. Sigma Sigma's AccuTaqÔ LA Polymerase Mix combines Sigma's Taq DNA polymerase with a small amount of proofreading enzyme, allowing amplification of genomic targets in excess of 20 kb. With less complex targets, such as bacterial and viral targets, amplifications of up to 40 kb have been achieved. The fidelity of AccuTaq is reportedly 6.5 times greater than that of Taq DNA polymerase alone. The KlenTaqÔ LA Polymerase Mix is a blend of KlenTaq-1 and a small amount of proofreading enzyme. KlenTaq-1 is a Klenow-fragment analog of Taq DNA polymerase that exhibits higher thermostability than Taq DNA polymerase. KlenTaq LA provides fidelity four times better than Taq and amplifications up to 10 kb for targets such as cDNA, bacterial DNA, and viral DNA. Stratagene provides a variety of high-fidelity PCR systems for a broad range of applications. Taq ExtenderTM PCR Aditive is designed to improve the reliability and yield of standard Taq-based PCR amplifications. Capable of handling difficult templates, Taq Extender can increase fidelity and yield of PCR products up to 35 kb in length. Users add the Taq Extender PCR additive to amplification reactions in an amount equal to that of Taq DNA polymerase and replace standard 10X Taq buffer with 10X Taq Extender buffer. The TaqPlus® Long PCR System is an optimized mixture of cloned Pfu and Taq2000TM DNA polymerases. By substituting standard Taq polymerase with the TaqPlus Long PCR System, high yields with greater fidelity can be achieved; synthesis of targets up to 35 kb in length from a variety of templates can be achieved. This system is used in place of Taq DNA polymerase in standard amplification reactions, and high yields of PCR products can be achieved with extension times as short as 30 seconds per kb per cycle. Two optimized buffers formulated for the length of template to be amplified are included with the system. Lastly, Stratagene supplies the TaqPlus® Precision PCR System, optimized for high replication accuracy. Utilizing a blend of cloned Pfu and Taq2000 polymerases, the TaqPlus Precision PCR System is ideally suited for PCR applications requiring high product yield, low error rate, and shorter PCR extension times than possible using Pfu DNA polymerase alone. Using 1 minute per kb per cycle extension times, the TaqPlus Precision PCR System can successfully amplify plasmid and l DNA templates up to 15 kb in length and single-copy genomic DNA templates up to 10 kb in length. TaKaRa Ex TaqTM DNA Polymerase is distributed in the United States by Oncor and PanVera Corporation. In routine PCR applications, Ex TaqTM Polymerase and Ex TaqÔ Buffer System will give higher yields and lower mutation rates (approximately four times lower as determined by the Kunkel method) than standard Taq polymerase. The system also allows amplification of longer products than Taq, with approximately 20 kb lengths possible from genomic DNA and up to 30 kb possible on l DNA. PCR products generated with Ex TaqTM contain a 3'-A overhang and are suitable for cloning into T vectors. PanVera Corporation also sells Premix Ex TaqTM, which is a mixture of TaKaRa Ex TaqTM DNA Polymerase, buffer, and dNTPs. Available through PanVera, TaKaRa LA TaqÔ DNA Polymerase is designed to overcome problems associated with efficient amplification of genomic DNA greater than 20 kb in length. PCR products generated from LA TaqTM also contain a 3'-A overhang and are suitable for cloning into T vectors. Both Oncor and PanVera distribute the TaKaRa LA PCR Kit Version 2 and the TaKaRa RNA LA PCR Kit Version 1.1. The TaKaRa RNA LA PCR Kit Version 1.1 is designed to achieve long and accurate PCR following reverse transcription. This kit allows reverse transcription of RNA to cDNA using AMV (Avian Myeloblastosis Virus) Reverse Transcriptase and subsequent amplification of long cDNA all in a single tube. The TaKaRa LA PCR Kit Version 2 includes TaKaRa LA TaqÔ and buffers for the amplification of long DNA fragments. *The PCR process is covered by patents owned by Hoffman-LaRoche, Inc. and F. Hoffman-LaRoche Ltd. The author can be reached online at sbeck@the-scientist.com. Chart 1, Chart 2

High Fidelity PCR: Enhancing the Accuracy of DNA Amplification

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