pfuturbo DNA polymerase for robust, high-fidelity PCR amplification
Holly Hogrefe Brad Scott Kirk Nielson Valerie
Hedden Connie Hansen Janice Cline Frances Bai Jeff Amberg Ronda
Allen Mark Madden
Stratagene announces the release of pfuturbo DNA polymerase,*, an improved version of Pfu DNA polymerase for high-fidelity PCR. PfuTurbo DNA polymerase is a blend of cloned Pfu DNA polymerase and a novel thermostable factor that enhances PCR product yields without altering the fidelity of DNA replication. PfuTurbo DNA polymerase can be used to amplify complex genomic DNA targets up to 10 kb in length and vector targets up to 15 kb in length. PfuTurbo DNA polymerase amplifies complex targets in higher yield than Taq DNA polymerase or other commercially available proofreading PCR enzymes. The error rate of PfuTurbo DNA polymerase is equal to that of Pfu DNA polymerase and is significantly lower than the error rates of other proofreading enzymes, DNA polymerase mixtures and Taq DNA polymerase. The enhanced performance of PfuTurbo DNA polymerase allows the use of shorter extension times, fewer PCR cycles and lower concentrations of DNA templates than are required for Pfu DNA polymerase. These attributes make PfuTurbo DNA polymerase ideally suited for both high-fidelity and higher-throughput PCR applications.
Pfu DNA polymerase exhibits the lowest error rate of any thermostable DNA polymerase characterized to date,1 making it the enzyme of choice for high-fidelity PCR applications. Pfu DNA polymerase can amplify high yields of genomic DNA targets up to 4 kb in length using PCR extension times of 2 minute per kb of target.2 Longer genomic targets may be successfully amplified with Pfu DNA polymerase using higher amounts of polymerase, longer extension times or modified PCR reaction buf the other hand, is recommended for amplifying genomic targets up to 18.5 kb and vector targets up to 35 kb when amplicon length and product yield are the primary considerations.
The discovery of a novel thermostable PCR-enhancing factor has led to the development of PfuTurbo DNA polymerase, an improved version of cloned Pfu DNA polymerase. PfuTurbo DNA polymerase exhibits the same high fidelity as Pfu DNA polymerase, making it ideally suited for amplifying DNA targets that are to be cloned, expressed and/or sequenced. Moreover, PfuTurbo DNA polymerase generates significantly higher yields of PCR product than can be obtained with other single PCR enzymes, and the enhanced performance of PfuTurbo DNA polymerase allows PCR amplifications to be conducted in a shorter time. The limitations in product yields, target length6 and PCR extension time2 encountered with Pfu DNA polymerase or other proofreading PCR enzymes are largely overcome by using PfuTurbo DNA polymerase.
PCR amplifications were aliquoted into 200-l, thin-walled PCR tubes and cycled using a RoboCycler Gradient 96 temperature cycler# fitted with a Hot Top Assembly. The amplified reaction products were electrophoresed through 1% agarose gels in 1X TBE for 60 minutes at 75 V, stained with ethidium bromide and imaged using he Eagle Eye II still video system.
In Figure 1, the temperature cycling parameters for the 0.9-kb target were as follows: 1 cycle at 95C for 1 minute; 30 cycles of 95C for 1 minute (denaturation), 58C for 1 minute (annealing) and 72C for 1 minute (extension) and a final extension cycle of 72C for 10 minutes. For the 5.2-kb target, an annealing temp- erature of 60C and an extension time of 6 minutes and 36 seconds were used.
In figur e 3, the temperature cycling parameters for the 1.9-kb target were as follows: 1 cycle at 95C for 1 minute; 20-30 cycles at 95C for 1 minute (denaturation), 50C for 1 minute (annealing) and 72C for 1 minute and 54 seconds (extension) and a final extension cycle of 72C for 10 minutes.
Cline, J., Braman, J.C., and Hogrefe, H.H. (1996) Nucleic Acids Res. 24: 3546-3551.
Nielson, K.B., Costa, G.L., and Braman, J. (1996) Strategies 9: 24-25.
Nielson, K.B., Braman, J., and Kretz, K. (1995) Strategies 8: 26.
Nielson, K.B., et al. (1994) Strategies 7: 64.
Nielson, K.B., Cline, J., Bai, F., McMullan, D., McGowen, B., and Hogrefe, H. (1997) Strategies 10: 29-32.
Barnes, W.M. (1994) Proc. Natl. Acad. Sci. USA 91: 2216-2220.
Guide to Pfu DNA Polymerase. (1996) Stratagene, La Jolla, California.
* Patent pending
** US Patent No. 5,556,772 and patents pending
The recent development of DNA polymerase mixtures has largely overcome the problem of reduced yields when amplifying longer targets (e.g., >4-kb genomic targets) with single DNA polymerases. For example, Stratagene has developed two DNA polymerase mixtures containing Taq and Pfu DNA polymerases: TaqPlus Long PCR system**,, for amplifying targets up to 35 kb in length4 and TaqPlus Precision PCR system**, for high-fidelity amplification of genomic targets up to 10 kb and cloned targets up to 15 kb.5 DNA polymerase mixtures typically consist of a blend of a nonproofreading DNA polymerase (e.g., Taq DNA polymerase) and a proofreading DNA polymerase (e.g., Pfu DNA polymerase). The addition of the proofreading enzyme is thought to enhance amplification of longer targets by repairing mispaired primer termini that Taq DNA polymerase cannot extend.6 However, because DNA polymerase mixtures are composed of a significant proportion of a nonproofreading DNA polymerase, error rates for mixtures are closer to that of Taq DNA polymerase and approximately 3- to 6-fold higher than the error rate of Pfu DNA polymerase.1,4,5
In a continuing effort to improve high-fidelity PCR performance, Stratagene has discovered a novel thermo- stable factor that enhances the performance of Pfu DNA polymerase in PCR amplifications without altering the enzymes high replication fidelity. This PCR enhancing factor was isolated from Pyrococcus and shown to lack both DNA polymerase and exonuclease activities (data not shown). PfuTurbo DNA polymerase is an optimized blend of cloned Pfu DNA polymerase and this PCR enhancing factor. PfuTurbo DNA polymerase has been shown to be ideally suited for both high-fidelity and higher-throughput PCR applications. In this article, we demonstrate that PCR product yields and replication fidelity are significantly higher with PfuTurbo DNA polymerase than with other PCR enzymes.
Figure 1 shows the results of amplifying 0.9-kb and 5.2-kb targets from human genomic DNA using PfuTurbo DNA polymerase and several other PCR enzymes. For these PCR amplifications, extension times of 1 minute per kb of target and varying amounts of each polymerase (from 0.5 to 5 U) were used. All of the enzymes tested successfully amplified the 0.9-kb target. The reaction products shown in lanes 1-7 were generated using 2 to 2.5 U of each DNA polymerase, with the exception that KOD DNA polymerase required 1.25 U to obtain sufficient yields with minimal smearing. Higher yields of PCR products were obtained when 5 U of Pfu, Pwo or Taq DNA polymerase was used (data not shown). Smearing or reduced yield was observed when higher amounts of KOD, VentR or Deep VentR DNA polymerases were used (data not shown).
The 5.2-kb target was also amplified with varying amounts (from 0.5 to 5 U) of each DNA polymerase. The reactions shown in Figure 1 (lanes 8-14) are those that exhibited the highest yields of PCR products (for PfuTurbo and Taq DNA polymerases) or those that were carried out using the highest possible number of polymerase units before excessive smearing was observed. Only PfuTurbo (lane 8) and Taq (lane 14) DNA polymerases successfully amplified the 5.2-kb target, and PCR product yields were significantly higher with PfuTurbo DNA polymerase. None of the other DNA polymerases could amplify the 5.2-kb genomic target despite the use of up to 5 U of enzyme or longer extension times (data not shown).
Lack of amplification of the 5.2-kb target by Pfu, Pwo, VentR, Deep VentR and KOD DNA polymerases was unrelated to the particular primer-template system used. None of these enzymes, including Taq DNA polymerase, could amplify a 6.6-kb target using a different primer set and mouse genomic DNA as template (data not shown). PfuTurbo DNA polymerase, however, was able to amplify this target.
PfuTurbo DNA polymerase has been used to amplify a variety of DNA targets, ranging in complexity from plasmid and lambda phage DNA templates to complex, single-copy amplicons from genomic DNA. The data in figure 2 demonstrate that PfuTurbo DNA polymerase synthesizes higher yields of PCR product than Pfu DNA polymerase alone, regardless of the primer-template system used. PfuTurbo DNA polymerase successfully amplified complex genomic targets of 0.9 to 5.2 kb in length using 1-minute-per-kb extension times (lanes 1-6) and genomic targets of 6.1 to 10 kb in length using 2-minute-per-kb extension times (lanes 7-12). Under the same conditions, Pfu DNA polymerase successfully amplified only the 0.9- and 1.9-kb targets (lanes 2 and 4), and product yields were significantly lower than those obtained using PfuTurbo DNA polymerase (lanes 1 and 3). To achieve higher yields of the 0.9- and 1.9-kb targets with Pfu DNA polymerase, greater amounts of polymerase or longer extension times (2 minutes per kb of target) must be used (data not shown).
PfuTurbo DNA pol ymerase was shown to successfully amplify vector targets up to 10 kb using 1-minute-per-kb extension times (lanes 13, 15 and 17) and a 14-kb lambda DNA target using 2-minute-per-kb extension times (lane 19). When relatively short vector targets were amplified, the most dramatic differences in PCR product yield between PfuTurbo and Pfu DNA polymerases were observed when limiting amounts of DNA template were used. For example, PfuTurbo DNA polymerase produced significantly more 1.9-kb PCR product when amplification was performed for 30 cycles using 1 pg of plasmid DNA (lane 13). PfuTurbo and Pfu DNA polymerases generated comparable yields of product when 10 pg of plasmid DNA was used (data not shown). Dramatic differences in yield between PfuTurbo and Pfu DNA polymerases were also observed when amplifying longer vector targets. For example, Pfu DNA polymerase amplified an 8-kb target from lambda phage DNA (lane 16), but not a 14-kb lambda DNA target (lane 20) nor a 10-kb, 40-copy amplicon from transgenic mouse genomic DNA (lane 18). In contrast, PfuTurbo DNA polymerase synthesized high yields of all of the longer vector targets (lanes 15, 17 and 19).
In total, the data demonstrate that PCR product yields generated with PfuTurbo DNA polymerase are higher than those generated with Pfu DNA polymerase (figure 2) or with other single PCR enzymes (Figure 1). PfuTurbo DNA polymerase can be used routinely in all PCR applications that utilize Taq DNA polymerase or a proof- reading PCR enzyme (e.g., Pfu, VentR, Deep VentR, Pwo, KOD and Ultma DNA polymerases). PfuTurbo DNA polymerase is particularly suited for amplifying longer (>2 kb) or complex genomic DNA targets, which are not readily amplified by single D NA polymerases using relatively short extension times and 25 to 35 PCR cycles.
The error rates of a number of DNA polymerases have been measured previously in a PCR-based forward mutation assay that utilizes the lacIOlacZa target gene.1 In these studies, Pfu DNA polymerase was found to exhibit an average error rate that was 2-fold lower than that of VentR and Deep VentR DNA polymerases,1 3- to 6-fold lower than those of DNA polymerase mixtures1,4,5,7 and 6-fold lower than that of Taq DNA polymerase.1
The effect of the PCR enhancing factor on the replication fidelity of Pfu DNA polymerase was investigated using the lacI-based PCR assay. The lacI mutational target gene was amplified from varying amounts of plasmid DNA and from a single-copy transgene in rodent genomic DNA, and error rates (mutation frequency per base pair per duplication) were found to be independent of the number of target duplications (data not shown). No significant difference in error rate was observed between PfuTurbo and Pfu DNA polymerases under several different PCR amplification conditions (table 1). In addition, the PCR enhancing factor did not alter the fidelity of Pfu DNA polymerase when added to PCR amplifications at concentrations ranging from 0.2- to 100-fold of the level present in PfuTurbo DNA polymerase (data not shown).
Performance Comparisons of Stratagenes PCR Enzymes
Extension time required
Recommended PCR target length
PfuTurbo DNA polymerase
Pfu DNA polymerase
TaqPlus Precision System
TaqPlus Long System
Taq DNA Polymerase
*Error rate equals mutation frequency per base pair per duplication.1
TaqPlus Long System
Taq DNA Polymerase
*Error rate equals mutation frequency per base pair per duplication.1
In addition to achieving higher PCR product yields, PfuTurbo DNA polymerase allows the use of shorter extension times, reduced cycle numbers or lower DNA template concentrations than are required in standard PCR protocols based on Pfu DNA polymerase. The data in Figure 1 and figure 2 demonstrate that PfuTurbo DNA polymerase synthesizes moderate-to-high yields of PCR product using extension times of 1 minute per kb of target, as compared to 2 minutes per kb of target, which is usually required for Pfu DNA polymerase.2 The utility of PfuTurbo DNA polymerase in PCR applications requiring fewer cycle numbers or lower amounts of DNA template is demonstrated in figure 3. Similar amounts of a 1.9-kb PCR product were amplified from genomic DNA after 30 cycles with Pfu DNA polymerase and after 24 cycles with PfuTurbo DNA polymerase. When the DNA template concentration was varied, PfuTurbo DNA polymerase generated the same amount of PCR product as Pfu DNA polymerase using approximately 75% less genomic DNA. These results suggest that PfuTurbo DNA polymerase is useful for both high-fidelity and higher-throughput PCR applications.
The features of Stratagenes PCR enzymes are summarized (table 1). As described in this article, PfuTurbo DNA polymerase is a robust PCR enzyme that amplifies genomic DNA targets up to 10 kb in length and cloned targets up to 15 kb in length with the highest replication fidelity possible. In general, PfuTurbo DNA polymerase amplifies complex targets in higher yield than Taq DNA polymerase or other commercially available proofreading PCR enzymes. Moreover, high-fidelity amplification of vector targets up to 10 kb in length and genomic targets up to 6 kb in length can be achieved using 1-minute-per-kb extension times rather than the 2-minute-per-kb extension times suggested for Pfu DNA polymerase.2
Stratagene recommends the TaqPlus Precision or TaqPlus Long
PCR systems when amplifying genomic DNA targets greater than 6 kb with
1-minute-per-kb extension times. The TaqPlus Precision PCR system
exhibits an error rate that is significantly lower than that of Taq DNA
polymerase and is the enzyme mixture of choice when replication fidelity is the
primary consideration. The TaqPlus Long PCR System, on