pfuturbo DNA polymerase is the superior choice for robust high-fidelity PCR amplification
Holly Hogrefe Frances Bai Janice Cline
In this update, we compare the PCR performances of pfuturbo DNA polymerase *, and Taq DNA polymerase. PCR product yields were higher (>3-kb targets) or comparable (0- to 2-kb targets) with PfuTurbo DNA polymerase using one-minute-per-kb extension times. The error rate of PfuTurbo DNA polymerase, like that of Pfu DNA polymerase, was significantly lower than the error rate of Taq DNA polymerase. Thus, PfuTurbo DNA polymerase is superior for both routine and high-performance, high-fidelity PCR applications.
Stratagene recently introduced PfuTurbo DNA polymerase for robust, high-fidelity PCR amplification.1 PfuTurbo DNA polymerase is a special formulation of cloned Pfu DNA polymerase and a newly discovered PCR-enhancing factor that dramatically improves PCR product yields without altering DNA replication fidelity. This novel PCR-enhancing factor was isolated from Pyrococcus sp. and lacks both DNA polymerase and exonuclease activities.**
PfuTurbo DNA polymerase has been shown to be ideally suited for amplifying genomic DNA targets up to 10 kb, and cloned targets up to 15 kb, with the highest replication fidelity possible. Initial studies revealed that PCR product yields are dramatically higher with PfuTurbo DNA polymerase than with Pfu DNA polymerase (alone) or other commercially available proofreading PCR enzymes.1 Moreover, high-fidelity amplification is achieved using one-minute-per-kb extension times with vector targets up to 10 kb and genomic targets up to 6 kb, rather than the two-minute-per-kb extension times recommended for Pfu DNA polymerase.2
In this update, additional PCR performance comparisons are presented. PfuTurbo DNA polymerase and Taq DNA polymerase are compared with respect to PCR product yield and amplification of complex targets.
Figure 1 shows results from amplifying a series of primer-template systems using PfuTurbo DNA polymerase or Taq DNA polymerase. The a1-antitrypsin gene was amplified from human genomic DNA, using a series of 3 staggered primers to determine the maximum target length that can be amplified by each enzyme (lanes 1-4, PfuTurbo DNA polymerase; lanes 7-10, Taq DNA polymerase). Both enzymes successfully amplified the 0.9- and 2.0-kb targets in high yield. PfuTurbo DNA polymerase amplified the longer 3.9- and 5.2-kb targets in significantly higher yield than Taq DNA polymerase, and little smearing was observed. In contrast, significant smearing was evident in amplifications of 3.9-kb targets with Taq DNA polymerase.
Superior performance of PfuTurbo DNA polymerase was also observed when additional primer-template systems were tested. For example, PfuTurbo DNA polymerase successfully amplified an 8-kb target from rodent genomic DNA (lane 5) and a 14-kb target from lambda phage DNA (lane 6). In contrast, Taq DNA polymerase had difficulty amplifying these relatively long amplicons (lanes 11 and 12).
The error rates of a number of DNA polymerases have been measured previously in a PCR-based forward mutation assay, which utilizes the lacIOlacZa target gene.3 In these studies, Pfu DNA polymerase exhibited an average error rate that was two-fold lower than that of Vent and Deep Vent DNA polymerases,3 three- to six-fold lower than those of DNA polymerase mixtures,3 -6 and six-fold lower than that of Taq DNA polymerase.3 PfuTurbo DNA polymerase exhibits the same high-fidelity benefits as Pfu DNA polymerase.1
In side-by-side comparisons, we determined that PfuTurbo DNA polymerase amplified longer complex targets (>3 kb) in higher yield than Taq DNA polymerase. High-fidelity amplification of genomic targets up to 10 kb, and cloned targets up to 15 kb, were previously achieved with PfuTurbo DNA polymerase.1 Although PfuTurbo and Taq DNA polymerases can amplify relatively easy targets (0 to 2 kb) with comparable yields, higher replication fidelity makes PfuTurbo DNA polymerase the better choice for amplifying DNA targets to be cloned, expressed, and/or sequenced. Thus, adding a novel PCR-enhancing factor makes PfuTurbo DNA polymerase superior to Taq DNA polymerase for both routine and high-performance PCR applications.
PCR amplifications were conducted with PfuTurbo DNA polymerase or Taq DNA polymerase in their recommended buffers, using 200 M each dNTP, 100 to 300 ng of genomic DNA or 15 ng of lambda phage DNA, and 100 ng of each oligonucleotide primer per 50 l reaction. PCR reactions were conducted in Stratagenes RoboCycler Gradient 96 temperature cycler fitted with a Hot Top assembly, using 200 l thin-walled PCR tubes. The temperature cycling parameters for all targets incorporated the following: one cycle at 95C for one minute, followed by 30 cycles at 95C for one minute (denaturation); 58 to 64C for one minute (annealing); 72C for one minute per kb of target amplified (extension); and one final extension cycle of 72C for 10 minutes. The amplified reaction products were electrophoresed on a 1% agarose/1X TBE gel, stained with ethidium bromide, and imaged using the Eagle Eye II Still Video System. Lanes labe led M contain the kb DNA markers.
Hogrefe, H. et al. (97) Strategies 10: 93-96.
Nielson, K.B., Costa, G.L., and Braman, J. (1996) Strategies 9: 24-25.
Cline, J., Braman, J.C., and Hogrefe, H. (1996) Nucleic Acids Res. 24: 3546-3551.
Nielson, K., Scott, B., Bauer, J.C., and Kretz, K. (1994) Strategies 7: 64.
Nielson, K.B., et al. (1997) Strategies 10: 29-32.
Guide to Pfu DNA Polymerase (1996) Stratagene, La Jolla, California.
Purchase of these enzymes is accompanied by a license to
use them in the Plymerase Chain Reaction (PCR) process in conjunction with an
Authorized Thermal Cycler. Stratagene's PCR products are sold under license
arrangements with Roche Molecular Systems Inc., F. Hoffmann-La Roche and Applied Biosystems.
* U.S. Patent No. 5,545,522 and patents pending.
** Patents pending.