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Novel PCR Enhancing Factor Improves Performance of Pfu DNA Polymerase

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.

Conclusions

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.

Methods

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.

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