Discovery Leads To Improved PCR Performance

Discovery Leads to Improved PCR Performance

• Greatly improves PCR performance of Pfu DNA polymerase
• Novel mechanism eliminates a potent PCR inhibitor
• Enhances yield, length, and overall PCR performance of blend formulations

Until the discovery of the ArchaeMaxx polymerase-enhancing factor, Pfu DNA polymerase generally required longer extension times and more stringent protocols than Taq DNA polymerase.

Stratagene’s unique ArchaeMaxx™ polymerase-enhancing factor* eliminates a PCR inhibitor and promotes shorter extension times, higher yield and greater target length. The ArchaeMaxx polymerase-enhancing factor is a key ingredient in Stratagene’s enzyme formulations, overcoming the limitations of proofreading DNA polymerases and making high-fidelity PCR routine in your lab.

Eliminates dUTP Poisoning

Stratagene scientists discovered the novel ArchaeMaxx™ polymerase-enhancing factor*, which they purified from the hyperthermophilic archae, Pyrococcus furiosus (Pfu).1 The ArchaeMaxx factor improves the yield of products amplified with Pfu DNA polymerase** by overcoming dUTP poisoning, which is caused by dUTP accumulation during PCR through dCTP deamination. This dUTP poisoning inhibits Pfu and many other archaeal proofreading polymerases, such as Vent® and Deep Vent® DNA polymerases, limiting their efficiency.1 The ArchaeMaxx factor functions as a dUTPase, converting poisonous dUTP to harmless dUMP and inorganic pyrophosphate.

Higher Yields, Longer Targets

Stratagene added the ArchaeMaxx factor to cloned Pfu DNA polymerase to create PfuTurbo® DNA polymerase‡, which retains the highest fidelity characteristics of Pfu polymerase.2,3 Compared to Pfu alone, PfuTurbo DNA polymerase produces higher product yields and amplifies a broader range of targets including genomic targets up to 19kb in length. Moreover, side-by-side comparisons with Taq and competitors' proofreading enzymes (Vent®, Deep Vent®, KOD, Pwo, Platinum® Pfx and native Pfu) have clearly shown that PfuTurbo produces superior product yields over a broad range of target lengths.1, 4-6
Pfu and PfuTurbo DNA polymerases exhibit the lowest error rate of any thermostable DNA polymerase characterized to date.2,3 A survey of published literature shows that the average error rate of Pfu polymerase is 2- to 60-fold lower than that of other proofreading enzymes and 6- to 100-fold lower than that of Taq, depending upon the assay methods employed.1 Recent comparisons reveal that PfuTurbo DNA polymerase is 2.7- and 1.7-fold more accurate compared to Pfx and Tgo DNA polymerases, respectively.3, unpublished PfuTurbo DNA polymerase provides both superior performance and accuracy compared to other proofreading enzymes.

Enhances Performance of Pfu Blends

The ArchaeMaxx factor also enhances the performance of DNA polymerase blends containing Pfu DNA polymerase. In the absence of dUTP poisoning, Pfu can make a greater contribution to blend performance (fidelity, yield), especially in later PCR cycles where high levels of dUTP would otherwise inhibit the proofreading component. Stratagene's unique blends, Herculase® Enhanced DNA polymerase§ and EXL™ DNA polymerase§, contain the ArchaeMaxx factor and a substantially higher proportion of proofreading activity compared to other commercial blends, which are composed predominantly of error-prone Taq DNA polymerase. In addition to superior accuracy, our novel Pfu-based blends provide higher yield and amplification of longer targets compared to competitors' blends, which are significantly compromised by dUTP poisoning in the absence of the ArchaeMaxx factor.

Herculase® DNA Polymerase for Difficult Targets

Herculase® Enhanced DNA polymerase has the highest proportion of proofreading activity, and hence the greatest accuracy, of any DNA polymerase blend on the market.7 Only Pfu and PfuTurbo DNA polymerases offer greater fidelity than Herculase DNA polymerase. The ArchaeMaxx factor1 included in the Herculase formulation extends its target-length capability and improves amplifications of complex and/or GC-rich
templates.7,8

EXL™ DNA Polymerase for Long Targets

EXL™ DNA polymerase owes its ability to amplify extremely long PCR templates to the ArchaeMaxx factor. Secondary structures and repetitive sequences are common within long DNA templates and can interfere with polymerization. The ArchaeMaxx factor enhances the target-length capability of EXL polymerase, while reaction buffer components and DMSO promote template denaturation. EXL DNA polymerase amplifies genomic targets up to 37 kb and lambda targets up to 50 kb in length. Because EXL DNA polymerase consists predominantly of Pfu, its accuracy is significantly higher than that of other vendors’ long PCR products.

The ArchaeMaxx™ Factor Advantage

Stratagene’s ArchaeMaxx factor vastly improves the PCR performance of Pfu DNA polymerase. The ArchaeMaxx factor is a key component of PfuTurbo DNA polymerase, as well as Stratagene's unique Pfu-based enzyme formulations—Herculase and EXL DNA polymerases. These are a new generation of enzymes, ideal for any PCR challenge.

REFERENCES

1. Hogrefe, H., et al. (2002) Proc Natl, Acad, Sci, USA 99: 596-601.
2. Cline, J., Braman, J.C., and Hogrefe, H. (1996) Nucleic Acids Res. 24: 3546-3551.
3. Borns, M. and Hogrefe, H. (2000) Strategies 13: 27-30.
4. Borns, M. and Hogrefe, H. (1997) Strategies 10: 93-96.
5. Hogrefe, H. and Cline, J. (1998) Strategies 11: 36-37.
6. Borns, M. and Hogrefe, H. (1999) Strategies 12: 40-42.
7. Borns, M. and Hogrefe, H. (2000) Strategies 13: 1-3.
8. Borns, M. and Hogrefe, H. (2000) Strategies 13: 76-79.

* U.S. Patent No. 6,379,553 and 6,333,165 and 6,183,997 and patents pending
** U.S. Patent Nos. 5,948,663, 5,866,395 and 5,545,552 and patents pending
‡ U.S. Patent Nos. 6,183,997 and 5,948,663 and 5,866,395 and 5,545,552 and patents pending
‡‡ U.S. Patent Nos. 6,379,553 and 6,333,165 and 6,183,997 and 5,948,663 and 5,866,395 and 5,545,552 and patents pending
§ U.S. Patent Nos. 6,379,553 and 6,333,165 and 6,183,997 and 5,948,663 and 5,866,395 and 5,556,772 and 5,545,552 and patents pending