Enzovera Pfu DNA Polymerase (EV-MOL-006) is a recombinant, thermostable DNA polymerase produced in E. coli Rosetta(DE3) from the cloned gene of the hyperthermophilic archaeon Pyrococcus furiosus, an organism that thrives in deep-sea hydrothermal vents at temperatures approaching 100°C. Its intrinsic thermostability and vigorous 3′→5′ proofreading exonuclease make it the standard high-fidelity polymerase for applications where PCR accuracy is paramount.

High-fidelity proofreading. Pfu DNA Polymerase's 3′→5′ exonuclease activity is substantially more robust than that of many other thermostable polymerases, resulting in an error rate of approximately 1.3 × 10⁻⁶ mutations per base pair per cycle — approximately 18-fold lower than Taq DNA Polymerase and among the lowest of any PCR polymerase in routine use. When errors of sequence accuracy are unacceptable — as in expression construct cloning, mutagenesis validation, or any application where PCR product sequence is directly submitted for functional use — Pfu DNA Polymerase is the appropriate choice.

Blunt-end product generation. Unlike Taq DNA Polymerase, which adds a non-templated adenine residue to the 3′ terminus of PCR products (A-tailing), Pfu DNA Polymerase generates blunt-ended products with 3′-OH and 5′-phosphate termini. Its proofreading exonuclease removes any misincorporated 3′ nucleotides including any adventitious non-templated additions, ensuring precisely blunt ends on both strands. Pfu PCR products can be directly ligated into blunt-end cloning vectors or used in Gibson Assembly without prior end-blunting. Note: Pfu products are not suitable for TA cloning without A-tailing with Taq or a dedicated A-tailing kit.

Thermostability and PCR cycling. Pfu DNA Polymerase is optimally active at 72–75°C for extension and is fully stable through standard PCR cycling conditions (denaturation at 95–98°C). The enzyme can withstand prolonged high-temperature incubation without significant loss of activity, making it compatible with long-range PCR protocols. Extension rate is approximately 1 kb/min at 72°C under standard conditions, and routine amplification of targets up to 5 kb is highly reliable; targets up to 10 kb can be amplified with optimized buffer conditions and extended elongation times.

Recommended applications:

High-fidelity PCR for cloning applications where insert sequence accuracy is critical

Amplification of expression constructs prior to subcloning into pET or other expression vectors

Site-directed mutagenesis by overlap extension PCR

Blunt-end PCR product generation for ligation into blunt-cloning vectors or Gibson Assembly

PCR for diagnostic and genotyping applications requiring low error rates

Amplification of GC-rich or AT-rich targets (with optimized buffer and DMSO additive)

Note: Pfu DNA Polymerase is supplied with 5× Pfu HF Buffer (EV-MOL-006-RB) containing 7.5 mM MgCl₂ at 1×. For targets requiring optimization, MgCl₂ concentration may be adjusted from 1.5 to 4 mM final. The enzyme has lower processivity than Taq-based polymerases; for superior processivity with retained fidelity, consider EV5™ High-Fidelity DNA Polymerase (EV-MOL-007).

‍