EV-MOL-007 Enzovera EV5™ High-Fidelity DNA Polymerase (EV-MOL-007) is Enzovera’s proprietary high-fidelity PCR enzyme — a rationally engineered fusion of the Sso7d processivity-enhancing domain from the hyperthermophilic archaeon Sulfolobus solfataricus and the Pfu DNA Polymerase from Pyrococcus furiosus. The result is a thermostable, high-fidelity enzyme that combines the accuracy of wild-type Pfu with dramatically superior processivity, speed, and long-range amplification capability — approaching Q5-class performance at a significantly lower price point.

The Sso7d fusion advantage. Wild-type Pfu DNA Polymerase, while highly accurate, exhibits limited processivity (~5–10 kb per binding event under standard conditions), requires slow extension rates, and struggles with GC-rich or structurally complex templates. The Sso7d domain is a small (7 kDa), thermostable, double-stranded DNA-binding protein that binds non-specifically and non-covalently to duplex DNA with sub-nanomolar affinity, dramatically reducing polymerase dissociation from the template. When fused to the N-terminus of Pfu polymerase via a flexible Gly-Ser linker, Sso7d tethers the holoenzyme to the DNA, increasing processivity by more than 10-fold over wild-type Pfu. This translates directly to faster extension rates (1 kb/min standard; 15 sec/kb in fast protocols), amplification of targets up to 20 kb, and superior performance on difficult templates including high-GC sequences, long homopolymer runs, and secondary-structure-forming regions.

Fidelity. EV5™ retains the full 3′→5′ proofreading exonuclease of Pfu polymerase. Under standard PCR conditions, the error rate is less than 1 mutation per 10⁷ base pairs amplified — among the lowest available for a commercial PCR polymerase. This level of accuracy means that, statistically, a 1 kb PCR product amplified for 30 cycles will contain an error in fewer than 1 in 30 molecules, ensuring that the majority of colonies from a cloning experiment carry the intended sequence. For maximum insert accuracy, gel-purify amplicons and screen a minimum of three colonies by sequencing.

Blunt-end products and downstream compatibility. EV5™ generates blunt-ended products free of non-templated overhangs. Products are directly compatible with blunt-end cloning vectors, Gibson Assembly, Golden Gate Assembly, SLIC, and ligation-independent cloning workflows. A-tailing with Taq is required for TA cloning. EV5™ amplicons are fully compatible with sequencing, restriction digestion, and all standard downstream processing.

Recommended applications:

High-fidelity PCR for cloning, expression construct amplification, and library generation

Long-range PCR of targets from 5 to 20 kb

Amplification of GC-rich, AT-rich, or secondary-structure-forming templates (use supplied GC enhancer buffer)

NGS library preparation requiring faithful amplification of genomic or cDNA templates

Site-directed mutagenesis by overlap extension or whole-plasmid inverse PCR

Amplification of expression constructs for cloning into pET-28a and related vectors

Any application where Taq-level error rates are unacceptable

Supplied with: 5× EV5 HF Reaction Buffer (EV-MOL-007-RB) and GC Enhancer Buffer. The GC Enhancer contains betaine and DMSO and should be added (up to 1× final) when amplifying sequences with GC content above 65% or with confirmed secondary structure. A fast-cycling protocol (15 sec/kb extension at 72°C) is recommended for targets below 3 kb.