Improved PCR performance and fidelity of double mutant Neq A523R/N540R DNA polymerase

• Nanoarchaeum eqitans (Neq) DNA polymerase has poor fidelity and PCR efficiency.
• To improve fidelity and PCR efficiency, a double mutant was created.
• The fidelity of Neq A523R/N540R DNA polymerase was approximately 9.1-fold higher than that of wild-type Neq.
• Neq A523R/N540R DNA polymerase amplified a long 12-kb PCR product in 3 min.

We previously reported that Neq A523R DNA polymerase is more efficient in PCR than wild-type Neq DNA polymerase, and amplifies products more rapidly. Neq A523R DNA polymerase also amplifies templates more rapidly than Pfu DNA polymerase, but has a lower fidelity than Pfu DNA polymerase. To improve product yield and the fidelity of amplification simultaneously, we constructed and characterized the double mutant Neq A523R/N540R. The yield of PCR products was greater for Neq A523R/N540R DNA polymerase than wild-type and other mutant DNA polymerases, and the Neq double mutant catalyzed amplification of a 12-kb PCR product from a lambda template with an extension time of 3 min. The PCR error rate of Neq A523R/N540R DNA polymerase (6.3 × 10−5) was roughly similar to that of Pfu DNA polymerase (4.8 × 10−5), but much lower than those of wild-type Neq DNA polymerase (57.2 × 10−5), Neq A523R DNA polymerase (13.1 × 10−5), and Neq N540R DNA polymerase (37.7 × 10−5). These results indicated that A523R and N540R mutations of Neq DNA polymerase had synergistic effects on its fidelity.