We previously reported that Neq A523RDNA 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 x 10(-5)) was roughly similar to that of Pfu DNA polymerase (4.8 x 10(-5)), but much lower than those of wild-type Neq DNA polymerase (57.2 x 10(-5)), Neq A523R DNA polymerase (13.1 x 10(-5)), and Neq N540R DNA polymerase (37.7 x 10(-5)). These results indicated that A523R and N540R mutations of Neq DNA polymerase had synergistic effects on its fidelity. (C) 2015 Elsevier Inc. All rights reserved.