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

Paper ID Volume ID Publish Year Pages File Format Full-Text
16915 42621 2016 8 PDF Available
Title
Improved PCR performance and fidelity of double mutant Neq A523R/N540R DNA polymerase
Abstract

•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.

Keywords
Nanoarchaeum equitans DNA polymerase (Neq); Neq A523R/N540R DNA polymerase; Polymerase chain reaction (PCR); PCR amplification; Fidelity
First Page Preview
Improved PCR performance and fidelity of double mutant Neq A523R/N540R DNA polymerase
Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 82, January 2016, Pages 197–204
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering