title: Improved PCR performance and fidelity of double mutant Neq A523R/N540R DNA polymerase.
abstract: 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.
authors.affiliation: Department of Biotechnology and Bioengineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea.
authors.forename: Hyewoo
authors.initials: H
authors.lastname: Ppyun
authors.affiliation: Department of Biotechnology and Bioengineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea.
authors.forename: Seung Hyun
authors.initials: SH
authors.lastname: Kim
authors.affiliation: Department of Biotechnology and Bioengineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea.
authors.forename: Man Hui
authors.initials: MH
authors.lastname: Youn
authors.affiliation: Department of Biotechnology and Bioengineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea.
authors.forename: Sung Suk
authors.initials: SS
authors.lastname: Cho
authors.affiliation: Department of Biological Science, Sungkyunkwan University, Suwon 440-746, Republic of Korea.
authors.forename: Kyung Min
authors.initials: KM
authors.lastname: Kwon
authors.affiliation: Department of Biotechnology and Bioengineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea.
authors.forename: Dae-Hyuk
authors.initials: DH
authors.lastname: Kweon
authors.affiliation: Department of Biotechnology and Bioengineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea. Electronic address: stkwon@skku.ac.kr.
authors.forename: Suk-Tae
authors.initials: ST
authors.lastname: Kwon
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D019843:Archaeal Proteins / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase / Q000235:genetics*; D005798:Genes, Bacterial; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D046588:Nanoarchaeota / Q000201:enzymology* / Q000235:genetics; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction / Q000379:methods; D011487:Protein Conformation; D015202:Protein Engineering; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
chemical_list: D019843:Archaeal Proteins; D004259:DNA-Directed DNA Polymerase
keywords: Fidelity; Nanoarchaeum equitans DNA polymerase (Neq); Neq A523R/N540R DNA polymerase; PCR amplification; Polymerase chain reaction (PCR)