Characterization of DNA polymerase from Pyrococcus sp. strain KOD1 and its application to PCR
| Autor: | Hiroaki Inoue, Hirofumi Kakihara, Masahiro Takagi, Masao Kitabayashi, Bunsei Tsuruga-shi Kawakami, Tadayuki Imanaka, Masanori Oka, Motomu Nishioka |
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| Rok vydání: | 1997 |
| Předmět: |
Pyrococcus
DNA clamp Ecology biology Pfu DNA polymerase DNA polymerase Inverse polymerase chain reaction DNA polymerase II Molecular Sequence Data DNA-Directed DNA Polymerase Polymerase Chain Reaction Applied Microbiology and Biotechnology Molecular biology Biochemistry biology.protein Amino Acid Sequence Cloning Molecular DNA polymerase I Polymerase Hot start PCR Research Article Food Science Biotechnology |
| Zdroj: | Applied and Environmental Microbiology. 63:4504-4510 |
| ISSN: | 1098-5336 0099-2240 |
| DOI: | 10.1128/aem.63.11.4504-4510.1997 |
| Popis: | The DNA polymerase gene from the archaeon Pyrococcus sp. strain KOD1 (KOD DNA polymerase) contains a long open reading frame of 5,013 bases that encodes 1,671 amino acid residues (GenBank accession no. D29671). Similarity analysis revealed that the DNA polymerase contained a putative 3'-5' exonuclease activity and two in-frame intervening sequences of 1,080 bp (360 amino acids; KOD pol intein-1) and 1,611 bp (537 amino acids; KOD pol intein-2), which are located in the middle of regions conserved among eukaryotic and archaeal alpha-like DNA polymerases. The mature form of the DNA polymerase gene was expressed in Escherichia coli, and the recombinant enzyme was purified and characterized. 3'-5' exonuclease activity was confirmed, and although KOD DNA polymerase's optimum temperature (75 degrees C) and mutation frequency (3.5 x 10(-3)) were similar to those of a DNA polymerase from Pyrococcus furiosus (Pfu DNA polymerase), the KOD DNA polymerase exhibited an extension rate (100 to 130 nucleotides/s) 5 times higher and a processivity (persistence of sequential nucleotide polymerization) 10 to 15 times higher than those of Pfu DNA polymerase. These characteristics enabled the KOD DNA polymerase to perform a more accurate PCR in a shorter reaction time. |
| Databáze: | OpenAIRE |
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