PCR performance of the highly thermostable proof-reading B-type DNA polymerase from Pyrococcus abyssi

• Published in: FEMS microbiology letters 2002-11, Vol.217 (1), p.89-94
• Main Authors: Dietrich, Jacques, Schmitt, Philippe, Zieger, Montserrat, Preve, Brigitte, Rolland, Jean-Luc, Chaabihi, Hassan, Gueguen, Yannick
• Format: Article
• Language: English
• Subjects: Archaea; Bacteriology; Biological and medical sciences; DNA polymerase; DNA Polymerase I - chemistry; DNA Polymerase I - genetics; DNA Polymerase I - metabolism; Enzyme Stability; Exonucleases - chemistry; Exonucleases - genetics; Exonucleases - metabolism; Fundamental and applied biological sciences. Psychology; Ions - metabolism; Kinetics; Magnesium Sulfate - metabolism; Metabolism. Enzymes; Microbiology; Mutation; Polymerase chain reaction; Polymerase Chain Reaction - instrumentation; Polymerase Chain Reaction - methods; Protein Conformation; Pyrococcus - enzymology; Pyrococcus - genetics; Pyrococcus - metabolism; Pyrococcus abyssi; Sensitivity and Specificity; Temperature
• ISSN: 0378-1097; 1574-6968
• DOI: 10.1016/S0378-1097(02)01037-6

DNA polymerase from the archaeon Pyrococcus abyssi strain Orsay was expressed in Escherichia coli. The recombinant DNA polymerase (Pab) was purified to homogeneity by heat treatment followed by 5 steps of chromatography and characterized for PCR applications. Buffer optimization experiments indicated that Pab PCR performance and fidelity parameters were highest in the presence of 20 mM Tris–HCl, pH 9.0, 1.5 mM MgSO 4, 25 mM KCl, 10 mM (NH 4) 2SO 4 and 40 μM of each dNTP. Under these conditions, the error rate was 0.66.10 −6 mutations/nucleotide/duplication. Pab DNA polymerase, having a half life of 5 h at 100°C, was demonstrated to be highly thermostable in PCR conditions compared to commercial Taq and Pfu DNA polymerases. These characteristics enable Pab to be one of the most efficient thermostable DNA polymerases described, exhibiting very high accuracy compared to other available commercial DNA polymerases and robust thermostable activity. This new DNA polymerase is currently on the market under the name Isis DNA Polymerase™ (Qbiogene Molecular Biology).