PCR performance of the highly thermostable proof-reading B-type DNA polymerase from Pyrococcus abyssi.
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 MgSO4, 25 mM KCl, 10 mM (NH4)2SO4 and 40 microM 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 degrees 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).
Dietrich
Jacques
J
Ifremer, Direction des Ressources Vivantes, France.
Schmitt
Philippe
P
Zieger
Montserrat
M
Preve
Brigitte
B
Rolland
Jean-Luc
JL
Chaabihi
Hassan
H
Gueguen
Yannick
Y
D004256:DNA Polymerase I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D005092:Exonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007477:Ions / Q000378:metabolism; D007700:Kinetics; D008278:Magnesium Sulfate / Q000378:metabolism; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods; D011487:Protein Conformation; D019711:Pyrococcus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity; D013696:Temperature
D007477:Ions; D008278:Magnesium Sulfate; D004256:DNA Polymerase I; D005092:Exonucleases