CHARACTERIZATION OF DNA-POLYMERASE FROM PYROCOCCUS SP. STRAIN KOD1 AND ITS APPLICATION TO PCR

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 puta tive 3'-5' exonuclease activity and two in-frame intervening sequences of 1,080 bp (360 amino acids; ROD pol intein-1) and 1,611 bp (537 ami no acids; KOD pol intein-2), which are located in the middle of region s conserved among eukaryotic and archaeal alpha-like DNA polymerases. The mature form of the DNA polymerase gene was expressed in Escherichi a 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 furios us (Pfu DNA polymerase), the KOD DNA polymerase exhibited an extension rate (100 to 130 nucleotides/s) 5 times higher and a processivity (pe rsistence of sequential nucleotide polymerization) 10 to 15 times high er than those of Pfu DNA polymerase . These characteristics enabled th e KOD DNA polymerase to perform a more accurate PCR in a shorter react ion time.