써모코커스 NA1의 열안정한 DNA polymerase 특성과 PCR 적용

Abstract

As a part of genomic research of a hyperthermophilic archaeal strain, Thermococcus sp. NA1 isolated from a deep-sea hydrothermal vent area at the PACMANUS field in the East Manus Basin and a gene encoding DNA polymerase was identified. To characterize the DNA polymerase, the gene encoding DNA polymerase was cloned into Escherichia coli by PCR. The DNA polymerase gene contains an open reading frame of 4,083 bases that encodes 1,360 amino acid residues. The amino acid sequence from the open reading frame had two conserved regions. One was the 3'-5' exonuclease motif (Exo Ⅰ, Ⅱ, Ⅲ) and the other region was the DNA polymerase domain consisting of six regions (region Ⅰ to Ⅵ) conserved among α-like DNA polymerases. One in frame intervening sequence of 1,761 bp (587 amino acid; NA1 pol intein) was found by similarity analysis. In a pairwise alignment, the amino acid sequence of the mature form of NA1 DNA polymerase showed highest identities to the following family B-type DNA polymerases of Thermococcus kodakaransis KOD1 (GeneBank accession no. D29671) (90% identity), of Pyrococcus sp. GB-D (GeneBank accession no. U00707) (81% identity). The mature form of the NA1 DNA polymerase gene was expressed in Escherichia coli, and the recombinant enzyme was purified. The purified NA1 DNA polymerase was further characterized and applied to the polymerase chain reaction (PCR).