Thermostable DNA polymerase from Thermus thermophilus B35: Cloning, sequence analysis, and gene expression

The nucleotide sequences of three thermostable DNA polymerase (Taq, Tth, an d Tfl) genes were analyzed and high conserved regions typical for this poly merase family were identified. Using primers for one of the conserved regio ns, the genomic DNA fragment of T. thermophilus B35 strain was amplified. T he resulting fragment was cloned into a plasmid and used as a hybridization probe with digests of T. thermophilus B35 DNA cleaved by different restric tion endonucleases. A restriction DNA fragment carrying the full-length Tte polymerase gene was found, cloned, and sequenced. The primary structures o f the Tte and Tth DNA polymerase genes were analyzed. The Tte-pol gene was recloned into an expression vector and recombinant protein was purified to homogeneity. The properties of Tte-pol in the polymerase chain reaction wer e investigated.