Thermostable DNA Polymerase from Thermus thermophilus B35: Cloning, Sequence Analysis, and Gene Expression

A. G. Akishev^1*, N. I. Rechkunova², N. A. Lebedeva², O. I. Lavrik², and S. Kh. Degtyarev¹

¹Institute of Molecular Biology and Biophysics, Siberian Branch, Russian Academy of Medical Sciences, ul. Timakova 2, Novosibirsk, 630117 Russia; fax: (3832) 33-6853; E-mail: degt@ma.nsc.ru

²Novosibirsk Institute of Bioorganic Chemistry, Siberian Branch, Russian Academy of Sciences, pr. Lavrent'eva 8, Novosibirsk, 630090 Russia; fax: (3832) 33-3677; E-mail: lavrik@niboch.nsc.ru

^* To whom correspondence should be addressed.

Received January 11, 1999; Revision received May 17, 1999

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The nucleotide sequences of three thermostable DNA polymerase (Taq, Tth, and Tfl) genes were analyzed and high conserved regions typical for this polymerase family were identified. Using primers for one of the conserved regions, the genomic DNA fragment of T. thermophilus B35 strain was amplified. The resulting fragment was cloned into a plasmid and used as a hybridization probe with digests of T. thermophilus B35 DNA cleaved by different restriction endonucleases. A restriction DNA fragment carrying the full-length Tte polymerase gene was found, cloned, and sequenced. The primary structures of 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 were investigated.

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KEY WORDS: thermostable DNA polymerase, DNA amplification, gene cloning, sequence analysis, gene expression

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