Abstract
Several native and engineered heat‐stable DNA polymerases from a variety of sources are used as powerful tools in different molecular techniques, including polymerase chain reaction, medical diagnostics, DNA sequencing, biological diversity assessments, and in vitro mutagenesis. The DNA polymerase from the extreme thermophile, Thermus scotoductus strain K1, (TsK1) was expressed in Escherichia coli, purified, and characterized. This enzyme belongs to a distinct phylogenetic clade, different from the commonly used DNA polymerase I enzymes, including those from Thermus aquaticus and Thermus thermophilus. The enzyme demonstrated an optimal temperature and pH value of 72–74°C and 9.0, respectively, and could efficiently amplify 2.5 kb DNA products. TsK1 DNA polymerase did not require additional K+ ions but it did need Mg2+ at 3–5 mM for optimal activity. It was stable for at least 1 h at 80°C, and its half‐life at 88 and 95°C was 30 and 15 min, respectively. Analysis of the mutation frequency in the amplified products demonstrated that the base insertion fidelity for this enzyme was significantly better than that of Taq DNA polymerase. These results suggest that TsK1 DNA polymerase could be useful in various molecular applications, including high‐temperature DNA polymerization.
Highlights
DNA polymerase is an omnipresent enzyme that synthesizes complementary DNA strands from an existing template in living cells
DNA polymerases are widely used for a variety of molecular techniques that rely on DNA manipulation, including polymerase chain reaction (PCR), molecular cloning, sequencing, DNA labeling, and mutagenesis among others (Gibbs et al, 2009; Killelea et al, 2009; Coulther et al, 2019)
Most thermostable DNA polymerases primarily used in PCR procedures belong to the A- and B-family polymerases from thermophilic bacteria and archaea, respectively
Summary
DNA polymerase is an omnipresent enzyme that synthesizes complementary DNA strands from an existing template in living cells. Several thermostable DNA polymerases have been isolated and studied in prokaryotes (Cho et al, 2012; Kim et al, 2007; Lee et al, 2009; Terpe, 2013; Zhang et al, 2015) These enzymes are grouped into eight families: A, B, C, D, X, Y, RT, and AEP based on their amino acid sequences (Killelea et al, 2009; Coulther et al, 2019). | 2 of 8 be used to create unique reagents (Aschenbrenner & Marx, 2017; Coulther et al, 2019; Reha-Krantz et al, 2014) This means that the search for novel DNA polymerases has been a major focus for the last couple of decades. The expression, purification, and characterization of a recombinant DNA polymerase I from Thermus scotoductus strain K1 (TsK1 DNA polymerase) originating from a geothermal spring in Karvachar, Nagorno-Karabakh (Saghatelyan et al, 2015) is described
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