Recombinant Thermostable DNA Polymerases: Current Approaches to Production, Molecular Engineering, and Applications in Biotechnology and Diagnostics.

DNA polymerases are crucial for the replication and repair of genetic material. Advances in recombinant DNA technology and protein engineering have enabled the production of enzymes with specific catalytic properties tailored to the requirements of molecular diagnostics, next-generation sequencing, and synthetic biology. This review discusses the classification and structural-functional organization of DNA polymerases, with an emphasis on the thermostable members of Families A and B, which are of great practical importance. The main systems for heterologous expression and methods for purifying recombinant polymerases are summarized. Molecular engineering approaches, including rational design, site-directed mutagenesis, directed evolution, and domain engineering, are also discussed, highlighting how enzymes with improved synthesis fidelity, processivity, inhibitor resistance, and broadened substrate specificity are created. Technologies for developing hot-start polymerases along with the creation of chimeric and multifunctional polymerases are reviewed. Information on commercial polymerases utilized in scientific research and molecular diagnostics is also provided. Furthermore, the current applications of recombinant DNA polymerases in conventional, quantitative, and digital PCR; isothermal amplification; sequencing; synthetic biology; and molecular diagnosis of infectious and hereditary diseases are summarized. Finally, we discuss how the integration of structural biology, computational modeling, and high-throughput screening creates new prospects for engineering next-generation specialized enzymes.