Strategies and procedures to generate chimeric DNA polymerases for improved applications

Chimeric DNA polymerase with notable performance has been generated for wide applications including DNA amplification and molecular diagnostics. This rational design method aims to improve specific enzymatic characteristics or introduce novel functions by fusing amino acid sequences from different proteins with a single DNA polymerase to create a chimeric DNA polymerase. Several strategies prove to be efficient, including swapping homologous domains between polymerases to combine benefits from different species, incorporating additional domains for exonuclease activity or enhanced binding ability to DNA, and integrating functional protein along with specific protein structural pattern to improve thermal stability and tolerance to inhibitors, as many cases in the past decade shown. The conventional protocol to develop a chimeric DNA polymerase with desired traits involves a Design-Build-Test-Learn (DBTL) cycle. This procedure initiates with the selection of a parent polymerase, followed by the identification of relevant domains and devising a strategy for fusion. After recombinant expression and purification of chimeric polymerase, its performance is evaluated. The outcomes of these evaluations are analyzed for further enhancing and optimizing the functionality of the polymerase. This review, centered on microorganisms, briefly outlines typical instances of chimeric DNA polymerases categorized, and presents a general methodology for their creation. KEY POINTS: • Chimeric DNA polymerase is generated by rational design method. • Strategies include domain exchange and addition of proteins, domains, and motifs. • Chimeric DNA polymerase exhibits improved enzymatic properties or novel functions.