Protein Engineering of Taq DNA Polymerase for Ultrafast PCR and Its Application in Molecular Diagnostics of...

To shorten the turnaround time of molecular diagnostics, an ultrafast polymerase chain reaction (PCR) technique has been developed in recent years, which requires thermostable DNA polymerases with high kinetic properties. In the present study, Thermus aquaticus (Taq) DNA polymerase was engineered through two strategies: fusion with CL7, a thermostable nonspecific DNA-binding tag, and rational design in its N-terminal exonuclease domain. The results indicated that the CL7 tag promoted the thermostability of this polymerase dramatically, while triple substitutions, L208I/E209 K/E210R, increased its polymerase activity by approximately 5-fold. Moreover, this engineered enzyme demonstrated a strong resistance to crude plant tissue lysate up to 30% (v/v) in the PCR system. Therefore, an ultrafast qPCR system was established to detect exogenous genes in genetically modified (GM) crops with this variant. The whole detection procedure was shortened to <30 min. Moreover, the limit of detection reached approximately 0.1 copies/μL. This study established a highly simple and accurate system suitable for rapid molecular diagnosis. More importantly, this is the first report about improving the kinetic properties of Taq polymerase through rational design of its exonuclease domain.