Structural Modification And Characterization Of Taq DNA Polymerase

This paper was devoted to the research of modification of Taq DNA polymerase, which is based on the structural and functional information and previous studies.The modified Taq DNA polymerase is more tolerant to PCR inhibitors, whole blood and soil extract, which could be applied to not only general PCR and real-time PCR system, but also direct amplification system of whole blood and soil extract.In the first chapter, this paper summarized the discovery, properties, structure and application of Taq DNA polymerase. Although natural Taq DNA polymerase had been widely used, there were still some deficiencies in its clinical application. Transforming and modifying of Taq DNA polymerase could reduce or eliminate the5’-31exonuclease activity, increase the heat resistance, processivity, fidelity, specificity, tolerance to inhibitors, and achieve hot-start PCR.In the second chapter, we modified Taq DNA polymerase using engineering technology. Firstly, we introduced three mutations by site-directed mutagenesis, namely E626K, I707L and E708Q. The two sites mutations, E626K and I707L, could improve the optimal temperature of enzyme, reducing non-specific amplification products. The E708Q mutation could significantly improve the tolerance to a variety of PCR inhibitors. Secondly, by deleting the278amino acids at the N side, we eliminated5’to3’exonuclease activity of Taq DNA polymerase. After transforming the Taq DNA polymerase, we also modified enzymes with citraconic anhydride, to achieve hot start activity, thus reducing the formation of PCR primer dimers and nonspecific amplification products.In the third chapter, we studied the character of the modified enzyme—KT-C3DNA polymerase. Firstly, we detected the thermal stability and optimal extension temperature of KT-C3DNA polymerase. It still had normal amplification activity after heating4h in95℃, showing high thermal stability. The optimal extesion temperature were higher than natural Taq DNA polymerase,which could reduce the formation PCR primer dimers and nonspecific amplification products. Secondly, we detected the application of KT-C3DNA polymerase in real-time PCR system. KT-C3 DNA polymerase had the same amplification effect and sensitivity as commercial Taq DNA polymerase and Taq-HS (Takara). Thirdly, we tested the tolerance of KT-C3DNA polymerase to ethanol, SDS, NaCl, whole blood and soil extract. We found that the enzyme could tolerate10%ethanol,0.048%SDS,100mM NaCl,more than35%whole blood and10μL/reaction soil extract.The detection limit could reach10copies per reaction in5%whole blood of KT-C3DNA polymerase and1copy per reaction in4μL/reaction soil extract using citraconic anhydride modified KT-C3DNA polymerase. The results showed that the tolerance of the modified DNA polymerase was much higher than general commercial enzymes, which could reduce the requirement of the PCR to templates, and had important significance on the development of DNA polymerase and application of PCR technique on molecular diagnosis.