Modification And Applications Of Taq DNA Polymerase

This dissertation consists of three parts. It focuses on modification and applications of Taq DNA polymerase. Starting from complete sequence of Taq DNA polymerase, we made a variety of modified Taq DNA polymerases and applied them to real-time PCR, pyrophosphorolysis-activated polymerization (PAP) and hot-start PCR.In chapter 1, we reviewed the wide applications of Taq DNA polymerase in PCR. Because natural Taq DNA polymerase has some deficiencies and thus many researcher had modifies Taq DNA polymerase to achieve higher fidelity, processivity, and specificity. Also, Taq DNA polymerase was modified to achieve hot-start PCR by using physical, chemical as well as antibody methods.In chapter 2, we first reduced or deleted the 5′-nuclease activity of Taq DNA polymerase by introducing G46D mutation and by deleting the 5′-nuclease domain. We also prepared and purified Taq-FS DNA polymerase that has no incorporation discrimination against different type of dNTPs.In chapter 3, we applied the modified Taq DNA polymerase in three different areas. Firstly, we used mutated Taq DNA polymerase in displacing probe-based real-time PCR. We found that the modified Taq DNA polymerase could avoid the false signal caused by 5' to 3' exonuclease activity of Taq DNA polymerase. Secondly, we used Taq-FS in PAP that could greatly improve the efficiency of PAP for rare mutation detection. Thirdly, we used prepared antibodies against Taq DNA polymerase to achieve hot-start PCR. Rabbit antibodies of Taq DNA polymerase could inhibit the activity of Taq DNA polymerase at low tempreture. By heating at high temperature, the antibody was denaturalised and the activity of Taq DNA polymerase was recovered. The result showed that much higher sencitivity and speciality were achieved using such hot-start PCR.