Genetically Engineering And Application Of A New Type Of DNA Polymerase

Nowadays, Polymerase chain reaction (PCR) is one of the most important tools in molecular biology research. In the PCR reaction, the activity characteristics of the heat-resistant DNA polymerase is related to the specificity, efficiency and fidelity of the PCR reaction. Taq DNA polymerase is a kind of the highly heat-stable DNA polymerase, that was isolated from thermophilic bacterium Thermus aquaticus. Because of its high thermostability, high specificity, sensitivity and other excellent features, Taq DNA polymerase is widely used in the PCR amplification. However, without 3'â†'5' exonuclease activity, the conventional Taq DNA polymerase lacks the proofreading activity in the PCR reaction. Compared with the high-fidelity Pfu DNA polymerase, Taq DNA polymerase'fidelity is much lower. In addition, the expansion efficiency of long DNA fragments is lower than others. Therefore, today, the modification of Taq DNA polymerase has become the focus of the field in PCR studies.In this study, in order to obtain a kind of new fidelity DNA polymerase Taq-omni DNA polymerase, genetic engineering technology is used to modify the original gene of Taq DNA polymerase at the level of gene. The new fidelity DNA polymerase both has high efficiency and fidelity.Firstly, with the means of genetic engineering, the protein Sso7d was merged together with Taq DNA polymerase. As a result, Sso7d fusion protein can accelerate the extension rate of Taq DNA polymerase in PCR and the resistance to inhibitors of PCR. Afterwards, site-directed mutagenesis was used to improve the specificity and the PCR amplification length of recombinant Taq DNA polymerase.Secondly, the recombinant Taq-omni DNA polymerase was induced with IPTG, and purified by Ni2+-NTA. The experiments of SDS-PAGE electrophoresis and gene sequencing were used to determine the molecular size of Taq-omni DNA polymerase.Finally, several characteristics of the new highly heat-resistant Taq-omni DNA polymerase were detected. The molecular weight of Taq-omni DNA polymerase is about 101KDa. After heated at 95℃for two hours, the remaining activity of Taq-omni DNA polymerase was more than 50% of the initial activity. The optimal PCR reaction buffer is: 50mmol/L Tris-HCl (pH 8.2),4mmol/L MgCl2 and 0.02% Triton X-100. In the PCR reaction, The amplification rate of Taq-omni DNA polymerase is higher than Taq and Pfu. When amplified to PCR with the 2.9kb fragment as the template, Taq-omni costs only 10 seconds with high better specificity, while Taq needs 40s and Pfu 80s. In the conventional PCR conditions, Taq-omni can amplify DNA template as long as 5.37Kb successfully, as well as the DNA template of soil DNA, mouse's serum, and high content(76%)of GC fragment, etc. Besides, it shows a higher resistance to the PCR inhibitors. Therefore, the results of this paper have a wide reference value.In this paper, the conflict between the efficiency and fidelity of Taq DNA polymerase has been initially solved. The methods and results in this research could provide a certain amount of research data for the study of DNA polymerase.