Gene Engineering And Application Of A New Type Of DNA Polymerase

The polymerase Chain Reaction (PCR) is one of the most important technologies in modern molecular biology, which amplifies special DNA fragment quickly in vitro same as in vivo. In 1985, Dr. Kary Mullis of Human Heredity Department, the Cetus Company set up this technology by using Klenow DNA polymerase. Once coming up, it was extensively used in biological areas because of its simple quick operation, high sensibility, strong specificity and good patience with sample product, and it has been developed and improved.There are two major confining factors in Polymerase Chain Reaction (PCR). One is the fidelity of amplification product, the other one is the length of the amplification product. They both have important effect on the efficiency of PCR. But the key factor is DNA polymerase which catalyzes PCR.The early DNA polymerase is Klenow fragment, but it quickly loses its activity at 60℃, so we have to increase Klenow DNA polymerase every circle, which makes PCR process more complicated and limits the development of PCR.Taq DNA polymerase is the first thermostable DNA polymerase used for PCR. The introduction of this enzyme made PCR automatic. But it has no 3' to 5' proofreading exonuclease activity, it can't cut off the false nucleotide introduced in PCR process. So it can't ensure the fidelity of amplificationproduct.After the discovery of Taq DNA polymerase, Vent, Deep, Pfu, Tli, Tgo and Kod, which are thermostable DNA polymerase and having edit function, were gradually discovered. But so far, there is contradiction between high efficiency and high fidelity in discovered and used thermostable DNA polymerase. Now the solution of this problem is to mix two enzymes with special proportion. That's to say, mix the enzyme (e.g. Taq), having elongation activity but no edit ability, with the enzyme (e.g. Pfu) with edit ability but weak elongation function. But compared with Pfu, the fidelity of the amplification product decreases.To solve the contradiction in DNA polymerase between high efficiency and high fidelity, we cloned Tgo DNA polymerase gene from thermophilous archaeobacterium Thermococcus gorgonarius. The amino acid sequence inferred from the base sequence of the enzyme is consistent with the opened sequence of Tgo DNA polymerase. The full length of the gene is 2.3 kb and encode 772 amino acids. We also used specific PCR technology, fused a specific factor gene with Tgo enzyme gene and gained a fused gene which is 2.5kb.E.coli. Expression system is the earliest developed and the most extensively used classic system in gene expression technology. It has advantages of good heredity background, short life circle and strong resistance to pollution. But it is middle-temperated bacteria. So it has low expression of thermostable enzyme. In order to express a high level of thermostable enzyme in E.coli., an expression plasmid pET101-Tgo was constructed by using high expression plasmid pET101 and a strong promoter T7, and a recombinant enzyme Tgo, which is 84kD., was expressed in expression bacteria BL21 Star(DE3).To set up PCR amplification program of Tgo DNA polymerase, wedetermined the enzyme's optimum PH is 8.75.The optimum concentration of Mg2+ is 2.0 mmol/L, the optimum extension temperature is 72°C. According to the results and the normal amplification program of Tgo enzyme and Pfu enzyme, we set up a general program of Tgo enzyme, and succeed in amplification plasmid pUC19 (2.98kb), NaVtT1" reverse transport protein gene (493bp) of wild barley, Cx37 gene (1.6kb) of mouse and CYT2C9 gene fragment(284bp) of human. Enzyme's specificity determines its amplification. This study examined partial specificity of Tgo DNA polymerase, the results revealed: Tgo enzyme has strong resistance to heat, it is heated by 95 °C for 10 min, compared with 95 "C 5 min, the amplification product has no change, if it is heated by 95 °C 40 min. compared with 95 °C 5 min., the product decrease a little; Tgo has the same amplification function as Taq enzyme, plasmid pUC19 gene, Na+/H+ reverse transport protein gene of wild barly, Cx37 gene of mouse, CYT2C9 gene fragment of human are used as template, compared with Taq's amplification, the two products are nearly the same; It has fidelity blue-white experiments examines its amplification fidelity. It is as 30 times high as Taq and 1.6 times as Pfu; It's used in long distant PCR, amplify 5.3kb Timeless gene of Arabidopsis. The creative points of this study:Reformed the gene of Tgo DNA polymerase by using the technology of gene engineering;Set up high expression line of E.coli, gained a new DNA polymerase of high fidelity, high activity and long distance;Solved the contradiction between high efficiency and high fidelity of thermostable DNA polymerase.The new DNA polymerase Tgo not only has the high efficiency of Taq, but also has high fidelity of Pfu. It has the two advantages, overcoming the contradiction between high efficiency and high loyalty.