Study On Preparation Of High-purity Taq DNA Polymerase

Polymerase chain reaction (PCR) is one of the most important tools in molecular biology research. DNA polymerase takes a critical role in the PCR technology. The high temperatures required for PCR necessitate a thermostable enzyme for DNA amplification. Taq DNA polymerase(Taqpol) derived from the thermophilic bacteria (Thermus aquaticus) is used most commonly.The cloning, expression and purification of Taqpol has been studied extensively. However, one of the most difficult issues surrounding the purification of Taqpol is how to remove the contaminating DNA. DNA contamination frequently occurs in the use of commercial Taqpol, in PCR reaction such as16S rDNA amplification, minute amounts of extraneous DNA can serve as PCR template and lead to confusion in interpreting results. To overcome this problem, methods such as PEG-Na2SO4system, use of poly ethyl eneimine (PEI) to precipitation DNA, Ni-NTA chromatography and Salecan-Na2SO4system have been used in the study to eliminate the contaminating DNA. Salecan, produced by Agrobacterium sp. ZX09, is a novel water-soluble extracellular (3-glucan. The results proved that DNA removal from Taqpol preparation using a biphasic system containing Salecan and Na2SO4is the optimum method, as Taqpol and DNA can be separated completely in the biphasic system, so as the other proteins.One of the common problems of conventional PCR is the presence of nonspecific products and primer-dimers formation, which could be due to several factors such as annealing temperature, primer concentration, and Taq DNA polymerase activity during setup of the PCR. Hot start PCR is one of the most important techniques to improve the PCR specificity. In this study, we use Taqpol antibody inhibits nonspecific products and primer dimers formation by blocking Taqpol activity until the reaction components are heated in the thermal cycles (hot start).Glucans are known to have obviously immunomodulatory effects, β-glucans can act as adjuvants and immunostimulants enhancing the activities of macrophages and natural killer cells. It has been confirmed that Salecan has functions such as suppressing steep increase in blood glucose concentration, protecting the liver acute injury and reducing liver fat, blood fat, body fat, body weight and cholesterol. Like other β-glucans, Salecan may also have biological activities, including immunomodulatory activities, so it may act as adjuvant to increase antibody titer. Our research had confirmed the hypothesis, the use of Salecan in preparation of Taqpol antibody can increase the Taqpol antibody titer. Our research provided the possibility of using Selecan as a novel immunologic adjuvant.