The Study Of The Improvement Of Various Nano-Materials On PCR And Their Mechanisms

Polymerase Chain Reaction (PCR) is an in vitro DNA amplification technology. PCR comes into one of the most common and important molecular biological techniques in the last twenty years. However, this technology still faces several"bottlenecks", e.g. the low yield, low sensitivity, the low specificity, etc. It is proven that by adding additives, some non-specific amplification problems could be solved to certain extent. However, finding new additives to enhance the specificity and efficiency of PCR is still valuable and remain a great challenge. Nano-material is defined as a solid material characterized by at least one dimension in the nanometer range. Nano-materials are significantly different with other materials because of their special properties such as reactivity, strength and electrical properties, optical characteristics. Since the middle of 1980s, nano-materials have been used in wide potential prospects about information storage, catalysts, photoabsorption, biomedicine, etc.In the previous work, our group reported the optimization of PCR by additive nano gold, which could conspicuously improve the key characters of PCR quality, e.g. the specificity. Whether nano-materials have the function of improving PCR process is worthy of discussion. In this thesis, we explore various nano-materials as specificity and efficiency enhancers in PCR, including hard nano-materials (Au-nanoparticles, carbon-nanoparticles, TiO2-nanoparticles, etc.) and soft nano-materials (dendrimer). Two test systems including an error-prone two-round PCR and a non-specific amplification system were employed in this study.We found that in the presence of the nano-materials with appropriate concentrations, PCR amplification can be optimized to enhance both specificity and efficiency. We identified nano carbon and one of dendrimers'derivatives (G5.50Ac) were especially potent enhancers, often outperforming conventional enhancer. In addition, we studied the interactions between nano-materials and PCR components in real PCR condition using many methods. We found that hard nano-materials were tend to interact with DNA polymerase, while soft nano-materials could have interaction with DNA polymerase, primers and template at the same time. We supposed that the concentration of DNA polymerase could be adjusted by interacting with hard nano-materials, thereby significantly improving the specificity of PCR. On the other hand, we thought soft nano-materials could greatly condense the concentrations of a variety of PCR components locally to increase the efficiency of amplification; it was also possible that competition might occur between primers, template, DNA polymerase and soft nano-materials during the annealing of the primers to the template or during formation of the synthesis complex consisting of primer, template and DNA polymerase, thereby enhancing the specificity of PCR. The thesis work shows that nano-materials are very useful in enhancing the specificity and efficiency of PCR, and has provided the experimental basis for broadening the application of nano-materials in clinical test and diagnosis.