各种纳米材料对聚合酶链式反应的优化及其机理的研究
摘要
聚合酶链式反应(Polymerase Chain Reaction,PCR)是一种在体外模拟体内DNA复制的核酸扩增技术。该技术是目前最常用、也最重要的分子生物学技术之一。然而,一些瓶颈问题限制了PCR技术的进一步发展,如复杂背景下产物特异性的问题、扩增效率的问题等。鉴于现有的优化剂的单效性及副作用,需由新的技术理论和途径寻找可改进PCR技术的物质。纳米材料是一类具有特殊性能的、尺度为纳米级的新型超细材料。纳米材料的概念形成于80年代中期,由于纳米材料会表现出特异的光、电、磁、热、力学、机械等性能,逐渐成为世界各国研究开发的重点,在信息存储、催化、光吸收、发光、医药等领域展示了广泛的应用前景。能否利用纳米结构催化或调控PCR反应是一个值得尝试的选择。
本研究组在前期研究中发现,纳米金颗粒参与的PCR(Nano-particle PCR,NP-PCR)能够显著提高产物特异性等PCR的关键指标,纳米材料是否均具有调控PCR过程的作用是个值得探讨的问题。基于这点考虑,本论文以再扩增体系和大鼠Thy-1基因体系为筛选平台,对多种纳米材料的优化作用进行了评估。
研究结果表明:1)纳米材料对PCR过程的调控具有普遍性,但随物质不同,调控能力有所差异。如金属单质纳米材料、金属氧化物纳米材料、非金属单质纳米材料、非金属氧化物纳米材料及树枝状聚合物(dendrimer)对PCR反应均有很好的优化作用,其中纳米碳粉和表面50%乙酰化的第五代树枝状聚合物优化效果尤其显著。2)纳米材料对PCR的调控作用主要是通过与生物分子之间相互作用实现的。在PCR环境中,采用目测胶体溶液颜色变化、PCR扩增、紫外吸收变化和PCS测量粒径变化等方法研究了纳米材料和PCR各组分的作用。并且分别研究了硬性纳米材料(纳米金、纳米碳粉、纳米二氧化钛等)和软性纳米材料(树枝状聚合物)可能的作用,提出了两种假设。硬性纳米材料更倾向于与DNA聚合酶的静电相互作用,我们认为这可以对PCR反应体系中参与扩增的酶的用量进行调节,从而实现对PCR非特异性扩增的优化。而软性纳米材料能同时与模板、引物、酶作用。我们推测dendrimer能提高局部PCR组分的浓度,从而增加了扩增反应的可能性,提高扩增效率;在引物退火或者延伸阶段初期“引物-酶-模板”复合物形成的时候,dendrimer可能与模板、引物、酶有竞争作用,促进了反应的特异性。
上述工作的完成将为今后拓展纳米材料在临床检测和诊断中的应用及纳米材料在其他更广泛的生物领域的应用提供实验基础。
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.
本研究组在前期研究中发现,纳米金颗粒参与的PCR(Nano-particle PCR,NP-PCR)能够显著提高产物特异性等PCR的关键指标,纳米材料是否均具有调控PCR过程的作用是个值得探讨的问题。基于这点考虑,本论文以再扩增体系和大鼠Thy-1基因体系为筛选平台,对多种纳米材料的优化作用进行了评估。
研究结果表明:1)纳米材料对PCR过程的调控具有普遍性,但随物质不同,调控能力有所差异。如金属单质纳米材料、金属氧化物纳米材料、非金属单质纳米材料、非金属氧化物纳米材料及树枝状聚合物(dendrimer)对PCR反应均有很好的优化作用,其中纳米碳粉和表面50%乙酰化的第五代树枝状聚合物优化效果尤其显著。2)纳米材料对PCR的调控作用主要是通过与生物分子之间相互作用实现的。在PCR环境中,采用目测胶体溶液颜色变化、PCR扩增、紫外吸收变化和PCS测量粒径变化等方法研究了纳米材料和PCR各组分的作用。并且分别研究了硬性纳米材料(纳米金、纳米碳粉、纳米二氧化钛等)和软性纳米材料(树枝状聚合物)可能的作用,提出了两种假设。硬性纳米材料更倾向于与DNA聚合酶的静电相互作用,我们认为这可以对PCR反应体系中参与扩增的酶的用量进行调节,从而实现对PCR非特异性扩增的优化。而软性纳米材料能同时与模板、引物、酶作用。我们推测dendrimer能提高局部PCR组分的浓度,从而增加了扩增反应的可能性,提高扩增效率;在引物退火或者延伸阶段初期“引物-酶-模板”复合物形成的时候,dendrimer可能与模板、引物、酶有竞争作用,促进了反应的特异性。
上述工作的完成将为今后拓展纳米材料在临床检测和诊断中的应用及纳米材料在其他更广泛的生物领域的应用提供实验基础。
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.
引文
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