【作者】 户敏；

【导师】 沈鹤柏；

【作者基本信息】 上海师范大学 ， 物理化学， 2006， 硕士

【摘要】 聚合酶链反应（PCR）是一种体外特定核酸序列扩增技术，是现代分子生物学的实验工作基础之一，在生命科学、遗传学和医学等领域发挥着重要作用。PCR通常在均一的溶液中进行，在纳米粒子界面上能否顺利进行PCR是一个具有探索性的问题。如果引物连接在纳米粒子的界面上能顺利进行PCR，将会把均相体系中的PCR技术扩展到纳米粒子的界面上，从而大大拓宽了PCR技术的应用范围，因此研究纳米粒子界面上的PCR具有重要的理论意义和应用价值。本文系统研究了纳米粒子界面上的聚合酶链反应及其应用，并取得了以下几个有意义的结果： （1）研究了金纳米粒子界面上的PCR。通过Au-S键将5′端修饰-SH-（CH₂）₆-的引物连接在金纳米粒子的界面上，采用质粒pBluescript SK作为模板，用琼脂糖凝胶电泳和透射电子显微镜（TEM）等技术对PCR产物进行分析和检测，研究了引物浓度、退火温度和循环次数对PCR的影响。实验研究结果表明：在优化的条件下，将一种引物连接在金纳米粒子的界面上以及将两种引物分别连接在金纳米粒子的界面上，均能顺利进行PCR。通过两种引物分别连接在金纳米粒子界面上的PCR，金纳米粒子能被dsDNA连接形成纳米粒子聚合体。 （2）研究了SiO₂纳米粒子界面上的PCR。采用微乳液法制得了粒度均一、分散性良好的球形SiO₂纳米粒子。首先在SiO₂纳米粒子界面上连接活性的巯基，再通过过硫键将5’端修饰-HO-（CH₂）₆-S-S-（CH₂）₆-O-（PO₃）-的引物共价键合在SiO₂纳米粒子的界面上，系统研究了SiO₂纳米粒子界面上的PCR，并用琼脂糖凝胶电泳和原子力显微镜（AFM）对PCR产物进行分析和检测。实验结果表明，引物连接在SiO₂纳米粒子的界面上，能顺利进行PCR，但受退火温度的影响较大；通过两种引物分别连接在SiO₂纳米粒子界面上的PCR，SiO₂纳米粒子也能被dsDNA连接形成纳米粒子聚合体。 （3）研究了基于纳米粒子界面上的PCR的纳米粒子聚合体的制备。当更多还原

【Abstract】 PCR, a technique for in vitro amplification of a specific nucleic acid fragment, is one of the most important experimental methods in molecular biology. It has been playing an important role in life science, genetics and medicine fields. Usually, PCR is carried out in liquid homogeneous system, whether it could proceed on the surface of nanoparticles or not becomes a challenging problem. If PCR based on the nanoparticle surface can proceed successfully, it will widely expand the applied range of PCR, from liquid homogeneous system to the nanoparticle surface. Therefore it is very significant to study PCR based on the nanoparticle surface. In this paper, we have systemically studied PCR based on the nanoparticle surface and its application, and obtain several significant results:（1） PCR based on the Au nanoparticle （AuNP） surface was studied. The 5’-SH- （CH₂）₆- modified primers were covalently attached to the AuNP surface via Au-S bonds. The linker - （CH₂）₆- between the AuNP surface and primers were used to reduce the steric effect caused by AuNPs. Plasmid pBluescript SK was used as templates. Agarose gel electrophoresis and transmission electron microscope （TEM） were used to detect and observe the PCR products. The effects of the concentration of AuNP-bound primers, annealing temperature and PCR cycle were evaluated, respectively. The results obtained indicate that PCR can proceed successfully with either one kind of primers or two kinds of primers bound to the AuNP surface. More interestingly, AuNPs can be interconnected by dsDNA and nanostructured aggregates are fabricated via PCR with two kinds of primers respectively bound to the AuNP surface.更多还原