基因单核苷酸多态性的均相高灵敏度光散射分析

项目名称： 基因单核苷酸多态性的均相高灵敏度光散射分析

项目编号： No.20875021

项目类型： 面上项目

立项/批准年度： 2009

项目学科： 金属学与金属工艺

项目作者： 李正平

作者单位： 河北大学

项目金额： 36万元

中文摘要： 在特异性PCR中加入生物素修饰的dATP，利用streptavidin修饰的Au纳米粒子检测PCR产物，实现了基因单核苷酸多态性（SNP）的高灵敏度可视性检测。发现了修饰DNA探针的Au纳米粒子自聚集的新现象，结合恒温指数扩增反应，实现了DNA突变的高灵敏度显色分析。利用电化学氧化方法研究制备了水溶性好的碳纳米粒子，基于其强烈荧光猝灭作用建立了快速、简便分析SNP的方法。 研究发现滚环扩增产物在酸性介质中可变性聚集为纳米粒子并产生强烈的光散射，由此建立了均相、无标记高灵敏度检测SNP的分析方法。基于PCR产物在酸性介质中变性聚集现象，建立了简便、快速、高灵敏度检测PCR产物的光散射分析方法。 基于核酸标志物的研究进展，开展了microRNA (miRNA)分析的研究工作，利用聚噻吩衍生物与DNA/ miRNA静电结合产生的构象及颜色变化，建立了miRNA可视性检测方法。实现了高灵敏度分析miRNA的连接酶链式反应。 针对miRNA分子小的特性，基于切口酶实现了miRNA的快速、恒温指数扩增，建立了miRNA高灵敏度分析方法，可检测0.1 zmol-1.0 pmol的miRN

中文关键词： 单核苷酸多态性； 光散射； 显色分析； 恒温扩增；microRNA

英文摘要： By using biotin-dATP in the specific PCR reaction, a colorimetric assay for highly sensitive detection of single nucleotide polymorphism (SNP) has been developed through detection of PCR products with Au nanoparticles modified by streptavidin. A new self-aggregation phenomenon of DNA modified Au nanoparticles has been demonstrated with the first time. Coupled with the isothermal exponential amplification reaction, the self-aggregation of DNA modified Au nanoparticles has been applied to highly sensitive detection of DNA mutation. Water-soluble carbon nanoparticles have been prepared with electrochemical oxidation. Based on the high efficiency to quench the fluorescence of the carbon nanoparticles, the carbon nanoparticle-based assay has been conveniently appled to analyze SNP rapidly. We have discovered that the long-chain DNA produced by rolling circle amplification (RCA) can be denatured and aggregated in acid medium, which results in a strong light scattering signal. Based on the light scattering detection, we have established a simple, homogeneous, and label-free method for SNP detection with high sensitivity. A fast, simple, and sensitive light scattering approach has also been developed to detect PCR products based on that the PCR products can be denatured and aggregate to form nanoparticles in acid medium. Based on the recent development of nucleic acid biomarker, we also study the detection of microRNA (miRNA). A colorimetric assay for convenient detection of miRNA has been established in a homogeneous and label-free manner based on conformational and colorimetric changes of a polythiophene derivative (PMNT) in the complex of DNA/miRNA/PMNT. We also design and realize the simple and sensitive miRNA assay based on the ligase chain reaction(LCR) .We have demonstrated that the isothermal exponential amplification reaction(EXPAR) based on the nicking enzyme is well suited to fast amplification of small miRNA. The EXPAR-based miRNA assay has been established ,in which 0.1 zmol-1.0 pmol miRNA can be detected. The miRNA assay is the one of most sensitive method for miRNA detection.

英文关键词： Single nucleotide polymorphism; light scattering; colorimetric assay; isothermal amplification; microRNA