【作者】 张亚莉；

【导师】 李正平；

【作者基本信息】 河北大学 ， 分析化学， 2009， 博士

【摘要】 核酸及其单核苷酸多态性(SNP)的分析检测在当前生化研究和临床诊断等领域都具有重要的意义。开发简便、快速、灵敏度高和特异性好的均相无标记的核酸和SNP的检测新方法具有广泛的应用前景。论文主要应用阳离子共轭聚合物,结合光散射、紫外吸收光谱和分子荧光等检测技术研究开发了一系列均相无标记检测核酸和SNP的新方法,主要内容如下：1、建立了一种利用共振光散射技术,通过阳离子共轭聚合物与核酸DNA作用后散射强度的变化来均相、无标记检测DNA杂交的方法。该方法是基于阳离子共轭聚合物与核酸通过静电结合使共振光散射强度增加而建立的,目标DNA链与探针杂交后的双链较单链探针所产生的共振光散射强度大大增加,增强的共振光散射强度与目标DNA在0.5-10nmol/L浓度范围内成正比。该方法对目标DNA链末端与探针之间存在一个不匹配碱基的检测同样具有很好的选择性。2、基于DNA诱导的聚噻吩衍生物(PMNT)构象变化,设计与突变型目标链相匹配的引物探针,加入反转录酶RTase M-MLV和dNTP,在适当的条件下进行延伸反应,突变型目标链的延伸产物就会形成双链,而野生型目标链的延伸产物依然是单链,根据不同的延伸产物所引发的聚噻吩衍生物的紫外吸收光谱和溶液颜色的不同,建立了一种基于阳离子共轭聚合物构象变化均相、无标记检测DNA单核苷酸多态性(SNP)的新方法,并且通过一步延伸反应在均相中同时检测SNP分型,结合特异性降解杂合体DNA/RNA中的RNA的核酸内切酶RNase H的降解反应,建立了均相、无标记RNA的SNP检测方法。3、基于DNA诱导的聚噻吩构象变化,根据PMNT在DNA/PMNT二聚体和DNA/miRNA/PMNT三元复合物中构象和颜色的变化可方便地检测序列特异性的miRNA。MiRNA浓度在0.05-1.0μmol/L范围内与吸收比率成线性关系。该法可用普通的分光光度计甚至用肉眼可视法检测,这可以大大降低分析成本、提高分析的效率。同时结合核酸内切酶RNase H的降解反应,通过测定适量的RNase H酶在一定的降解时间内的吸收比率,建立了RNase H酶活性的检测。4、利用阳离子共轭聚芴(PFP)的光学信号放大作用,结合引物延伸反应,建立了高灵敏度、高选择性地检测miRNA的新方法。该方法以DNA为模板,miRNA直接作为引物,加入DNA聚合酶和标记荧光素的dATP引发延伸反应,延伸产物与PFP之间发生能量共振转移,大大增强了荧光素的荧光信号,实现高灵敏度地检测miRNA。通过控制DNA聚合酶的用量,实现了区别同一家族miRNA分子间单碱基的差别。更多还原

【Abstract】 Detection of nucleic acids and single-nucleotide polymorphism (SNP) is very important in the field of biochemical research and clinical diagnosis. Developing simple, rapid, highly sensitive and specific methods for detecting nucleic acids and SNP have great potential applications, especially, in homogeneous and label-free fashion. This dissertation presents a series of studies for homogeneous and label-free detection of nucleic acids and SNP with Cation Conjugated Polymers (CCPs) by using resonance light scattering, spectrophotometer and molecular fluorescence. The main contents are as follows:1. A label-free detection method for DNA hybridization in homogeneous solution with CCP by using a light scattering technique was established. This method is based on the observation of greatly enhanced light scattering resulted from the hybridization between the ssDNA combined with CCP and its complementary DNA target. The enhancement of light scattering is linearly depended on the concentration of the DNA target in the range of 0.5～10 nmol/L. Moreover, it was also found that the enhanced light scattering intensity has well selectivity against one-base mismatch at the DNA duplex terminal.2. Based on the conformational changes of polythiophene derivative (PMNT) resulted from the formation of interpolyeletrolyte complex with DNA strands, a novel methodology for SNP genotyping assay of DNA and RNA targets has been developed. With the proposed method, all three kinds of SNP genotypes can be colorimetrically identified with one primer extension reaction in homogeneous solution. This procedure could provide simple operation without requirement of any separation steps and inexpensive instrumentation for reliable and robust SNP detection.3. Based on the conformational changes of the conjugate backbone of PMNT, we have developed a rapid and convenient method for detection of miRNA and RNase H activity in a label-free and homogeneous manner. The conformational and colorimetric changes of PMNT in the DNA/PMNT duplex and DNA/miRNA/PMNT triplex are used to conveniently detect the sequence-specific miRNA. The absorbance ratio linearly depend on the concentration of the miRNA in the range of 0.05-1.0μmol/L. This assay can be accomplished with common spectrophotometer, even with visualization by "naked eye", which would significantly reduce the analysis cost and increase the analysis throughput. Combined with the RNase H to digest the miRNA of the DNA/miRNA hybrids, a colorimetric method for RNase H activity has also been developed.4. Combining the miRNA-primed extension reaction and the optical amplification of cationic conjugated polymer, the sensitive, selective and homogeneous method for miRNA determination has been developed based on fluorescence resonance energy transfer (FRET) mechanism. By controlling the amount of DNA polymerase, the proposed method can discriminate one-base difference between miRNA molecules. The homogeneous platform for miRNA assay eliminates the use of labeling probes and the requirement of isolation steps, which greatly simplifies the assay procedure and results in rapid and low-cost detection.更多还原