信号放大新策略与DNA检测新方法研究

发布时间：2018-01-16 02:37

本文关键词：信号放大新策略与DNA检测新方法研究　出处：《南京大学》2012年硕士论文　论文类型：学位论文

【摘要】：随着人类基因组计划的完成,人类已经进入基因革命的时代。特定序列DNA的检测对于简单的疾病诊断和重大的医疗评估都有着巨大的研究价值,因此,需要发展一系列简单、灵敏、便携的DNA诊断设备。这些设备不仅可以在实验室分析中起作用,而且在临床诊断、环境监测、食品污染、法医等领域也发挥着重要的作用。本文致力于核酸检测新方法研究,发展了两种核酸分析新方法与一种端粒酶活性检测新技术。 1金纳米聚沉的银放大方法用于DNA可视化比色分析方法发展了一种新的DNA可视化比色分析方法。该方法巧妙地结合金属离子稳定的分子信标与基于金纳米粒子增强的银放大方法。这种检测策略利用目标DNA与汞离子对连接DNA的竞争反应。当不存在目标DNA的时候,连接DNA可以与汞离子结合,形成一个分子信标。这个分子信标可以避免腺苷酸功能化的金纳米粒子的聚沉。而当存在目标DNA时,它与连接DNA的结合能力大于汞离子,目标DNA与连接DNA的结合就会打开分子信标。这时,打开的分子信标就会与金纳米粒子上的腺苷酸杂交反应,造成金纳米粒子的聚沉。而金纳米粒子的聚沉会减弱银增强的效果从而减小玻板上的点的相对灰度值。最后利用扫描法检测,就可以方便地检测目标DNA的浓度,线性范围从1.0-30nM。在相同汞离子的浓度下,单碱基错配的DNA相对灰度值只有完全匹配DNA的22%,显示了良好的选择性。这种利用金属离子稳定的分子信标的新型DNA比色检测方法,具有简单、价廉、方便的优点,在临床应用上具有巨大的潜力。 2.基于目标增强放大与滚环扩增的超灵敏核酸检测新方法用量子点作为电化学标记物,结合内切酶放大技术和滚环扩增放大技术,发展了一种超灵敏的电化学检测方法。目标DNA、帮助DNA及分子信标三者会生成-个稳定的Y型结构,这种结构可以被切口内切酶所识别。被内切酶剪开的分子信标可以作为滚环扩增反应的初始引物。而DNA功能化的量子点可以通过杂交反应连接上滚环扩增的产物。这样,通过稀硝酸溶解量子点,与目标浓度相关的电化学信号可以方便地用方波伏安法读出。由于采用了特异性好的标记DNA方法与级联信号放大策略,本方法的检测下限可达11aM并有六个数量级的线性范围(1×10-17到1×10-11M),而且可以特异性地识别错配DNA。这种灵敏特异的DNA检测方法在基因研究中有着巨大的应用潜力。 3.基于酶放大细胞中端粒酶活性检测结合分子信标技术和内切酶放大技术发展了一个简单方便的Hela细胞中端粒酶活性检测的新方法。通过引物在端粒酶的存在下会扩增出特定序列的性质,再结合酶放大技术,可以方便地通过荧光方法来检测端粒酶的活性。本方法可以检测出10-1000个Hela细胞中端粒酶的活性,是一种方便、简单的端粒酶活性检测方法。
[Abstract]:With the completion of the Human Genome Project, humans have entered the era of genetic revolution. The detection of specific sequences of DNA has great research value for simple disease diagnosis and major medical evaluation. There is a need to develop a series of simple, sensitive, portable DNA diagnostic devices that can be used not only in laboratory analysis, but also in clinical diagnosis, environmental monitoring, and food contamination. In this paper, two new methods for nucleic acid analysis and a new technique for detecting telomerase activity have been developed. Application of silver amplification method to DNA visual colorimetric analysis A new visual colorimetric analysis method for DNA is developed. The method combines metal ion stabilized molecular beacon with silver amplification method based on gold nanoparticles enhancement. The detection strategy uses target DNA. The competitive reaction with mercury ions to connect to DNA. When there is no target DNA. DNA binds to mercury ions to form a molecular beacon that avoids the accumulation of gold nanoparticles functionalized by adenylate. When the target DNA exists. Its binding ability to DNA is greater than that of mercury ions, and the binding of target DNA to DNA will open molecular beacons. In this case, the open molecular beacons will be hybridized with adenylic acid on gold nanoparticles. The gold nanoparticles will weaken the silver enhancement effect and reduce the relative gray value of the points on the glass plate. Finally, the scanning method is used to detect the gold nanoparticles. The concentration of target DNA can be easily detected. The linear range is from 1.0-30nM. under the same concentration of mercury ions, the relative gray value of DNA with single base mismatch is only 22% of that of DNA. This new DNA colorimetric method using molecular beacon stabilized by metal ions has the advantages of simple, inexpensive and convenient, and has great potential in clinical application. 2. A novel method for detection of hypersensitive nucleic acid based on target enhanced amplification and rolling amplification A highly sensitive electrochemical detection method, target DNA, was developed by using quantum dots as electrochemical markers, combined with endonuclease amplification technique and roller-ring amplification technique. Help DNA and molecular beacons will generate a stable Y-shaped structure. This structure can be recognized by the incisional endonuclease. The molecular beacons cut by the endonuclease can be used as the initial primers for the ring-amplification reaction. The DNA functionalized quantum dots can be linked to the ring-up-loop amplification by hybridization reaction. The product. Like this. The electrochemical signals related to the target concentration can be easily read by square wave voltammetry by dilute nitric acid dissolved quantum dots. Due to the use of specific labeling DNA method and cascade signal amplification strategy. The detection limit of this method is up to 11am and has six linear ranges of 1 脳 10-17 to 1 脳 10-11M). This sensitive and specific DNA detection method has great potential in gene research. 3. Detection of telomerase activity based on enzyme amplification A simple and convenient method for detecting telomerase activity in Hela cells was developed by combining molecular beacons and endonuclease amplification techniques. The specific sequence properties could be amplified by primers in the presence of telomerase. Combined with enzyme amplification technique, telomerase activity can be easily detected by fluorescence method. This method can detect telomerase activity in 10-1000 Hela cells, which is a convenient method. A simple method for detecting telomerase activity.
【学位授予单位】：南京大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R346

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