双色比率荧光寡核苷酸探针的设计及其在生物分析中的应用
发布时间：2019-03-06 19:16
【摘要】：端粒酶作为一种核糖蛋白酶,可以催化合成真核细胞生物染色体末端的端粒重复序列,延长端粒的长度,以至达到细胞的“永生”。端粒酶是重要的肿瘤标志物之一,近些年来端粒酶无疑成为研究的热点,根据组织细胞内是否有可检测的端粒酶活性,可以判定组织细胞癌变与否。无论体外还是体内检测,对于端粒酶检测的方法都相当成熟,并且仍有大量的报道在探索新的方法。人体端粒酶RNA是端粒酶重要组成之一,充当端粒酶催化反应过程中的逆转录模板。人体端粒酶RNA在正常细胞和肿瘤细胞中都普遍存在,可根据其在细胞内表达水平对细胞进行肿瘤分级。然而,对于人体端粒酶RNA检测的热度远不及端粒酶。荧光手段是一种简便快捷的检测核酸的方法,但往往很多方法都没有有效的避免假阳性信号的策略。因此找到一种能高效便捷且具有稳定可靠信号的方法检测人体端粒酶RNA成为本论文的研究重点。本论文中,我们基于荧光共振能量转移原理设计了一种双色比率荧光寡核苷酸探针,实现了对于人体端粒酶RNA的定量检测。本文主要架构如下:第一章,主要综述了端粒酶和人体端粒酶RNA并列举当前对于二者比较经典的检测方法。同时也简单介绍了荧光共振能量转移的原理及其在化学和生物学领域的应用。第二章,论述了双色比率荧光寡核苷酸探针的设计及检测原理。合成出探针,并对探针本身的信号稳定性、选择性、可逆性等性质进行了研究与探讨,用凝胶电泳技术对探针检测原理进行了验证。结果表明,该探针具有很好的信号稳定性好并且对目标物具有较高的特异选择性,检测过程中还呈现肉眼可见的颜色变化。最后,对于细胞提取液中人体端粒RNA,探针也表现出良好的检测效果。第三章,介绍了几种合成介孔二氧化硅及其表面修饰、制备不同粒径金纳米粒子、银包金纳米粒子、二氧化硅包金纳米粒子和纳米四氧化三铁的方法。介孔二氧化硅表面修饰后与金、银包金、四氧化三铁粒子一样,都具有表面吸附单链寡核苷酸的能力,基于此性质,可以设计丰富的对端粒酶或人体端粒酶RNA检测的探针。此举有望为日后的研究提供一种可选择的新思路。
[Abstract]:Telomerase, as a ribonuclease, can catalyze the synthesis of telomere repeats at the end of the chromosome of eukaryotic cells, prolonging the length of telomere and reaching the "immortality" of cells. Telomerase is one of the important tumor markers. In recent years telomerase has undoubtedly become the focus of research. According to whether there is detectable telomerase activity in the tissue cells, it can be used to judge the carcinogenesis of tissue cells or not. Both in vitro and in vivo detection, telomerase detection methods are quite mature, and there are still a large number of reports are exploring new methods. Human telomerase RNA is one of the important components of telomerase, which acts as a reverse transcription template in the process of telomerase catalytic reaction. Human telomerase RNA is common in normal cells and tumor cells. It can be used to grade human telomerase cells according to their intracellular expression level. However, human telomerase RNA detection is far less hot than telomerase. Fluorescence is a simple and rapid method for nucleic acid detection, but many methods do not have effective strategies to avoid false positive signals. Therefore, finding a high efficient, convenient and reliable signal detection method for human telomerase RNA has become the focus of this paper. In this thesis, we designed a dual-color ratio fluorescent oligonucleotide probe based on the principle of fluorescence resonance energy transfer, and realized the quantitative detection of human telomerase RNA. The main structure of this paper is as follows: in chapter one, telomerase and human telomerase RNA are reviewed, and some classical detection methods for telomerase and human telomerase are listed. At the same time, the principle of fluorescence resonance energy transfer and its application in chemistry and biology are also briefly introduced. In chapter 2, the design and detection principle of dichroism ratio fluorescent oligonucleotide probe are discussed. The probe was synthesized, and the signal stability, selectivity and reversibility of the probe itself were studied and discussed. The principle of probe detection was verified by gel electrophoresis. The results show that the probe has good signal stability and high selectivity to the target, and the color change is visible to the naked eye in the detection process. Finally, the human telomere RNA, probe in the cell extract also showed a good detection effect. In chapter 3, several methods of synthesizing mesoporous silica and its surface modification are introduced, and the methods of preparing gold nanoparticles with different particle sizes, silver-coated gold nanoparticles, silica-coated gold nanoparticles and nano-Fe _ 2O _ 4 are introduced. The surface modification of mesoporous silica, like gold, silver-coated gold and iron trioxide particles, has the ability to adsorb single-stranded oligonucleotides on the surface. Based on this property, we can design abundant probes for detection of telomerase or human telomerase RNA. This is expected to provide a new alternative for future research.
【学位授予单位】：中国科学技术大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78;O657.3
本文编号：2435819
[Abstract]:Telomerase, as a ribonuclease, can catalyze the synthesis of telomere repeats at the end of the chromosome of eukaryotic cells, prolonging the length of telomere and reaching the "immortality" of cells. Telomerase is one of the important tumor markers. In recent years telomerase has undoubtedly become the focus of research. According to whether there is detectable telomerase activity in the tissue cells, it can be used to judge the carcinogenesis of tissue cells or not. Both in vitro and in vivo detection, telomerase detection methods are quite mature, and there are still a large number of reports are exploring new methods. Human telomerase RNA is one of the important components of telomerase, which acts as a reverse transcription template in the process of telomerase catalytic reaction. Human telomerase RNA is common in normal cells and tumor cells. It can be used to grade human telomerase cells according to their intracellular expression level. However, human telomerase RNA detection is far less hot than telomerase. Fluorescence is a simple and rapid method for nucleic acid detection, but many methods do not have effective strategies to avoid false positive signals. Therefore, finding a high efficient, convenient and reliable signal detection method for human telomerase RNA has become the focus of this paper. In this thesis, we designed a dual-color ratio fluorescent oligonucleotide probe based on the principle of fluorescence resonance energy transfer, and realized the quantitative detection of human telomerase RNA. The main structure of this paper is as follows: in chapter one, telomerase and human telomerase RNA are reviewed, and some classical detection methods for telomerase and human telomerase are listed. At the same time, the principle of fluorescence resonance energy transfer and its application in chemistry and biology are also briefly introduced. In chapter 2, the design and detection principle of dichroism ratio fluorescent oligonucleotide probe are discussed. The probe was synthesized, and the signal stability, selectivity and reversibility of the probe itself were studied and discussed. The principle of probe detection was verified by gel electrophoresis. The results show that the probe has good signal stability and high selectivity to the target, and the color change is visible to the naked eye in the detection process. Finally, the human telomere RNA, probe in the cell extract also showed a good detection effect. In chapter 3, several methods of synthesizing mesoporous silica and its surface modification are introduced, and the methods of preparing gold nanoparticles with different particle sizes, silver-coated gold nanoparticles, silica-coated gold nanoparticles and nano-Fe _ 2O _ 4 are introduced. The surface modification of mesoporous silica, like gold, silver-coated gold and iron trioxide particles, has the ability to adsorb single-stranded oligonucleotides on the surface. Based on this property, we can design abundant probes for detection of telomerase or human telomerase RNA. This is expected to provide a new alternative for future research.
【学位授予单位】：中国科学技术大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78;O657.3
【参考文献】
相关期刊论文 前2条
1 张霞菲;成锐;时志路;金燕;;基于链置换循环无标记检测端粒酶RNA的荧光法[J];高等学校化学学报;2016年01期
2 ZHANG LingHui;TANG Zhuo;;RNA-primed allele-specific PCR[J];Science China(Chemistry);2014年07期
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