雙色比率熒光寡核苷酸探針的設(shè)計(jì)及其在生物分析中的應(yīng)用
發(fā)布時(shí)間：2019-03-06 19:16
【摘要】：端粒酶作為一種核糖蛋白酶,可以催化合成真核細(xì)胞生物染色體末端的端粒重復(fù)序列,延長(zhǎng)端粒的長(zhǎng)度,以至達(dá)到細(xì)胞的“永生”。端粒酶是重要的腫瘤標(biāo)志物之一,近些年來(lái)端粒酶無(wú)疑成為研究的熱點(diǎn),根據(jù)組織細(xì)胞內(nèi)是否有可檢測(cè)的端粒酶活性,可以判定組織細(xì)胞癌變與否。無(wú)論體外還是體內(nèi)檢測(cè),對(duì)于端粒酶檢測(cè)的方法都相當(dāng)成熟,并且仍有大量的報(bào)道在探索新的方法。人體端粒酶RNA是端粒酶重要組成之一,充當(dāng)端粒酶催化反應(yīng)過(guò)程中的逆轉(zhuǎn)錄模板。人體端粒酶RNA在正常細(xì)胞和腫瘤細(xì)胞中都普遍存在,可根據(jù)其在細(xì)胞內(nèi)表達(dá)水平對(duì)細(xì)胞進(jìn)行腫瘤分級(jí)。然而,對(duì)于人體端粒酶RNA檢測(cè)的熱度遠(yuǎn)不及端粒酶。熒光手段是一種簡(jiǎn)便快捷的檢測(cè)核酸的方法,但往往很多方法都沒(méi)有有效的避免假陽(yáng)性信號(hào)的策略。因此找到一種能高效便捷且具有穩(wěn)定可靠信號(hào)的方法檢測(cè)人體端粒酶RNA成為本論文的研究重點(diǎn)。本論文中,我們基于熒光共振能量轉(zhuǎn)移原理設(shè)計(jì)了一種雙色比率熒光寡核苷酸探針,實(shí)現(xiàn)了對(duì)于人體端粒酶RNA的定量檢測(cè)。本文主要架構(gòu)如下:第一章,主要綜述了端粒酶和人體端粒酶RNA并列舉當(dāng)前對(duì)于二者比較經(jīng)典的檢測(cè)方法。同時(shí)也簡(jiǎn)單介紹了熒光共振能量轉(zhuǎn)移的原理及其在化學(xué)和生物學(xué)領(lǐng)域的應(yīng)用。第二章,論述了雙色比率熒光寡核苷酸探針的設(shè)計(jì)及檢測(cè)原理。合成出探針,并對(duì)探針本身的信號(hào)穩(wěn)定性、選擇性、可逆性等性質(zhì)進(jìn)行了研究與探討,用凝膠電泳技術(shù)對(duì)探針檢測(cè)原理進(jìn)行了驗(yàn)證。結(jié)果表明,該探針具有很好的信號(hào)穩(wěn)定性好并且對(duì)目標(biāo)物具有較高的特異選擇性,檢測(cè)過(guò)程中還呈現(xiàn)肉眼可見(jiàn)的顏色變化。最后,對(duì)于細(xì)胞提取液中人體端粒RNA,探針也表現(xiàn)出良好的檢測(cè)效果。第三章,介紹了幾種合成介孔二氧化硅及其表面修飾、制備不同粒徑金納米粒子、銀包金納米粒子、二氧化硅包金納米粒子和納米四氧化三鐵的方法。介孔二氧化硅表面修飾后與金、銀包金、四氧化三鐵粒子一樣,都具有表面吸附單鏈寡核苷酸的能力,基于此性質(zhì),可以設(shè)計(jì)豐富的對(duì)端粒酶或人體端粒酶RNA檢測(cè)的探針。此舉有望為日后的研究提供一種可選擇的新思路。
[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.
【學(xué)位授予單位】：中國(guó)科學(xué)技術(shù)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q78;O657.3
本文編號(hào)：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.
【學(xué)位授予單位】：中國(guó)科學(xué)技術(shù)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q78;O657.3
【參考文獻(xiàn)】
相關(guān)期刊論文 前2條
1 張霞菲;成銳;時(shí)志路;金燕;;基于鏈置換循環(huán)無(wú)標(biāo)記檢測(cè)端粒酶RNA的熒光法[J];高等學(xué)�；瘜W(xué)學(xué)報(bào);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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