基于聚合酶的核酸扩增技术及其在microRNA检测中的应用研究

发布时间：2018-07-02 22:56

本文选题：MicroRNAs(miRNAs) + 核酸扩增技术　；参考：《南昌大学》2016年博士论文

【摘要】：MicroRNAs(miRNAs)是一类非蛋白编码的短小的内源性RNAs,是重要的基因表达的后转录调节因子,它在细胞发育、新陈代谢、细胞凋亡和肿瘤的发生等生物过程中都起着至关重要的作用。核酸扩增检测技术主要是通过生成大量的目标物的复制序列或者是将目标物转化为其他的特殊的核酸序列或信号分子,达到扩增检测目标物的目的。目标物或信号的扩增过程致使该检测技术具有较高的灵敏度和选择性。然而,大多核酸扩增检测技术的实验设计复杂,不便于操作,而且荧光标记探针和特殊的DNA或RNA酶的使用大大增加了检测成本,这限制了它们的应用范围。因此,迫切需要研发新的经济方便、灵敏度高且选择性好的miRNA检测方法。本论文致力于研究基于聚合酶的核酸扩增检测技术,主要设计了一系列简单、快速、灵敏且经济的miRNA检测新方法,为癌症的早期诊断、介入治疗以及抗癌药物的发现提供了研究基础。主要研究内容如下:1、围绕本论文的研究内容,对miRNAs、末端脱氧核苷酸转移酶(TdTase)和用于miRNA检测的核酸扩增技术进行了综述。首先,对miRNAs的起源、作用机制和生物学功能进行了总结,并着重论述了miRNAs在细胞发育、细胞分化以及在免疫系统、癌症和作为致癌基因或抑癌基因方面的重要作用和意义。然后,简要介绍了DNA聚合酶的重要性和分类,着重论述了TdTase聚合酶的活性分析。最后,综述了几种常用的miRNA核酸扩增检测技术,包括聚合酶链反应扩增检测技术、滚环扩增检测技术、链置换扩增检测技术、双链特异性酶扩增检测技术以及杂交链反应扩增检测技术等。2、提出一种新颖简单通用的分支级联酶扩增(branched cascade enzymatic amplification,BCEA)技术,旨在通过目标miRNA和捕获探针DNA的直接杂交启动聚合酶的催化反应,并通过第二引物的引入产生大量的双链分支进而提高扩增效率,利用SYBR Green?(SG)作为荧光信号对miRNAs进行定量检测。本方法中SG的荧光强度随目标miRNA浓度(1 fM-10 pM)的增加呈指数扩增,对miRNA的检测下限为0.1 fM。本方法不仅可以有效区分miRNAs之间的单碱基错配,还可以直接用于癌症细胞提取物中mi RNAs的灵敏检测。本方法的优势在于:首先,降低了实验操作的复杂性和分析成本;其次,对mi RNAs的检测限非常低且选择性高,并可直接用于癌症细胞提取物中mirnas的高灵敏检测,在临床诊断中具有很大的应用潜力;最后,本文提出的beca方法简单、扩增效率高且通用性好,可拓展应用于其他传感技术。3、发展一种基于hg2+辅助的双聚合酶等温核酸扩增技术(hg2+-assistantdualpolymeraseisothermalnucleicacidamplification,hg2+-dpina)的mirna检测方法。目标mirna与模板dna杂交,然后在klenow片段和tdtase两种聚合酶的作用下催化生成聚t碱基序列,加入hg2+形成t-hg2+-t双链结构,sg荧光染料可插入到此双链结构中使sg的荧光显著增强,而sg荧光增强的程度与mirnas的浓度呈正相关,据此实现mirnas的灵敏检测。本方法对mirnas检测的线性范围为10pm-10nm,其检测下限为5pm。本方法的优势在于:首先,hg2+的引入可以直接形成t-hg2+-t双链结构,避免了因杂交引起的错配或发生链置换反应,提高了双链的结合效率;其次,本方法采用的“turn-on”模式可有效提高检测灵敏度和减少假阳性信号;最后,本方法可用于识别检测细胞溶解产物中的mirna序列,具有良好的实际应用价值。4、将双聚合酶催化延伸胸腺嘧啶(enzymaticallyengineeredprimerextensionpoly-thymine,epept)机制与纳米材料的原位生成技术相结合,发展了一种以聚胸腺嘧啶为模板原位生成荧光cunps的mirna检测方法。本方法基于聚合酶klenow片段和tdtase的共同催化作用,将mirna与模板dna杂交后有效延伸成长度较长的聚胸腺嘧啶序列,加入cu2+和抗坏血酸钠,即以此聚胸腺嘧啶序列为模板原位生成红色荧光的polyt-cunps,polyt-cunps的荧光强度与mirna浓度的对数成正比,据此实现mirnas的低背景和高灵敏检测。本方法对mirna检测的线性范围为1pm-1nm,检测下限为100fm,应用于多种细胞溶解产物中mirnas的检测,获得满意结果。此外,由于本方法生成的polyt-cunps可发射强红色荧光并具有良好的stokes位移,因此在紫外灯照射下可以实现mirnas的可视化检测,在荧光成像、临床诊断和生物化学分析等领域具有广阔的应用前景。5、将富g碱基序列与tb3+之间的共振能量转移效应应用于mirna检测中,提出一种新颖的基于稀土离子荧光增强的mirna分析方法,并用于复杂样品中mirnas的良好检测。利用配体敏化tb3+优良的stokes位移和强而尖锐的发射光谱等光学性质,构建基于富鸟嘌呤dna敏化tb3+荧光检测mirnas的方法。首先由目标miRNA诱导启动聚合反应,通过两个聚合酶的联合作用,生成大量的长链富G碱基DNA序列,富G碱基DNA序列通过共振能量转移效应增强Tb3+的荧光。随着目标miRNA浓度的增加(0-1 nM),Tb3+在545 nm处的荧光发射光谱强度逐渐增强,据此可实现对miRNAs的检测,检测下限为100 fM。在以上基础上,建立了基于Tb3+和半导体QDs双发射比率荧光传感平台用于miRNAs的灵敏检测。先利用偶联技术将QDs与捕获探针pDNA耦合形成QDs-pDNA复合物,并以QDs的荧光发射峰为内参比荧光,目标miRNA诱导启动聚合反应生成大量的长链富G碱基DNA序列,并增强Tb3+的荧光强度,根据Tb3+和半导体QDs的荧光强度比值(I545/I610)可实现miRNAs的宽范围和灵敏检测。双信号比率荧光传感技术中的内参比荧光保持不变,可以避免样品环境、仪器和人工操作等外界因素对实验的影响。
[Abstract]:MicroRNAs (miRNAs) is a kind of non protein encoding short endogenous RNAs, which is an important transcriptional regulator of gene expression. It plays a vital role in biological processes such as cell development, metabolism, cell apoptosis and tumor occurrence. The nucleic acid amplification test technique is mainly by producing a large number of targets. The sequence or the transformation of the target into other special nucleic acid sequences or signal molecules to achieve the purpose of amplification and detection of target objects. The amplification process of target or signal causes the detection technology to have high sensitivity and selectivity. However, most of the experimental design of nucleic acid amplification detection techniques is complex and inconvenient to operate, and the fluorescence is not easy to be operated. The use of labeled probes and special DNA or RNA enzymes greatly increases the detection cost, which limits their application. Therefore, there is an urgent need to develop new miRNA detection methods, which are convenient, sensitive and selective. This paper is devoted to the study of polymerase chain nucleic acid amplification detection technology, which mainly designed a series of simple methods. A fast, sensitive and economical new method of miRNA detection provides the basis for the early diagnosis of cancer, interventional therapy and the discovery of anticancer drugs. The main contents are as follows: 1, the contents of this paper are summarized in this paper. MiRNAs, terminal deoxynucleotidyl transferase (TdTase) and nucleic acid amplification technology for miRNA detection are reviewed. First, the origin, mechanism and biological function of miRNAs were summarized, and the important role and significance of miRNAs in cell development, cell differentiation and in immune system, cancer and carcinogenic gene or tumor suppressor gene were discussed. Then, the importance and classification of DNA polymerase were briefly introduced, and TdTase polymerization was emphasized. In the end, several common miRNA nucleic acid amplification detection techniques, including polymerase chain reaction amplification detection, rolling ring amplification detection, chain replacement amplification detection, double chain specific enzyme amplification detection and hybridization chain reaction amplification detection, are reviewed. A new and simple and simple branch grade is proposed. Branched cascade enzymatic amplification (BCEA) technology, which aims to initiate the polymerase chain reaction by direct hybridization between target miRNA and the capture probe DNA, and produces a large number of double stranded branches by the introduction of second primers to increase the amplification efficiency. SYBR Green? (SG) is used as a fluorescent signal for quantitative detection of miRNAs. In this method, the fluorescence intensity of SG is amplified exponentially with the increase of target miRNA concentration (1 fM-10 pM), and the detection limit for miRNA is 0.1 fM.. This method can not only effectively distinguish the single base mismatch between miRNAs, but also can be used directly for the detection of MI RNAs in cancer cell extract. The advantage of this method is that first, reduce the fact. Secondly, the detection limit of MI RNAs is very low and high selectivity, and can be used directly for the high sensitivity detection of miRNAs in cancer cell extract. It has great potential in clinical diagnosis. Finally, the beca method proposed in this paper is simple, high efficiency and good generality, and can be extended to the others. Sensing technology.3, developing a miRNA detection method based on hg2+ assisted double polymerase isothermal amplification (hg2+-assistantdualpolymeraseisothermalnucleicacidamplification, hg2+-dpina). Target miRNA is hybridized with template DNA, and then catalyzes the formation of poly t base sequences under the action of Klenow fragment and tdtase two polymerase. The t-hg2+-t double stranded structure is formed into the hg2+, and the SG fluorescent dye can be inserted into the double stranded structure to enhance the fluorescence of the SG, and the degree of SG fluorescence enhancement is positively correlated with the concentration of miRNAs. Accordingly, the sensitive detection of miRNAs is realized. The linear range of this method is 10pm-10nm for miRNAs detection, and the advantage of the detection limit for 5pm. is the first of the 5pm. method. First, the introduction of hg2+ can directly form a t-hg2+-t double stranded structure, which avoids the mismatch caused by hybridization or chain replacement, and improves the binding efficiency of the double chain. Secondly, the "turn-on" model used in this method can effectively improve the detection sensitivity and reduce false positive signals. Finally, this method can be used to identify and detect the dissolving products of cells. The miRNA sequence, which has good practical application value.4, combines the mechanism of enzymaticallyengineeredprimerextensionpoly-thymine (epept) and the in situ formation of nanomaterials, and develops a miRNA detection method using polythymine as a template in situ to generate fluorescence cunps. Based on the co catalysis of polymerase Klenow fragment and tdtase, miRNA and template DNA are hybridized with the template DNA to extend the polythymine sequence effectively, and add cu2+ and sodium ascorbate, that is, the polythymine sequence is used as the template to produce the red fluorescence in situ, and the fluorescence intensity of polyt-cunps is logarithmic with the miRNA concentration. This method is proportional to the low background and high sensitivity detection of miRNAs. This method has a linear range of 1pm-1nm and a low detection limit of 100fM, which is applied to the detection of miRNAs in a variety of dissolving products. In addition, the polyt-cunps generated by this method can shoot strong red fluorescence and have good Stokes displacement, because the method has good Stokes displacement. The visual detection of miRNAs can be realized under ultraviolet light..5 is widely used in the fields of fluorescence imaging, clinical diagnosis and biochemical analysis. The resonance energy transfer effect between the rich g base sequence and tb3+ is applied to the miRNA detection, and a new glume based on the rare earth ion fluorescence enhanced miRNA analysis is proposed. Method and good detection of miRNAs in complex samples. By using ligand sensitized tb3+ Stokes displacement and strong and sharp emission spectra, a method based on DNA sensitized tb3+ fluorescence detection of miRNAs was constructed. First, the polymerization reaction was induced by the target miRNA, and the combination of two polymerase chain reaction was used to generate large amounts of miRNAs. The long chain rich G base DNA sequence, the rich G base DNA sequence enhanced the fluorescence of Tb3+ through the resonance energy transfer effect. With the increase of the target miRNA concentration (0-1 nM), the fluorescence emission spectrum intensity of Tb3+ at 545 nm increased gradually, thus the miRNAs detection could be realized. The detection limit was 100 fM. on the basis of Tb3+ and half. The QDs dual emission ratio fluorescence sensing platform is used for the sensitive detection of miRNAs. First, the coupling technology is used to combine the QDs with the capture probe pDNA to form a QDs-pDNA complex, and the fluorescence emission peak of QDs is used as the internal reference ratio fluorescence. The target miRNA induces a startup polymerization to generate a large number of long chain G base DNA sequences and enhance the fluorescence intensity of Tb3+. According to the fluorescence intensity ratio (I545/I610) of Tb3+ and semiconductor QDs, the wide range and sensitive detection of miRNAs can be realized. The internal reference ratio fluorescence in the dual signal ratio fluorescence sensing technology remains unchanged, and the influence of the external factors such as sample environment, instrument and manual operation on the experiment can be avoided.
【学位授予单位】：南昌大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q789

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