本文選題：MicroRNAs(miRNAs) + 核酸擴(kuò)增技術(shù)�。� 參考：《南昌大學(xué)》2016年博士論文

【摘要】：MicroRNAs(miRNAs)是一類非蛋白編碼的短小的內(nèi)源性RNAs,是重要的基因表達(dá)的后轉(zhuǎn)錄調(diào)節(jié)因子,它在細(xì)胞發(fā)育、新陳代謝、細(xì)胞凋亡和腫瘤的發(fā)生等生物過程中都起著至關(guān)重要的作用。核酸擴(kuò)增檢測技術(shù)主要是通過生成大量的目標(biāo)物的復(fù)制序列或者是將目標(biāo)物轉(zhuǎn)化為其他的特殊的核酸序列或信號分子,達(dá)到擴(kuò)增檢測目標(biāo)物的目的。目標(biāo)物或信號的擴(kuò)增過程致使該檢測技術(shù)具有較高的靈敏度和選擇性。然而,大多核酸擴(kuò)增檢測技術(shù)的實(shí)驗(yàn)設(shè)計復(fù)雜,不便于操作,而且熒光標(biāo)記探針和特殊的DNA或RNA酶的使用大大增加了檢測成本,這限制了它們的應(yīng)用范圍。因此,迫切需要研發(fā)新的經(jīng)濟(jì)方便、靈敏度高且選擇性好的miRNA檢測方法。本論文致力于研究基于聚合酶的核酸擴(kuò)增檢測技術(shù),主要設(shè)計了一系列簡單、快速、靈敏且經(jīng)濟(jì)的miRNA檢測新方法,為癌癥的早期診斷、介入治療以及抗癌藥物的發(fā)現(xiàn)提供了研究基礎(chǔ)。主要研究內(nèi)容如下:1、圍繞本論文的研究內(nèi)容,對miRNAs、末端脫氧核苷酸轉(zhuǎn)移酶(TdTase)和用于miRNA檢測的核酸擴(kuò)增技術(shù)進(jìn)行了綜述。首先,對miRNAs的起源、作用機(jī)制和生物學(xué)功能進(jìn)行了總結(jié),并著重論述了miRNAs在細(xì)胞發(fā)育、細(xì)胞分化以及在免疫系統(tǒng)、癌癥和作為致癌基因或抑癌基因方面的重要作用和意義。然后,簡要介紹了DNA聚合酶的重要性和分類,著重論述了TdTase聚合酶的活性分析。最后,綜述了幾種常用的miRNA核酸擴(kuò)增檢測技術(shù),包括聚合酶鏈反應(yīng)擴(kuò)增檢測技術(shù)、滾環(huán)擴(kuò)增檢測技術(shù)、鏈置換擴(kuò)增檢測技術(shù)、雙鏈特異性酶擴(kuò)增檢測技術(shù)以及雜交鏈反應(yīng)擴(kuò)增檢測技術(shù)等。2、提出一種新穎簡單通用的分支級聯(lián)酶擴(kuò)增(branched cascade enzymatic amplification,BCEA)技術(shù),旨在通過目標(biāo)miRNA和捕獲探針DNA的直接雜交啟動聚合酶的催化反應(yīng),并通過第二引物的引入產(chǎn)生大量的雙鏈分支進(jìn)而提高擴(kuò)增效率,利用SYBR Green?(SG)作為熒光信號對miRNAs進(jìn)行定量檢測。本方法中SG的熒光強(qiáng)度隨目標(biāo)miRNA濃度(1 fM-10 pM)的增加呈指數(shù)擴(kuò)增,對miRNA的檢測下限為0.1 fM。本方法不僅可以有效區(qū)分miRNAs之間的單堿基錯配,還可以直接用于癌癥細(xì)胞提取物中mi RNAs的靈敏檢測。本方法的優(yōu)勢在于:首先,降低了實(shí)驗(yàn)操作的復(fù)雜性和分析成本;其次,對mi RNAs的檢測限非常低且選擇性高,并可直接用于癌癥細(xì)胞提取物中mirnas的高靈敏檢測,在臨床診斷中具有很大的應(yīng)用潛力;最后,本文提出的beca方法簡單、擴(kuò)增效率高且通用性好,可拓展應(yīng)用于其他傳感技術(shù)。3、發(fā)展一種基于hg2+輔助的雙聚合酶等溫核酸擴(kuò)增技術(shù)(hg2+-assistantdualpolymeraseisothermalnucleicacidamplification,hg2+-dpina)的mirna檢測方法。目標(biāo)mirna與模板dna雜交,然后在klenow片段和tdtase兩種聚合酶的作用下催化生成聚t堿基序列,加入hg2+形成t-hg2+-t雙鏈結(jié)構(gòu),sg熒光染料可插入到此雙鏈結(jié)構(gòu)中使sg的熒光顯著增強(qiáng),而sg熒光增強(qiáng)的程度與mirnas的濃度呈正相關(guān),據(jù)此實(shí)現(xiàn)mirnas的靈敏檢測。本方法對mirnas檢測的線性范圍為10pm-10nm,其檢測下限為5pm。本方法的優(yōu)勢在于:首先,hg2+的引入可以直接形成t-hg2+-t雙鏈結(jié)構(gòu),避免了因雜交引起的錯配或發(fā)生鏈置換反應(yīng),提高了雙鏈的結(jié)合效率;其次,本方法采用的“turn-on”模式可有效提高檢測靈敏度和減少假陽性信號;最后,本方法可用于識別檢測細(xì)胞溶解產(chǎn)物中的mirna序列,具有良好的實(shí)際應(yīng)用價值。4、將雙聚合酶催化延伸胸腺嘧啶(enzymaticallyengineeredprimerextensionpoly-thymine,epept)機(jī)制與納米材料的原位生成技術(shù)相結(jié)合,發(fā)展了一種以聚胸腺嘧啶為模板原位生成熒光cunps的mirna檢測方法。本方法基于聚合酶klenow片段和tdtase的共同催化作用,將mirna與模板dna雜交后有效延伸成長度較長的聚胸腺嘧啶序列,加入cu2+和抗壞血酸鈉,即以此聚胸腺嘧啶序列為模板原位生成紅色熒光的polyt-cunps,polyt-cunps的熒光強(qiáng)度與mirna濃度的對數(shù)成正比,據(jù)此實(shí)現(xiàn)mirnas的低背景和高靈敏檢測。本方法對mirna檢測的線性范圍為1pm-1nm,檢測下限為100fm,應(yīng)用于多種細(xì)胞溶解產(chǎn)物中mirnas的檢測,獲得滿意結(jié)果。此外,由于本方法生成的polyt-cunps可發(fā)射強(qiáng)紅色熒光并具有良好的stokes位移,因此在紫外燈照射下可以實(shí)現(xiàn)mirnas的可視化檢測,在熒光成像、臨床診斷和生物化學(xué)分析等領(lǐng)域具有廣闊的應(yīng)用前景。5、將富g堿基序列與tb3+之間的共振能量轉(zhuǎn)移效應(yīng)應(yīng)用于mirna檢測中,提出一種新穎的基于稀土離子熒光增強(qiáng)的mirna分析方法,并用于復(fù)雜樣品中mirnas的良好檢測。利用配體敏化tb3+優(yōu)良的stokes位移和強(qiáng)而尖銳的發(fā)射光譜等光學(xué)性質(zhì),構(gòu)建基于富鳥嘌呤dna敏化tb3+熒光檢測mirnas的方法。首先由目標(biāo)miRNA誘導(dǎo)啟動聚合反應(yīng),通過兩個聚合酶的聯(lián)合作用,生成大量的長鏈富G堿基DNA序列,富G堿基DNA序列通過共振能量轉(zhuǎn)移效應(yīng)增強(qiáng)Tb3+的熒光。隨著目標(biāo)miRNA濃度的增加(0-1 nM),Tb3+在545 nm處的熒光發(fā)射光譜強(qiáng)度逐漸增強(qiáng),據(jù)此可實(shí)現(xiàn)對miRNAs的檢測,檢測下限為100 fM。在以上基礎(chǔ)上,建立了基于Tb3+和半導(dǎo)體QDs雙發(fā)射比率熒光傳感平臺用于miRNAs的靈敏檢測。先利用偶聯(lián)技術(shù)將QDs與捕獲探針pDNA耦合形成QDs-pDNA復(fù)合物,并以QDs的熒光發(fā)射峰為內(nèi)參比熒光,目標(biāo)miRNA誘導(dǎo)啟動聚合反應(yīng)生成大量的長鏈富G堿基DNA序列,并增強(qiáng)Tb3+的熒光強(qiáng)度,根據(jù)Tb3+和半導(dǎo)體QDs的熒光強(qiáng)度比值(I545/I610)可實(shí)現(xiàn)miRNAs的寬范圍和靈敏檢測。雙信號比率熒光傳感技術(shù)中的內(nèi)參比熒光保持不變,可以避免樣品環(huán)境、儀器和人工操作等外界因素對實(shí)驗(yàn)的影響。
[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.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：Q789

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8 王澤坤;cIAP2蛋白抑制乙型肝炎病毒復(fù)制的分子機(jī)制研究[D];復(fù)旦大學(xué);2011年

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