本文選題：酶循環(huán)擴增法 + T7核酸外切酶��； 參考：《重慶醫(yī)科大學》2017年碩士論文

【摘要】：MicroRNA(miRNA)是一類長度為18-23個堿基的內(nèi)源性、非編碼RNA,它與真核細胞的基因調(diào)控有著重要的關系。研究表明,miRNA參與了疾病發(fā)生發(fā)展過程中的信號調(diào)節(jié),并且miRNA的異常表達與癌癥,神經(jīng)系統(tǒng)疾病及糖尿病等多種疾病有著緊密的聯(lián)系。作為一種新型的生物標志物,miRNA具有長度短、豐度低及家族同源性高等特性,使得臨床上實現(xiàn)高靈敏檢測miRNA面臨著巨大的挑戰(zhàn)。因此,建立高靈敏的miRNA定量分析方法是目前亟需解決的難題。本課題建立了一種基于T7核酸外切酶“信號關閉”的策略,在均相體系中成功實現(xiàn)了對miRNA的可視化檢測。在無靶物質(zhì)miRNA存在時,單鏈捕獲探針無法被T7核酸外切酶剪切,打開含有G4序列的發(fā)夾結(jié)構(gòu)探針,吸光度達到最大值;在靶物質(zhì)miRNA存在時,miRNA與捕獲探針雜交形成DNA/RNA雙鏈,利用T7核酸外切酶,剪切雙鏈中的捕獲探針,使得捕獲探針濃度大大減小,被打開的發(fā)夾探針數(shù)量減少,從而導致信號降低。在最優(yōu)的實驗條件下,miRNA最低檢測限可達到0.6 p M(S/N=3),在10 p M到100 n M的線性范圍內(nèi),吸光度的與miRNA的濃度呈線性關系。該方法對同源miRNA具有很高的區(qū)分度,同時在復雜基質(zhì)中對miRNA有著良好的分析性能。因此,該傳感策略將有可能為miRNA的檢測提供了新方法,為臨床相關疾病的診斷及治療提供了技術支持。
[Abstract]:MicroRNAs miRNAs are a class of endogenous, non-coding RNAs with a length of 18-23 bases, which have an important relationship with the gene regulation of eukaryotic cells. Studies have shown that miRNAs are involved in signal regulation in the process of disease development, and the abnormal expression of miRNA is closely related to many diseases, such as cancer, nervous system diseases, diabetes mellitus and so on. As a new biomarker, miRNA has the characteristics of short length, low abundance and high homology. Therefore, it is an urgent problem to establish a sensitive miRNA quantitative analysis method. In this paper, a strategy of signal shutoff based on T7 nucleic acid exonuclease was established, and the visual detection of miRNA was successfully realized in homogeneous system. In the absence of target material miRNA, the single strand capture probe could not be cut by the T7 nucleic acid exonuclease, and the hairpin structure probe containing G4 sequence was opened, and the absorbance reached the maximum. In the presence of the target material miRNA, the miRNA was hybridized with the capture probe to form a double strand of DNA/RNA. By using T7 nucleic acid exonuclease, the capture probe in the double strand is cut down, the concentration of the capture probe is greatly reduced and the number of hairpin probes opened is reduced, which leads to the decrease of the signal. Under the optimal experimental conditions, the minimum detection limit of miRNA can reach 0.6 p MN / N ~ (3 +). In the linear range of 10 pm to 100nM, the absorbance has a linear relationship with the concentration of miRNA. This method has high discrimination for homologous miRNA and good analytical performance for miRNA in complex matrix. Therefore, this sensing strategy may provide a new method for the detection of miRNA and provide technical support for the diagnosis and treatment of clinically-related diseases.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R440

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相關期刊論文 前1條

1 成永強;李正平;王愈聰;范永山;;MicroRNA分析方法進展[J];化學進展;2010年08期

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本文編號：1875061