【摘要】：蛋白激酶與MicroRNA分子在生命過程中發(fā)揮著至關重要的調控作用,它們的異常與癌癥、糖尿病及心臟病等許多重大疾病有著密切聯(lián)系。因此,對激酶以及MicroRNA進行高靈敏度檢測對于臨床診斷、藥物篩選及疾病靶向治療方面都具有重要意義。本文分別以microRNA和T4 PNK為研究對象,構建了兩種簡單的恒溫核酸指數(shù)擴增機制,實現(xiàn)了靶標分子的高靈敏度檢測。具體研究內容如下：一、基于微球表面鏈式雜交反應體系的T4多核苷酸激酶(T4 PNK)活性分析基于寡核苷酸探針5'端磷酸化引發(fā)的λ核酸外切酶(λ exo)的切割反應,以及微球表面的雜交鏈式反應擴增與流式微球分析手段,我們建立起了一種簡單、靈敏的用于檢測T4 PNK活性的新型傳感策略。首先,我們將一條5'末端生物素標記的HCR引發(fā)序列連接至微球表面,隨后加入與之互補的封閉DNA序列以形成雙螺旋結構。如果存在T4 PNK, T4 PNK就會將封閉DNA的5'末端磷酸化；λexo酶會特異性識別DNA的5'磷酸化末端并對其進行5'-3'的切割,從而被封閉的HCR引發(fā)探針得以釋放,并在微球表面與熒光標記的H1和H2 DNA 探針交替反應形成HCR擴增。這樣,就會在微球表面進行熒光的富集；如果體系中沒有T4 PNK,就沒有被磷酸化的5'末端,那么,λ exo就無法進行識別與切割,這樣也就無法通過HCR反應將熒光富集在微球表面。因此,微球表面的熒光信號與T4 PNK的活性成正比,本論文創(chuàng)新性采用流式細胞儀來快速分析每個微球表面。我們通過微球的信號富集與HCR反應的信號放大顯著提高了測定T4 PNK的靈敏度,該方法可以檢測到1×10-5U/mL (3σ)的T4 PNK,是目前已報道的T4PNK檢出限最低的方法之一。而且該方法可以用于復雜生物體系的分析,在今后相關多核苷酸激酶的生理過程以及其抑制劑藥物篩選中都有著非常大的潛力。二、基于DSN酶切與TdT恒溫放大反應檢測MicroRNA我們創(chuàng)新性的將雙鏈特異性核酸酶(DSN)與末端脫氧核糖核酸轉移酶(TdT)的恒溫放大反應相結合,構建了一種用于檢測MicroRNA的新方法。DSN酶只會特異性的切割雙鏈DNA或者DNA與RNA雜合體中的DNA；TdT酶可將3'末端為羥基的DNA延伸至上千個堿基,而對MicroRNA是沒有延伸效果的。我們針對靶標miRNA序列設計了3'PO4修飾的寡核苷酸,該反應體系中若存在MicroRNA,其就會與互補的DNA雜交,DSN酶會將DNA切成3'為羥基的多個碎片,而釋放出來MicroRNA會不斷與DNA進行雜交、切割,形成第一步的循環(huán)放大。而被切成小碎片的DNA會在TdT酶的作用下延伸至上千個堿基,形成第二步放大。最后加入A50與SG以實現(xiàn)信號的檢測。該方法可檢測到500fM的MicroRNA。由于MicroRNA是生物重要的調控基因,調控著生物體中大約30%的基因,與許多疾病都有著密切的關系。因此,實現(xiàn)MicroRNA的高靈敏度檢測對以后其相關疾病的發(fā)現(xiàn)與治療都有著至關重要的作用。
[Abstract]:Protein kinase and MicroRNA molecules play a vital role in the regulation of life. Their abnormalities are closely related to many major diseases, such as cancer, diabetes, heart disease and so on. Therefore, high sensitivity detection of kinase and MicroRNA is of great significance in clinical diagnosis, drug screening and disease targeting therapy. In this paper, two simple isothermal nucleic acid index amplification mechanisms were constructed using microRNA and T4 PNK, respectively, and the target molecules were detected with high sensitivity. The main contents are as follows: firstly, the activity of T4 polynucleotide kinase (T4 PNK) based on microsphere surface chain hybridization system was used to analyze the cleavage reaction of 位 nucleic acid exonuclease (位 exo) initiated by oligonucleotide probe 5'terminal phosphorylation. As well as the hybridization chain reaction amplification and flow microsphere analysis on the surface of the microspheres, we have established a simple and sensitive sensing strategy for detecting the activity of T4 PNK. Firstly, a 5'-terminal biotinylated HCR initiation sequence was connected to the surface of the microspheres, and then a complementary closed DNA sequence was added to form a double helix structure. In the presence of T4 PNK, T4 PNK, the 5 'terminal phosphorylation of DNA was blocked. 位 exo can specifically recognize the 5'phosphorylated end of DNA and dissect the 5'-3', which is released by the closed HCR initiation probe, and alternately react with the fluorescent labeled H1 and H2 DNA probes on the surface of the microspheres to form HCR amplification. In this way, fluorescence enrichment will be carried out on the surface of the microspheres. If there is no phosphorylated 5'terminal in the system without T4 PNK, then 位 exo cannot be recognized and cut, thus the fluorescence can not be enriched on the surface of the microspheres by HCR reaction. Therefore, the fluorescence signal on the surface of the microspheres is directly proportional to the activity of T4 PNK. In this paper, flow cytometry is used to analyze the surface of each microsphere rapidly. The sensitivity of T4 PNK can be significantly improved by signal enrichment of microspheres and signal amplification of HCR reaction. T4 PNK, with 1 脳 10-5U/mL (3 蟽) can be detected as one of the lowest detection limits of T4PNK reported at present. This method can be used in the analysis of complex biological systems and has great potential in the physiological process of polynucleotide kinase and the screening of its inhibitor drugs. Secondly, based on DSN digestion and TdT constant temperature amplification reaction, we creatively combine double strand specific nuclease (DSN) with terminal deoxyribonucleic acid transferase (TdT) constant temperature amplification reaction. A new method for the detection of MicroRNA was constructed. The DSN enzyme only specifically cleans double-stranded DNA or DNA; in DNA / RNA hybrids. TdT enzyme can extend DNA with 3 '-terminal hydroxyl group to thousands of bases, but it has no effect on MicroRNA. We designed 3'PO4 modified oligonucleotides for the target miRNA sequence. If there is MicroRNA, in this reaction system, it will be hybridized with complementary DNA, and the DSN enzyme will cut DNA into 3 '-hydroxyl groups. The release of MicroRNA will continue to hybridize with DNA, cutting, forming the first step of cyclic amplification. DNA, which is cut into small fragments, extends to thousands of bases under the action of the TdT enzyme, forming a second step of amplification. Finally, A 50 and SG are added to realize signal detection. The MicroRNA. of 500fM can be detected by this method. Because MicroRNA is an important biological regulatory gene, it regulates about 30% of the genes in organisms, which is closely related to many diseases. Therefore, high sensitivity detection of MicroRNA plays an important role in the discovery and treatment of related diseases.
【學位授予單位】：陜西師范大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：O629.8;O652

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1 張越誠;核酸恒溫擴增技術在多核苷酸激酶及MicroRNA分析中的應用[D];陜西師范大學;2016年

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本文編號：2379040