核酸恒溫?cái)U(kuò)增技術(shù)在多核苷酸激酶及MicroRNA分析中的應(yīng)用
發(fā)布時(shí)間：2018-12-14 17:42
【摘要】：蛋白激酶與MicroRNA分子在生命過(guò)程中發(fā)揮著至關(guān)重要的調(diào)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(duì)激酶以及MicroRNA進(jìn)行高靈敏度檢測(cè)對(duì)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(duì)象,構(gòu)建了兩種簡(jiǎn)單的恒溫核酸指數(shù)擴(kuò)增機(jī)制,實(shí)現(xiàn)了靶標(biāo)分子的高靈敏度檢測(cè)。具體研究?jī)?nèi)容如下：一、基于微球表面鏈?zhǔn)诫s交反應(yīng)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(yīng),以及微球表面的雜交鏈?zhǔn)椒磻?yīng)擴(kuò)增與流式微球分析手段,我們建立起了一種簡(jiǎn)單、靈敏的用于檢測(cè)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(biāo)記的HCR引發(fā)序列連接至微球表面,隨后加入與之互補(bǔ)的封閉DNA序列以形成雙螺旋結(jié)構(gòu)。如果存在T4 PNK, T4 PNK就會(huì)將封閉DNA的5'末端磷酸化；λexo酶會(huì)特異性識(shí)別DNA的5'磷酸化末端并對(duì)其進(jìn)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(biāo)記的H1和H2 DNA 探針交替反應(yīng)形成HCR擴(kuò)增。這樣,就會(huì)在微球表面進(jìn)行熒光的富集；如果體系中沒(méi)有T4 PNK,就沒(méi)有被磷酸化的5'末端,那么,λ exo就無(wú)法進(jìn)行識(shí)別與切割,這樣也就無(wú)法通過(guò)HCR反應(yīng)將熒光富集在微球表面。因此,微球表面的熒光信號(hào)與T4 PNK的活性成正比,本論文創(chuàng)新性采用流式細(xì)胞儀來(lái)快速分析每個(gè)微球表面。我們通過(guò)微球的信號(hào)富集與HCR反應(yīng)的信號(hào)放大顯著提高了測(cè)定T4 PNK的靈敏度,該方法可以檢測(cè)到1×10-5U/mL (3σ)的T4 PNK,是目前已報(bào)道的T4PNK檢出限最低的方法之一。而且該方法可以用于復(fù)雜生物體系的分析,在今后相關(guān)多核苷酸激酶的生理過(guò)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(yīng)檢測(cè)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(zhuǎn)移酶(TdT)的恒溫放大反應(yīng)相結(jié)合,構(gòu)建了一種用于檢測(cè)MicroRNA的新方法。DSN酶只會(huì)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(gè)堿基,而對(duì)MicroRNA是沒(méi)有延伸效果的。我們針對(duì)靶標(biāo)miRNA序列設(shè)計(jì)了3'PO4修飾的寡核苷酸,該反應(yīng)體系中若存在MicroRNA,其就會(huì)與互補(bǔ)的DNA雜交,DSN酶會(huì)將DNA切成3'為羥基的多個(gè)碎片,而釋放出來(lái)MicroRNA會(huì)不斷與DNA進(jìn)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(huì)在TdT酶的作用下延伸至上千個(gè)堿基,形成第二步放大。最后加入A50與SG以實(shí)現(xiàn)信號(hào)的檢測(cè)。該方法可檢測(cè)到500fM的MicroRNA。由于MicroRNA是生物重要的調(diào)控基因,調(diào)控著生物體中大約30%的基因,與許多疾病都有著密切的關(guān)系。因此,實(shí)現(xiàn)MicroRNA的高靈敏度檢測(cè)對(duì)以后其相關(guān)疾病的發(fā)現(xiàn)與治療都有著至關(guān)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(xué)位授予單位】：陜西師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：O629.8;O652
本文編號(hào)：2379040
[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(xué)位授予單位】：陜西師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：O629.8;O652
【相似文獻(xiàn)】
相關(guān)碩士學(xué)位論文 前1條
1 張?jiān)秸\(chéng);核酸恒溫?cái)U(kuò)增技術(shù)在多核苷酸激酶及MicroRNA分析中的應(yīng)用[D];陜西師范大學(xué);2016年
，本文編號(hào)：2379040
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