核酸恒温扩增技术在多核苷酸激酶及MicroRNA分析中的应用
发布时间：2018-12-14 17:42
【摘要】：蛋白激酶与MicroRNA分子在生命过程中发挥着至关重要的调控作用,它们的异常与癌症、糖尿病及心脏病等许多重大疾病有着密切联系。因此,对激酶以及MicroRNA进行高灵敏度检测对于临床诊断、药物筛选及疾病靶向治疗方面都具有重要意义。本文分别以microRNA和T4 PNK为研究对象,构建了两种简单的恒温核酸指数扩增机制,实现了靶标分子的高灵敏度检测。具体研究内容如下：一、基于微球表面链式杂交反应体系的T4多核苷酸激酶(T4 PNK)活性分析基于寡核苷酸探针5'端磷酸化引发的λ核酸外切酶(λ exo)的切割反应,以及微球表面的杂交链式反应扩增与流式微球分析手段,我们建立起了一种简单、灵敏的用于检测T4 PNK活性的新型传感策略。首先,我们将一条5'末端生物素标记的HCR引发序列连接至微球表面,随后加入与之互补的封闭DNA序列以形成双螺旋结构。如果存在T4 PNK, T4 PNK就会将封闭DNA的5'末端磷酸化；λexo酶会特异性识别DNA的5'磷酸化末端并对其进行5'-3'的切割,从而被封闭的HCR引发探针得以释放,并在微球表面与荧光标记的H1和H2 DNA 探针交替反应形成HCR扩增。这样,就会在微球表面进行荧光的富集；如果体系中没有T4 PNK,就没有被磷酸化的5'末端,那么,λ exo就无法进行识别与切割,这样也就无法通过HCR反应将荧光富集在微球表面。因此,微球表面的荧光信号与T4 PNK的活性成正比,本论文创新性采用流式细胞仪来快速分析每个微球表面。我们通过微球的信号富集与HCR反应的信号放大显著提高了测定T4 PNK的灵敏度,该方法可以检测到1×10-5U/mL (3σ)的T4 PNK,是目前已报道的T4PNK检出限最低的方法之一。而且该方法可以用于复杂生物体系的分析,在今后相关多核苷酸激酶的生理过程以及其抑制剂药物筛选中都有着非常大的潜力。二、基于DSN酶切与TdT恒温放大反应检测MicroRNA我们创新性的将双链特异性核酸酶(DSN)与末端脱氧核糖核酸转移酶(TdT)的恒温放大反应相结合,构建了一种用于检测MicroRNA的新方法。DSN酶只会特异性的切割双链DNA或者DNA与RNA杂合体中的DNA；TdT酶可将3'末端为羟基的DNA延伸至上千个碱基,而对MicroRNA是没有延伸效果的。我们针对靶标miRNA序列设计了3'PO4修饰的寡核苷酸,该反应体系中若存在MicroRNA,其就会与互补的DNA杂交,DSN酶会将DNA切成3'为羟基的多个碎片,而释放出来MicroRNA会不断与DNA进行杂交、切割,形成第一步的循环放大。而被切成小碎片的DNA会在TdT酶的作用下延伸至上千个碱基,形成第二步放大。最后加入A50与SG以实现信号的检测。该方法可检测到500fM的MicroRNA。由于MicroRNA是生物重要的调控基因,调控着生物体中大约30%的基因,与许多疾病都有着密切的关系。因此,实现MicroRNA的高灵敏度检测对以后其相关疾病的发现与治疗都有着至关重要的作用。
[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.
【学位授予单位】：陕西师范大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：O629.8;O652
本文编号：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.
【学位授予单位】：陕西师范大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：O629.8;O652
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1 张越诚;核酸恒温扩增技术在多核苷酸激酶及MicroRNA分析中的应用[D];陕西师范大学;2016年
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