恒温扩增法检测甲基化酶活性的研究

发布时间：2018-05-11 04:25

本文选题：DNA + 甲基化酶　；参考：《河北大学》2017年硕士论文

【摘要】：DNA甲基化是最主要的表观遗传方式之一。生物体中基因表达、蛋白质相互作用以及肿瘤的产生发展等与DNA甲基化水平和DNA甲基化酶活性密切相关。发展简便、灵敏的甲基化酶检测新方法对于生化分析和临床相关疾病的诊断研究具有重要意义。本文基于恒温扩增技术,发展了两种简便、灵敏检测甲基化酶活性的新方法。具体内容如下:一、基于恒温指数扩增反应(EXPAR)检测甲基化酶活性。我们设计了无标记的发夹探针,该发夹探针的茎部有Dam甲基化酶的特异识别序列。当溶液中存在甲基化酶时,发夹探针被甲基化。然后加入限制性内切酶特异切割发夹探针,释放出来的环部序列可作为引物引发EXPAR反应。当无甲基化酶时,发夹探针未被切断,不能引发EXPAR反应。最后,EXPAR反应产物利用DNA特异荧光染料直接检测,实现了甲基化酶活性的测定。方法简便、快速、成本低,并可实现抑制甲基化酶活性的药物筛选,为抗癌药物的筛选提供了一种新方法。二、滚环扩增反应结合水溶性阳离子共轭聚合物均相检测甲基化酶活性的研究。我们新设计发夹探针,其在甲基化酶作用下,茎部的酶识别序列被甲基化。再经限制性内切酶切割后,释放的序列将作为引物引发滚环扩增反应(RCA)。在扩增过程中,我们加入荧光素修饰的dUTP(dUTP-FITC),它随扩增反应的进行而添加到DNA产物中。当加入PFP后,DNA与PFP通过静电力结合拉近荧光素与PFP之间的距离,并发生有效的荧光共振能量转移(FRET)。当无甲基化酶时,该发夹探针结构保持完整,不会引发RCA。溶液中游离的dUTP-FITC经碱性磷酸酶降解,降解产物与PFP相互作用很小,不能形成有效的FRET。基于FRET效率的不同,实现了甲基化酶活性的检测。该方法仪器简单、灵敏度高,检出限为0.36 U/mL。方法对于相关疾病的临床诊断及药物筛选具有潜在的应用价值。
[Abstract]:DNA methylation is one of the most important epigenetic methods. Gene expression protein interaction and tumor development are closely related to the level of DNA methylation and the activity of DNA methylase. To develop a simple and sensitive method for detection of methylase is of great significance for biochemical analysis and the diagnosis of clinically related diseases. Based on the technique of constant temperature amplification, two simple and sensitive methods for the detection of methylase activity were developed. The main contents are as follows: firstly, the activity of methylase was detected based on isothermal index amplification reaction (EXPAR). We designed an unlabeled hairpin probe with a specific recognition sequence of Dam methylase in the stem. When methylase exists in the solution, the hairpin probe is methylated. Then the restriction endonuclease specific hairpin probe was added and the released ring sequence could be used as a primer to trigger the EXPAR reaction. When there was no methylase, the hairpin probe was not cut off and could not initiate EXPAR reaction. Finally, the activity of methylase was determined by direct detection of DNA specific fluorescent dyes. The method is simple, rapid, low cost and can be used to screen drugs that inhibit the activity of methylase, which provides a new method for screening anticancer drugs. Second, the detection of methylase activity by ring-ring amplification reaction combined with water-soluble cationic conjugated polymer homogeneously. We designed a new hairpin probe, which methylated the enzyme recognition sequence of stem under the action of methylase. After restriction endonuclease cleavage, the released sequence will be used as a primer to trigger the ring-ring amplification reaction (RCAA). In the process of amplification, we added the fluorescein modified dUTPDUTP-FITCX, which was added to the DNA product with the amplification reaction. After the addition of PFP, the distance between PFP and PFP was reduced by electrostatic binding, and an effective fluorescence resonance energy transfer was observed. When there is no methylase, the hairpin probe remains intact and does not trigger RCA. The free dUTP-FITC in the solution was degraded by alkaline phosphatase, and the degradation product had little interaction with PFP and could not form an efficient fret. Based on the different efficiency of FRET, the activity of methylase was detected. The method is simple and sensitive, and the detection limit is 0.36 U / mL. Methods have potential application value for clinical diagnosis and drug screening of related diseases.
【学位授予单位】：河北大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O657.3

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