本文選題：DNA + 甲基化酶　； 參考：《河北大學(xué)》2017年碩士論文

【摘要】：DNA甲基化是最主要的表觀遺傳方式之一。生物體中基因表達(dá)、蛋白質(zhì)相互作用以及腫瘤的產(chǎn)生發(fā)展等與DNA甲基化水平和DNA甲基化酶活性密切相關(guān)。發(fā)展簡(jiǎn)便、靈敏的甲基化酶檢測(cè)新方法對(duì)于生化分析和臨床相關(guān)疾病的診斷研究具有重要意義。本文基于恒溫?cái)U(kuò)增技術(shù),發(fā)展了兩種簡(jiǎn)便、靈敏檢測(cè)甲基化酶活性的新方法。具體內(nèi)容如下:一、基于恒溫指數(shù)擴(kuò)增反應(yīng)(EXPAR)檢測(cè)甲基化酶活性。我們?cè)O(shè)計(jì)了無標(biāo)記的發(fā)夾探針,該發(fā)夾探針的莖部有Dam甲基化酶的特異識(shí)別序列。當(dāng)溶液中存在甲基化酶時(shí),發(fā)夾探針被甲基化。然后加入限制性內(nèi)切酶特異切割發(fā)夾探針,釋放出來的環(huán)部序列可作為引物引發(fā)EXPAR反應(yīng)。當(dāng)無甲基化酶時(shí),發(fā)夾探針未被切斷,不能引發(fā)EXPAR反應(yīng)。最后,EXPAR反應(yīng)產(chǎn)物利用DNA特異熒光染料直接檢測(cè),實(shí)現(xiàn)了甲基化酶活性的測(cè)定。方法簡(jiǎn)便、快速、成本低,并可實(shí)現(xiàn)抑制甲基化酶活性的藥物篩選,為抗癌藥物的篩選提供了一種新方法。二、滾環(huán)擴(kuò)增反應(yīng)結(jié)合水溶性陽離子共軛聚合物均相檢測(cè)甲基化酶活性的研究。我們新設(shè)計(jì)發(fā)夾探針,其在甲基化酶作用下,莖部的酶識(shí)別序列被甲基化。再經(jīng)限制性內(nèi)切酶切割后,釋放的序列將作為引物引發(fā)滾環(huán)擴(kuò)增反應(yīng)(RCA)。在擴(kuò)增過程中,我們加入熒光素修飾的dUTP(dUTP-FITC),它隨擴(kuò)增反應(yīng)的進(jìn)行而添加到DNA產(chǎn)物中。當(dāng)加入PFP后,DNA與PFP通過靜電力結(jié)合拉近熒光素與PFP之間的距離,并發(fā)生有效的熒光共振能量轉(zhuǎn)移(FRET)。當(dāng)無甲基化酶時(shí),該發(fā)夾探針結(jié)構(gòu)保持完整,不會(huì)引發(fā)RCA。溶液中游離的dUTP-FITC經(jīng)堿性磷酸酶降解,降解產(chǎn)物與PFP相互作用很小,不能形成有效的FRET。基于FRET效率的不同,實(shí)現(xiàn)了甲基化酶活性的檢測(cè)。該方法儀器簡(jiǎn)單、靈敏度高,檢出限為0.36 U/mL。方法對(duì)于相關(guān)疾病的臨床診斷及藥物篩選具有潛在的應(yīng)用價(jià)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.
【學(xué)位授予單位】：河北大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：O657.3

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 Pei Liu;Xiao-Hai Yang;Qing Wang;Jing Huang;Jian-Bo Liu;Ying Zhu;Lei-Liang He;Ke-Min Wang;;Sensitive detection of DNA methyltransferase activity based on rolling circle amplification technology[J];Chinese Chemical Letters;2014年07期

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本文編號(hào)：1872410