本文關鍵詞：信號放大新策略與DNA檢測新方法研究　出處：《南京大學》2012年碩士論文　論文類型：學位論文

  更多相關文章： 信號 大新 策略 檢測 方法研究

【摘要】：隨著人類基因組計劃的完成,人類已經(jīng)進入基因革命的時代。特定序列DNA的檢測對于簡單的疾病診斷和重大的醫(yī)療評估都有著巨大的研究價值,因此,需要發(fā)展一系列簡單、靈敏、便攜的DNA診斷設備。這些設備不僅可以在實驗室分析中起作用,而且在臨床診斷、環(huán)境監(jiān)測、食品污染、法醫(yī)等領域也發(fā)揮著重要的作用。本文致力于核酸檢測新方法研究,發(fā)展了兩種核酸分析新方法與一種端粒酶活性檢測新技術。 1金納米聚沉的銀放大方法用于DNA可視化比色分析方法 發(fā)展了一種新的DNA可視化比色分析方法。該方法巧妙地結合金屬離子穩(wěn)定的分子信標與基于金納米粒子增強的銀放大方法。這種檢測策略利用目標DNA與汞離子對連接DNA的競爭反應。當不存在目標DNA的時候,連接DNA可以與汞離子結合,形成一個分子信標。這個分子信標可以避免腺苷酸功能化的金納米粒子的聚沉。而當存在目標DNA時,它與連接DNA的結合能力大于汞離子,目標DNA與連接DNA的結合就會打開分子信標。這時,打開的分子信標就會與金納米粒子上的腺苷酸雜交反應,造成金納米粒子的聚沉。而金納米粒子的聚沉會減弱銀增強的效果從而減小玻板上的點的相對灰度值。最后利用掃描法檢測,就可以方便地檢測目標DNA的濃度,線性范圍從1.0-30nM。在相同汞離子的濃度下,單堿基錯配的DNA相對灰度值只有完全匹配DNA的22%,顯示了良好的選擇性。這種利用金屬離子穩(wěn)定的分子信標的新型DNA比色檢測方法,具有簡單、價廉、方便的優(yōu)點,在臨床應用上具有巨大的潛力。 2.基于目標增強放大與滾環(huán)擴增的超靈敏核酸檢測新方法 用量子點作為電化學標記物,結合內(nèi)切酶放大技術和滾環(huán)擴增放大技術,發(fā)展了一種超靈敏的電化學檢測方法。目標DNA、幫助DNA及分子信標三者會生成-個穩(wěn)定的Y型結構,這種結構可以被切口內(nèi)切酶所識別。被內(nèi)切酶剪開的分子信標可以作為滾環(huán)擴增反應的初始引物。而DNA功能化的量子點可以通過雜交反應連接上滾環(huán)擴增的產(chǎn)物。這樣,通過稀硝酸溶解量子點,與目標濃度相關的電化學信號可以方便地用方波伏安法讀出。由于采用了特異性好的標記DNA方法與級聯(lián)信號放大策略,本方法的檢測下限可達11aM并有六個數(shù)量級的線性范圍(1×10-17到1×10-11M),而且可以特異性地識別錯配DNA。這種靈敏特異的DNA檢測方法在基因研究中有著巨大的應用潛力。 3.基于酶放大細胞中端粒酶活性檢測 結合分子信標技術和內(nèi)切酶放大技術發(fā)展了一個簡單方便的Hela細胞中端粒酶活性檢測的新方法。通過引物在端粒酶的存在下會擴增出特定序列的性質(zhì),再結合酶放大技術,可以方便地通過熒光方法來檢測端粒酶的活性。本方法可以檢測出10-1000個Hela細胞中端粒酶的活性,是一種方便、簡單的端粒酶活性檢測方法。
[Abstract]:With the completion of the Human Genome Project, humans have entered the era of genetic revolution. The detection of specific sequences of DNA has great research value for simple disease diagnosis and major medical evaluation. There is a need to develop a series of simple, sensitive, portable DNA diagnostic devices that can be used not only in laboratory analysis, but also in clinical diagnosis, environmental monitoring, and food contamination. In this paper, two new methods for nucleic acid analysis and a new technique for detecting telomerase activity have been developed. Application of silver amplification method to DNA visual colorimetric analysis A new visual colorimetric analysis method for DNA is developed. The method combines metal ion stabilized molecular beacon with silver amplification method based on gold nanoparticles enhancement. The detection strategy uses target DNA. The competitive reaction with mercury ions to connect to DNA. When there is no target DNA. DNA binds to mercury ions to form a molecular beacon that avoids the accumulation of gold nanoparticles functionalized by adenylate. When the target DNA exists. Its binding ability to DNA is greater than that of mercury ions, and the binding of target DNA to DNA will open molecular beacons. In this case, the open molecular beacons will be hybridized with adenylic acid on gold nanoparticles. The gold nanoparticles will weaken the silver enhancement effect and reduce the relative gray value of the points on the glass plate. Finally, the scanning method is used to detect the gold nanoparticles. The concentration of target DNA can be easily detected. The linear range is from 1.0-30nM. under the same concentration of mercury ions, the relative gray value of DNA with single base mismatch is only 22% of that of DNA. This new DNA colorimetric method using molecular beacon stabilized by metal ions has the advantages of simple, inexpensive and convenient, and has great potential in clinical application. 2. A novel method for detection of hypersensitive nucleic acid based on target enhanced amplification and rolling amplification A highly sensitive electrochemical detection method, target DNA, was developed by using quantum dots as electrochemical markers, combined with endonuclease amplification technique and roller-ring amplification technique. Help DNA and molecular beacons will generate a stable Y-shaped structure. This structure can be recognized by the incisional endonuclease. The molecular beacons cut by the endonuclease can be used as the initial primers for the ring-amplification reaction. The DNA functionalized quantum dots can be linked to the ring-up-loop amplification by hybridization reaction. The product. Like this. The electrochemical signals related to the target concentration can be easily read by square wave voltammetry by dilute nitric acid dissolved quantum dots. Due to the use of specific labeling DNA method and cascade signal amplification strategy. The detection limit of this method is up to 11am and has six linear ranges of 1 脳 10-17 to 1 脳 10-11M). This sensitive and specific DNA detection method has great potential in gene research. 3. Detection of telomerase activity based on enzyme amplification A simple and convenient method for detecting telomerase activity in Hela cells was developed by combining molecular beacons and endonuclease amplification techniques. The specific sequence properties could be amplified by primers in the presence of telomerase. Combined with enzyme amplification technique, telomerase activity can be easily detected by fluorescence method. This method can detect telomerase activity in 10-1000 Hela cells, which is a convenient method. A simple method for detecting telomerase activity.
【學位授予單位】：南京大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R346

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