本文選題：納米金　切入點(diǎn)：DNA　出處：《山東大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：基因是遺傳的物質(zhì)基礎(chǔ),控制著人類的生老病死�；驒z測對一些疾病的診斷和預(yù)防具有重要的意義。乳腺癌是女性最常見的惡性腫瘤之一,嚴(yán)重威脅著女性的健康。乳腺癌易感基因(BRCA1)在乳腺細(xì)胞周期的調(diào)控、基因轉(zhuǎn)錄調(diào)節(jié)和維持基因穩(wěn)定性中起著重要的作用。BRCA1的突變則會削弱這些功能,從而增加乳腺癌發(fā)病的危險度。因此對該基因進(jìn)行檢測對乳腺癌的早期診斷和癌癥預(yù)測具有重要的意義。 近年來,納米材料的迅速發(fā)展為分子生物學(xué)提供了新的途徑。納米材料的特殊性質(zhì)如表面效應(yīng)、小尺寸效應(yīng)、量子效應(yīng)和宏觀量子隧道效應(yīng)等使其在疾病診斷、環(huán)境監(jiān)控、藥物研究及法醫(yī)鑒定等領(lǐng)域起著重要的作用。納米金作為一種重要的納米材料,在DNA的檢測中被作為檢測探針、固定載體和熒光猝滅劑廣泛使用。 方法的靈敏度和簡便性是DNA檢測中的重要部分。本論文建立了兩種不同的DNA熒光檢測方法,兩種方法均利用單雙鏈DNA在納米金表面不同的吸附性質(zhì)和納米金作為熒光猝滅劑的特性,探索提高DNA檢測靈敏度和實(shí)現(xiàn)快速檢測的新途徑。 論文共分為三個部分： 一、概述了DNA的結(jié)構(gòu)及常見的檢測方法。著重介紹了DNA熒光探針、納米金的特性、合成方法及其在DNA檢測中的應(yīng)用。 二、建立了一種基于納米金猝滅羅丹明B熒光的免標(biāo)記法檢測DNA。該方法同時利用了單雙鏈DNA在納米金表面的不同吸附性質(zhì)和不同聚集程度的納米金對染料猝滅能力不同的性質(zhì),基于靶目標(biāo)的加入引起的體系熒光信號的增強(qiáng),實(shí)現(xiàn)了DNA的高靈敏檢測。在最佳實(shí)驗(yàn)條件下,該方法的檢出限為2.9×10-13 molL-1。該方法無需進(jìn)行任何標(biāo)記,檢測過程簡單快速。 三、建立了一種基于納米金和核酸外切酶Ⅲ的循環(huán)熒光放大法檢測DNA。該方法同樣將納米金作為猝滅劑,不僅利用了單雙鏈DNA在納米金表面不同的吸附性質(zhì),同時還引入了核酸外切酶Ⅲ對雙鏈DNA的水解作用實(shí)現(xiàn)了熒光信號的放大,從而提高DNA檢測的靈敏度。一方面,納米金的引入簡化了降低體系的背景信號的過程；另一方面在核酸外切酶的作用下,靶目標(biāo)被釋放后重新參與雜交,使得體系的熒光信號從恢復(fù)到增強(qiáng),實(shí)現(xiàn)DNA的信號放大檢測。在最佳實(shí)驗(yàn)條件下,在2.5×10-11mol L-1～1.0×10-9mol L-1的濃度范圍內(nèi),體系的△F值和目標(biāo)DNA的濃度呈現(xiàn)良好的線性關(guān)系,檢測限為1.0×10-11mol L-1。
[Abstract]:Gene is the material basis of heredity and controls the birth, old and death of human beings. Gene detection is of great significance in the diagnosis and prevention of some diseases. Breast cancer is one of the most common malignant tumors in women. Breast cancer susceptibility gene BRCA1) plays an important role in the regulation of breast cell cycle, gene transcription regulation and maintaining gene stability. The mutation of BRCA1 weakens these functions. Therefore, detection of this gene is of great significance for early diagnosis and cancer prediction of breast cancer. In recent years, the rapid development of nanomaterials has provided a new way for molecular biology. The special properties of nanomaterials such as surface effect, small size effect, quantum effect and macroscopic quantum tunneling effect make them in disease diagnosis and environmental monitoring. As an important nanomaterial, gold nanoparticles are widely used as probe, stationary carrier and fluorescence quenching agent in the detection of DNA. The sensitivity and simplicity of the method is an important part of DNA detection. In this paper, two different DNA fluorescence detection methods were established. The two methods use the different adsorption properties of single and double stranded DNA on the surface of gold nanoparticles and the characteristics of gold nanoparticles as fluorescence quenching agents to explore a new way to improve the detection sensitivity of DNA and to realize rapid detection. The thesis is divided into three parts:. The main contents are as follows: 1. The structure and common detection methods of DNA are summarized, especially the fluorescent probe of DNA, the characteristics of nanocrystalline gold, the synthetic method and its application in the detection of DNA. II. An immunolabelling method based on the fluorescence quenching of Rhodamine B by nanocrystalline gold was developed. The different adsorption properties of single and double stranded DNA on the surface of gold nanoparticles and the different properties of the quenching ability of gold nanoparticles with different aggregation degree on the dye surface were used in this method. The high sensitivity detection of DNA is realized based on the enhancement of fluorescence signal caused by the addition of target. Under the optimum experimental conditions, the detection limit of this method is 2.9 脳 10 ~ (-13) mol ~ (-1) mol 路L ~ (-1). This method does not need any labeling, and the detection process is simple and rapid. Thirdly, a cyclic fluorescence amplification method based on nanocrystalline gold and nucleic acid exonuclease 鈪，

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