【摘要】：肽核酸(PNA)是以中性酰胺鍵為骨架的一種DNA類似物。用PNA分子做基因芯片的探針,有望解決基因芯片應(yīng)用在分子生物學(xué)和生物醫(yī)學(xué)等領(lǐng)域中存在的探針特異性與靈敏度之間的矛盾等問(wèn)題。本文合成了PNA單體,并制備出高密度的PNA芯片,主要工作如下： 對(duì)PNA芯片合成的虛擬掩模光刻系統(tǒng)進(jìn)行了介紹,同時(shí)給出了實(shí)驗(yàn)平臺(tái)中基于熒光試劑染色的曝光聚焦平面調(diào)節(jié)方法； 改進(jìn)現(xiàn)存的超序列算法,提出翻轉(zhuǎn)算法,并通過(guò)實(shí)驗(yàn)證明該算法能有效減少芯片原位合成制備循環(huán)次數(shù)；分析曝光過(guò)程中的衍射負(fù)效應(yīng),提出一種“拉伸”數(shù)學(xué)模型,降低由于光衍射引起的芯片合成中的堿基插入風(fēng)險(xiǎn)；在此基礎(chǔ)上,開(kāi)發(fā)了一套掩模圖生成軟件,可以輸出制備PNA芯片所需的虛擬掩模圖； 設(shè)計(jì)合成2-(2-硝基苯)丙氧羰基(NPPOC)基團(tuán)作為光敏保護(hù)基團(tuán),堿敏性的酰基為堿基環(huán)外氨基的保護(hù)基團(tuán)的PNA單體,并通過(guò)質(zhì)譜和核磁共振波譜對(duì)所得化合物的結(jié)構(gòu)進(jìn)行表征； 依照光脫保護(hù)法制備PNA芯片的流程開(kāi)展條件優(yōu)化實(shí)驗(yàn),制備出PNA微陣列,密度大于每平方厘米10000條探針。PNA芯片與熒光標(biāo)記的靶序列雜交實(shí)驗(yàn)結(jié)果顯示出正確的堿基正錯(cuò)配識(shí)別信息,證明了虛擬掩模光脫保護(hù)法制備PNA芯片技術(shù)的可行性,為推動(dòng)PNA芯片的應(yīng)用研究打下基礎(chǔ)。
[Abstract]:Peptide nucleic acid (PNA) is a DNA analogue with neutral amide bond as its skeleton. Using PNA molecule as probe of gene chip is expected to solve the contradiction between probe specificity and sensitivity in molecular biology and biomedicine. In this paper, PNA monomers are synthesized, and high density PNA chips are fabricated. The main work is as follows: the virtual mask lithography system synthesized by PNA chip is introduced. At the same time, the adjustment method of the exposure focusing plane based on fluorescent reagent staining in the experimental platform is given, and the existing supersequence algorithm is improved, and the flipping algorithm is proposed. The experiments show that the algorithm can effectively reduce the number of cycles of in-situ synthesis of the chip, analyze the negative effect of diffraction in the exposure process, and put forward a "stretch" mathematical model. On the basis of reducing the risk of base insertion in chip synthesis caused by optical diffraction, a mask map generation software is developed, which can output the virtual mask map needed for the fabrication of PNA chip. The 2- (2-nitrobenzene) propyl carbonyl (NPPOC) group was designed and synthesized as Guang Min protection group, and the alkali-sensitive acyl group was a PNA monomer with a protective group of base extracyclic amino groups. The structure of the compound was characterized by mass spectrometry and nuclear magnetic resonance spectroscopy, and the PNA microarray was prepared by optimizing the process of preparing PNA chip by optical protection method. The hybridization results of 10000 probes per square centimeter with fluorescence labeled target sequences show that the correct recognition information of base positive mismatch is obtained, which proves the feasibility of fabricating PNA chip by virtual mask photopolysis. It lays a foundation for the application research of PNA chip.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R318.6

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相關(guān)期刊論文 前1條

1 陳鳴,府偉靈,蔡國(guó)儒,劉明華,張波,陳慶海,俞凡;一種新型肽核酸基因傳感器陣列檢測(cè)系統(tǒng)的構(gòu)建[J];中華醫(yī)院感染學(xué)雜志;2004年01期

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