虚拟掩模光脱保护法制备高密度肽核酸芯片
发布时间：2018-10-08 12:04
【摘要】：肽核酸(PNA)是以中性酰胺键为骨架的一种DNA类似物。用PNA分子做基因芯片的探针,有望解决基因芯片应用在分子生物学和生物医学等领域中存在的探针特异性与灵敏度之间的矛盾等问题。本文合成了PNA单体,并制备出高密度的PNA芯片,主要工作如下： 对PNA芯片合成的虚拟掩模光刻系统进行了介绍,同时给出了实验平台中基于荧光试剂染色的曝光聚焦平面调节方法； 改进现存的超序列算法,提出翻转算法,并通过实验证明该算法能有效减少芯片原位合成制备循环次数；分析曝光过程中的衍射负效应,提出一种“拉伸”数学模型,降低由于光衍射引起的芯片合成中的碱基插入风险；在此基础上,开发了一套掩模图生成软件,可以输出制备PNA芯片所需的虚拟掩模图； 设计合成2-(2-硝基苯)丙氧羰基(NPPOC)基团作为光敏保护基团,碱敏性的酰基为碱基环外氨基的保护基团的PNA单体,并通过质谱和核磁共振波谱对所得化合物的结构进行表征； 依照光脱保护法制备PNA芯片的流程开展条件优化实验,制备出PNA微阵列,密度大于每平方厘米10000条探针。PNA芯片与荧光标记的靶序列杂交实验结果显示出正确的碱基正错配识别信息,证明了虚拟掩模光脱保护法制备PNA芯片技术的可行性,为推动PNA芯片的应用研究打下基础。
[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.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R318.6
本文编号：2256677
[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.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R318.6
【参考文献】
相关期刊论文 前1条
1 陈鸣,府伟灵,蔡国儒,刘明华,张波,陈庆海,俞凡;一种新型肽核酸基因传感器阵列检测系统的构建[J];中华医院感染学杂志;2004年01期
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