摘要
随着第一台核酸合成仪的诞生和DNA化学合成、标记技术取得的重大突破,核酸分子探针的应用得到了飞速发展。由于核酸探针设计简单、稳定性好、易于合成、信号转导机制灵活,因而被广泛的应用到化学、医学和生物学等领域。至今,核酸探针已经发展出许多类型,如经典的RNA探针、cDNA探针及基因组DNA探针等,新型的阴阳探针、TaqMan探针、Padlock探针、核酸识体(aptamer)探针、分子信标探针等。自从Tyagi和Krammer于1996年发明分子信标探针以来,发夹型的核酸探针作为一种高选择性和高灵敏性的新型荧光探针得到了广泛的应用。随着对发夹型核酸探针研究的深入及新型发夹型核酸探针的出现,人们发现发夹型核酸探针不仅适用于体外蛋白质和核酸的分析,同样也适用于活体分析。它还可以用于核酸与药物小分子、蛋白质等相互作用的研究,是一种极好的研究核酸与蛋白质相互作用的工具,也是一种极具发展前景的新型核酸探针。发夹型核酸探针与传统的核酸探针相比,能很好的分辨单碱基错配,且能用于实时监测。核酸探针的标记物有生物素、放射性物质和荧光基团等。其中,荧光分子标记由于对生物分子影响较小、灵敏度高、且分析检测手段简单,成为最常用的标记物。本论文着眼于发夹型DNA荧光探针新方法的研究,主要开展以下三个方面的工作:
(1)基于时间分辨荧光技术,设计了一种利用铕离子配合物作为荧光基团的发夹型荧光探针。由于铕离子配合物的荧光寿命长,用于生物样品检测时能有效消除背景荧光,提高了探针检测的灵敏度。
(2)基于汞离子与DNA的T碱基的特异性结合作用和荧光共振能量转移(fluorescence resonance energy transfer, FRET),设计了以T-Hg2+-T结构为茎部的发夹型DNA荧光探针,并用此DNA荧光探针实现了生物硫的高灵敏度检测。
(3)基于一些杂环荧光小分子能与DNA缺失位点中的碱基形成氢键结合的特性,设计了一种免标记发夹型荧光核酸探针,考察了这种核酸探针的实验条件,并对其合理性进行了验证。
With the birth of the first nucleic acid synthesis and DNA chemical synthesis, markers get break through, a nucleic acid molecule probe have developed rapidly. Because the nucleic acid probe is simple in design, good stability, easy synthesis, signal mechanism flexible, by widespread application to chemistry, medicine and biology, etc. So far, many types of nucleic acid probe has developed, such as classic ucleic acid probe including RNA probe, cDNA probes and genomic DNA probe etc, the new-style nucleic acid probe including Yin Yang probe,TaqMan probe, Padlock probe, Aptamer probe, molecules beacon probe , etc.
Since Tyagi and Krammer invent the molecular beacon probe in 1996, hairpin nucleic acid probe as a high selectivity and high sensitivity of new fluorescent probe been widely used. Along with the further research of hairpin nucleic acid probe and the emergence of new types hairpin nucleic acid probe, people found hairpin nucleic acid probe not only applicable in the analysis of protein and nucleic acid in vitro, also suitable for living analysis. It can also be used to analyt the interaction of drug small molecules, protein and nucleic acid, it is a powerful tool to research nucleic acid and protein interaction, also is a kind of extremely promising new nucleic acid probe. Contract with traditional nucleic acid probe, the hairpin probe can be used in detecting single base mismatch and real-time monitoring. The nucleic acid probe marker is biotin, radioactive substances and fluorescence group, etc. Among them, the fluorescent signal is less influence of biological molecules, high sensitivity and simple, become the most commonly markers. This paper focus on hairpin type of DNA fluorescence probe new method research, mainly in the following three areas:
Based on room-temperature phosphorescence, designed a hairpin probe which using Eu~(3+) ion complexes as fluorescence group. Due to the Eu~(3+) ion complexes' have a long luminescence lifetime, when used in biological samples can effectively reduce the background, improves the sensitivity.
Based on mercury ion competitive ligation and fluorescence resonance energy transfer (FRET), designed a hairpin probe which used the structure of T-Hg2+-T for stem, and it is used to detection biological sulfur with high sensitivity.
Based on some heterocyclic fluorescence small molecules can inset in Abasic site, designed a label-free hairpin fluorescence nucleic acid probe, and investigated experimental conditions' s effort, also verified the rationality.
引文
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