核酸探针的制备及初步应用

发布时间：2018-10-05 09:36

【摘要】：核酸是生命遗传信息的携带者和传递者,它不仅对生物的延续、生物物种遗传特性的保持、生长发育、细胞分化等起着重要的作用。而且与生物变异,如肿瘤、遗传病等也密切相关,因此研究核酸,尤其是核酸的结构和功能,将有助于人们从分子水平上了解生命现象的本质。核酸探针技术是研究核酸结构与功能的重要手段。核酸探针的制备和标记是核酸探针技术发展的关键,因此研究建立新的制备探针技术具有重要的理论意义和应用价值。本论文针对核酸分子“光开关”和磁性纳米粒子-ssDNA这两种核酸探针的制备和应用进行了较为系统的研究,取得了以下几个有意义的结果: (1) 制备了一种具有核酸分子“光开关”性质的荧光试剂-Ru(bipy)_2(dppz)~(2+),并选用Ru(bipy)_2(dppz)~(2+)和苯胺红T 分别作为探针,研究PNA-DNA、dsDNA 之间的差异性。通过相同条件下同一荧光探针与PNA-DNA、dsDNA 两者之间的不同作用,来说明PNA-DNA、dsDNA 结构具有一定的差异,并对引起差异性的原因进行了初步的探讨。 (2) 建立了一种基于磁性纳米粒子的不对称PCR 技术,用于制备磁性纳米粒子-ssDNA 探针。将一对引物中的一条引物共价键合到磁性纳米粒子表面,限制另一条引物的量,一定条件下能够进行PCR 扩增,并且可以获得大量连有纳米粒子的目标ssDNA。磁性纳米粒子的存在可以方便扩增产物的分离,在外磁场中即可实现产物的分离和纯化。所得的磁性纳米粒子-ssDN 可以作为探针,应用于生物芯片、基因测序、基因导弹等方面的研究。 (3) 由于纳米粒子对结合在表面的DNA 有保护作用,可以有效防止核酸酶的降解,因此将核酸探针结合到纳米粒子表面后可以很好地用于细胞或活体内的分析、检测,同时,纳米粒子的存在还可以实现探针大量、定向地导入其中。本文设计了针对端粒酶RNA 的反义脱氧核苷酸,将其键合到核壳型磁性纳米粒子表面后,导入HL-60 细胞中。原子力学显微镜观察的结果表明:通过细胞吞噬作用,磁性纳米粒子-ssDNA 可以大量地导入HL-60 细胞中,并能有效地诱导HL-60细胞凋亡。
[Abstract]:Nucleic acid is the carrier and transporter of life genetic information. It not only plays an important role in the continuation of organisms, the maintenance of genetic characteristics of biological species, growth and development, cell differentiation and so on. Moreover, it is closely related to biological variation, such as tumor, genetic disease, etc. Therefore, studying the structure and function of nucleic acid, especially nucleic acid, will help people to understand the nature of life phenomenon at molecular level. Nucleic acid probe technique is an important method to study the structure and function of nucleic acid. The preparation and labeling of nucleic acid probe is the key to the development of nucleic acid probe technology. In this thesis, the preparation and application of two nucleic acid probes, called "optical switch" and magnetic nanoparticles (-ssDNA), have been studied systematically. Some significant results have been obtained as follows: (1) A fluorescent reagent Ru (bipy) _ 2 (dppz) ~ (2) with the properties of nucleic acid "optical switch" was prepared, and Ru (bipy) _ 2 (dppz) ~ (2) and aniline red T were used as probes, respectively. To study the differences between PNA-DNA,dsDNA. The difference of PNA-DNA,dsDNA structure between the same fluorescent probe and PNA-DNA,dsDNA under the same conditions was proved, and the causes of the difference were discussed. (2) an asymmetric PCR technique based on magnetic nanoparticles was developed to prepare magnetic nanoparticles-ssDNA probes. Covalently bonding one pair of primers to the surface of magnetic nanoparticles, limiting the amount of another primer, PCR amplification can be carried out under certain conditions, and a large number of target ssDNA. with nanoparticles can be obtained. The existence of magnetic nanoparticles can facilitate the separation of amplified products, and the separation and purification of the products can be realized in the external magnetic field. The magnetic nanoparticles-ssDN can be used as probes in the research of biochip, gene sequencing, gene missile and so on. (3) because nanoparticles can protect the surface bound DNA, it can effectively prevent the degradation of nuclease, so the nucleic acid probe can be well used in cell or in vivo analysis, detection, at the same time, The existence of nanoparticles can also achieve a large number of probes, directed into them. Antisense deoxynucleotides (antisense deoxynucleotides) targeting telomerase RNA were designed and bonded to the surface of core-shell magnetic nanoparticles and transfected into HL-60 cells. The results of atomic mechanics microscope showed that magnetic nanoparticles-ssDNA could be introduced into HL-60 cells by phagocytosis and induce apoptosis of HL-60 cells effectively.
【学位授予单位】：上海师范大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：Q78

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