基于发卡型DNA自组装及纳米金比色一步法检测核酸的新技术研究

发布时间：2018-12-07 08:17

【摘要】：核酸作为生命体重要的遗传物质,它的检测尤其重要,实现对核酸快速、简单、可视化的检测在生化分析领域具有重要的意义。无论是变温的核酸扩增反应,还是等温的核酸扩增反应,都是酶促反应,这类型的反应对实验条件的高要求限制了核酸检测的适用范围。所以无酶DNA自组装反应的提出,不仅在核酸检测领域,同样在DNA纳米结构和体内检测领域具有一定的应用价值。首先,本论文的第一章在无酶的指数发卡自组装反应的基础上,结合芘分子,展开指数发卡自组装反应的荧光检测,并对方法进行了验证,但方案不能满足核酸检测的要求。因此,调整实验方案,选用指数发卡自组装反应结合荧光共振能量转移对Cy3/Cy5,进行单链核酸的检测。为获得最佳的能量传递效率,对实验条件进行了优化。对方法的灵敏度进行的验证结果表明,方法的检测限为10 pM;利用添加10%胎牛血清的细胞培养基对方法的抗干扰能力进行检测,结果表明方法具有很好的抗干扰能力;另外,通过对不同错配靶标的检测,证明了该方法具有良好的特异性。其次,本课题组的研究发现,PEG 200能显著提高链置换的速率,而DNA自组装反应是DNA链之间自发的置换与杂交过程。因此我们设想PEG 200也可能促进DNA自组装反应。在论文的第三章中,选取六种常用于核酸扩增的小分子物质,将它们用于杂交链式反应反应并比较了小分子物质对杂交链式反应的影响,结果表明PEG 200为最佳的促进杂交链式反应的小分子物质,然后将PEG200用于指数发卡自组装反应,结果显示PEG 200同样能促进指数发卡自组装反应。而且,在20%的PEG 200的促进下,杂交链式反应的速率被提高100倍左右,指数发卡自组装反应的速率被提高10倍左右。最后,论文的第四章以纳米金比色为基础,从修饰后的纳米金的抗盐能力以及在加入靶标链后的变色时间及颜色变化情况比较了全修饰和不对称修饰两种纳米金修饰方式,根据实验结果确定全修饰的纳米金适合用于核酸扩增反应。然后将全修饰的纳米金用于聚合酶链式反应、环介导核酸等温扩增反应中。结果显示全修饰的纳米金对核酸扩增反应有抑制作用,通过添加BSA可减少纳米金对反应的抑制作用。但是实验的比色结果表明,该方法还不能完成明显的纳米金比色。我们认为,可以通过优化实验条件,找到集纳米金比色技术与核酸扩增反应于一体的一步核酸比色检测法。
[Abstract]:Nucleic acid is an important genetic material in organism, and its detection is especially important. It is of great significance to detect nucleic acid quickly, easily and visually in the field of biochemical analysis. Both the reaction of nucleic acid amplification at varying temperature and the reaction of isothermal nucleic acid amplification are enzymatic reactions. The high requirements of these reactions limit the scope of application of nucleic acid detection. Therefore, the self-assembly reaction of non-enzymatic DNA not only in the field of nucleic acid detection, but also in the field of DNA nanostructure and in vivo detection has certain application value. In the first chapter of this paper, based on the enzymatic exponential card self-assembly reaction and pyrene molecule, the fluorescence detection of exponential card self-assembly reaction is carried out, and the method is verified, but the scheme can not meet the requirements of nucleic acid detection. Therefore, the single strand nucleic acid detection of Cy3/Cy5, was carried out by adjusting the experimental scheme and using exponential card self-assembly reaction and fluorescence resonance energy transfer. In order to obtain the best energy transfer efficiency, the experimental conditions were optimized. The test results show that the detection limit of the method is 10 pM; and the anti-interference ability of the method is detected by adding 10% fetal bovine serum to the cell culture medium. The results show that the method has good anti-interference ability. In addition, the detection of different mismatch targets proves that the method has good specificity. Secondly, our study shows that PEG 200 can significantly increase the rate of chain substitution, while the DNA self-assembly reaction is a spontaneous substitution and hybridization process between DNA strands. Therefore, we assume that PEG 200 may also promote DNA self-assembly reaction. In the third chapter of the thesis, six kinds of small molecules which are often used in nucleic acid amplification are selected and used in hybridization chain reaction and the effects of small molecules on hybridization chain reaction are compared. The results show that PEG 200 is the best small molecule to promote hybrid chain reaction, and then PEG200 is used in exponential card self-assembly reaction. The results show that PEG 200 can also promote exponential card self-assembly reaction. Moreover, with the promotion of 20% PEG 200, the rate of hybrid chain reaction was increased by about 100 times, and the rate of exponential hairpin self-assembly reaction was increased by about 10 times. Finally, the fourth chapter of the thesis based on nano-gold colorimetry, from the modified nano-gold salt resistance, after adding the target chain color change time and color changes, compared the full modification and asymmetric modification of two kinds of nano-gold modification methods. According to the experimental results, the fully modified gold nanoparticles are suitable for nucleic acid amplification. Then the fully modified gold nanoparticles were used in polymerase chain reaction and cyclized nucleic acid isothermal amplification reaction. The results showed that the fully modified gold nanoparticles could inhibit the nucleic acid amplification reaction, and the addition of BSA could reduce the inhibition effect of gold nanoparticles on the reaction. However, the experimental colorimetric results show that the method can not complete the apparent gold nanocrystalline colorimetry. We think that the one step nucleic acid colorimetry method can be found by optimizing the experimental conditions and combining the gold colorimetric technique with nucleic acid amplification.
【学位授予单位】：青岛科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78

【参考文献】