三芳基甲烷类化合物对凝胶分离DNA检测的机理研究

Gel electrophoresis is one of the most reliable methods available for the research of DNA, making the detection techniques for gel separated DNA play an important role in life science. In our previous experiments, we have applied victoria pure blue BO (VPBBO), one of the triarylmethane dyes, to DNA visualization in agarose and polyacrylamide gels. The VPBBO detection method is convenient and safe, and its sensitivity is similar to, or higher than that of ethidium bromide (EB). Since the lack of in-depth study of the mechanism, the bottleneck of gel separated DNA detection technology still couldn't be overcomed. In the present project, ultraviolet-visible spectroscopy, fluorescence spectroscopy, electrochemical technique, viscosity method, mass spectrometry, NMR and molecular docking will be used to investigate the detection mechanism. Furthermore, the structure-activity relationship between triarylmethane dyes and DNA will also be established and series of VPBBO analogues will be synthesized in this project. Finally, based on the established detection mechanism, we will develop novel DNA detection method with more advantages. The results of our research will be helpful to break the technical bottleneck of gel separated DNA detection method in theory, and then provide strong support to the rapid development of life science.

凝胶电泳是研究DNA最常用手段之一，使得凝胶上DNA的检测技术对生命科学的相关研究起着极为重要的作用。前期我们已在凝胶上成功建立了以碱性艳蓝BO（VPBBO）为代表的三芳基甲烷类染料检测DNA的方法，该方法操作简单、生物安全性好，灵敏度可与溴化乙锭（EB）相媲美，甚至更高。然而由于缺乏对检测机理的深入研究，尚无法突破当前凝胶分离DNA检测的技术瓶颈。本课题拟在分子水平上借助紫外吸收光谱法、荧光光谱法、电化学法、黏度法、质谱法、核磁共振法、分子对接等技术手段对以VPBBO为代表的三芳基甲烷类染料检测DNA的机理进行深入研究；找出三芳基甲烷类化合物与DNA结合的构效关系；同时在此基础上设计合成系列VPBBO类似物。最后在上述检测机理的指导下，摸索更具优势的新型DNA检测方法。本项目的实施将从理论上突破DNA检测的技术瓶颈，为生命科学研究的飞速发展提供有力的支撑。

目的：（1）研究开发快速、灵敏、亲环境的DNA检测方法，为聚丙烯酰胺凝胶和琼脂糖凝胶上DNA的检测提供合适的方法。（2）探究凝胶上DNA的检测机理，阐明VPBBO染料与核酸的相互作用，探讨了其作用方式和作用机理，将有助于人们从分子水平上了解检测机制。.方法：（1）DNA染料检测方法：选用碱性艳蓝BO（VPBBO）染料，考察影响检测结果的各个因素，如染色液的浓度及组成、染色时间、脱色时间及各种盐离子对检测结果的影响等，建立DNA染料检测操作步骤。（2）为了能更深入的研究染料检测的机理，通过采用光谱法、粘度法、分子对接模拟、质谱法等多种研究手段来共同研探究染料与DNA的相互作用。.结果：（1）分别在琼脂糖凝胶和聚丙烯酰胺凝胶上建立了DNA染料检测方法，灵敏度分别为0.4-0.8 ng/band和0.8-1.6 ng/band，与EB荧光染色法相似。（2）通过采用光谱法、粘度法、分子对接模拟法、质谱法等多种研究手段来共同研探究染料与DNA的结合的机制，VPBBO染料和DNA以沟槽形式相结合。通过阐明VPBBO染料与核酸的相互作用，探讨了其作用方式和作用机理，将有助于人们从分子水平上了解检测机制。.成果：本项目已发表SCI论文9篇，申请发明专利4项，培养硕士研究生3名。综上所述，本项目已完成了课题规定的任务。