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化学学报
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化学学报 ›› 2011, Vol. 69 ›› Issue (12): 1450-1456. 上一篇    下一篇

研究论文

基于Hoechst33258荧光染料检测单链DNA的方法研究

曾国平1,2，向东山2，何治柯*,2   

  1. (1 湖北第二师范学院 武汉 430205)
    (2 武汉大学化学与分子科学学院 生物医学分析化学教育部重点实验室 武汉 430072)
  • 投稿日期:2010-09-26 修回日期:2010-12-29 发布日期:2011-02-25
  • 通讯作者: 何治柯 E-mail:zhkhe@whu.edu.cn
  • 基金资助:

    国家自然科学基金项目

Fluorimetric Method for the Determination of Sequence-Specific DNA with the Fluorescent Dye Hoechst 33258

Zeng Guoping1,2 Xiang Dongshan2 He Zhike*,2   

  1. (1 Hubei University of Education, Wuhan 430205)
    (2 Key Laboratory of Analytical Chemistry for Biology and Medicine, Ministry of Education, College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072)
  • Received:2010-09-26 Revised:2010-12-29 Published:2011-02-25

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2229

被引次数

21

根据Hoechst与ssDNA作用时不产生荧光或荧光很弱, 而与dsDNA作用时荧光增强的原理, 将待测ssDNA与互补ssDNA杂交形成dsDNA, 实现Hoechst染料对ssDNA的检测. 文中研究了不同序列、不同长度、互补链及碱基错配链等杂交产物与Hoechst染料作用的荧光强度的变化规律. 同时以H1N1禽流感病毒DNA特定序列为例建立了定量检测ssDNA的新方法. 线性范围为5.6×10－10～3.0×10－8 mol/L, 线性关系: y＝24.005x＋0.4858, R2＝0.9967, 检出限为4.8×10－10 mol/L. 该方法具有操作简单, 检测快速, 灵敏度高和选择性好等优点.

关键词: 荧光染料Hoechst, ssDNA, 杂交, 荧光光谱, 定量分析

A fluorimetric method is developed for the sequence-specific DNA detection with the Hoechst, which displays no photoluminescence in the presence of ssDNA but enhances great in the presence of dsDNA. The interactions between Hoechst and ssDNA with different base sequence and different sequence length were investigated in detail. On the basis of that, the detection of sequence-specific DNA related to the avian influenza A H1N1 virus was established. Under the optimum conditions, the linear range is 5.6× 10－10～3.0×10－8 mol/L, the calibration curve is y＝24.005x＋0.4858, R2＝0.9967. The detection limit of 4.8×10－10 mol/L could be estimated using 3σ (where σ was the relative standard deviation of a blank solution, n＝11). This method is simple, rapid, sensitive and selective.

Key words: fluorescent dye hoechst, sequence-specific DNA, hybrid, fluorescence spectra, quantitative detection

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