基于杂交链式反应信号放大和磁分离技术荧光检测端粒酶活性

doi:10.6023/A16030136

化学学报 ›› 2016, Vol. 74 ›› Issue (6): 513-517.DOI: 10.6023/A16030136 上一篇下一篇

研究论文

基于杂交链式反应信号放大和磁分离技术荧光检测端粒酶活性

张佳玉, 周晓毓, 周曼, 贾红霞

河北大学化学与环境科学学院保定 071002

投稿日期:2016-03-18 发布日期:2016-05-13
通讯作者: 贾红霞 E-mail:jia123renren@126.com
基金资助:
项目受国家自然科学基金青年基金(No. 21405032)和教育部博士点基金(No. 20121301120006)资助.

Detection of Telomerase Activity Based on Signal Amplification of Hybridization Chain Reaction Combining with Magnetic Separation

Zhang Jiayu, Zhou Xiaoyu, Zhou Man, Jia Hongxia

College of Chemistry and Environmental Science, Hebei University, Baoding 071002, China

Received:2016-03-18 Published:2016-05-13
Supported by:
Project supported by the National Natural Science Foundation of China (No. 21405032) and the Specialized Research Fund for the Doctoral Program of Higher Education of China (No. 20121301120006).

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摘要/Abstract

端粒酶是由RNA和蛋白质组成的一种核糖核蛋白酶, 它一般在癌细胞中被激活. 它与端粒DNA的不断复制以及癌细胞的不断增殖密切相关. 所以检测端粒酶的活性对癌症的早期诊断以及以端粒酶为靶标分子的抗癌药物的开发具有重要意义. 利用杂交链式反应(HCR)无酶放大检测信号, 建立了一种简单、快速的端粒酶活性检测方法. 端粒酶延伸产物是一条末端具有(ggttag)_n重复序列的DNA. 在实验过程中, 通过链霉亲合素与生物素的特异性作用将端粒酶延伸产物连接在磁性微球上. 设计一条端粒酶延伸产物特异性的DNA探针I作为杂交链式反应的引发探针. DNA探针I的3'-端与端粒酶延伸产物的重复序列匹配, 通过杂交, DNA探针I被固定在磁球上; DNA探针I的5'-端引发DNA探针II和探针III发生杂交链式反应. DNA探针II和探针III上都标记有荧光基团, 可以利用荧光直接进行信号检测. 在反应过程中, 通过磁分离去除多余未反应的三种DNA探针. 在优化条件下, 可以检测到1.0×10⁵个Hela细胞中的端粒酶活性. 该方法简单、快速、检测成本低, 分析全程无酶参与, 在肿瘤或癌症的临床诊断以及以端粒酶为靶标分子的抗癌药物的筛选上具有广阔的应用前景.

关键词: 端粒酶活性, 杂交链式反应, 无酶信号放大, 荧光检测, 磁分离

Telomerase is a ribonucleoprotein complex that is usually activated in the cancer cells and is closely related to telomere maintenance and immortalization of cancer cells. Telomerase activity detection is important for early diagnosis of human cancers as well as the screening of telomerase-target anti-cancer drugs. A new simple and fast method to detect the telomerase activity has been developed based on hybridization chain reaction (HCR) and magnetic separation. In the assay of experiment, the biotin-labeled telomerase substrate is elongated by telomerase generating a special DNA with repeated sequences-(ggttag)_n at their terminals. These telomerase elongated products are fixedly connected with streptavidin-coated magnetic beads through the specific combination of streptavidin with biotin. At the same time, other cell extracts are removed by magnetic separation. A specific DNA probe I is designed as the initiator of HCR. 3'-Terminus of DNA probe I is complementary with three repeated sequences of telomerase elongated product. So, DNA probe I could be fixed on magnetic bead through hybridization. 5'-Terminus of DNA probe I is in charge of triggering HCR with DNA probe II and probe III. DNA probe II and probe III are modified with fluorophores. So, the HCR amplification results can be easily detected by fluorescence. All of excessive DNA probes can be removed by magnetic separation. Under the optimal conditions, telomerase activity in 1.0×10⁵ Hela cells has been obviously detected. Because no enzyme involves in the signal amplification process of HCR, our proposed method can effectively avoid the interference of nonspecific amplification which usually exists in the polymerase amplification processes and increase the accuracy of the test results. Furthermore, enzyme-free signal amplification can effectively avoid the potential interference of telomerase inhibitor to the enzyme activity and improve the reliability of screening of telomerase inhibitors.

Key words: telomerase activity, hybridization chain reaction, enzyme-free signal amplification, fluorescence detection, magnetic separation

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张佳玉, 周晓毓, 周曼, 贾红霞. 基于杂交链式反应信号放大和磁分离技术荧光检测端粒酶活性[J]. 化学学报, 2016, 74(6): 513-517.

Zhang Jiayu, Zhou Xiaoyu, Zhou Man, Jia Hongxia. Detection of Telomerase Activity Based on Signal Amplification of Hybridization Chain Reaction Combining with Magnetic Separation[J]. Acta Chim. Sinica, 2016, 74(6): 513-517.

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基于杂交链式反应信号放大和磁分离技术荧光检测端粒酶活性

Detection of Telomerase Activity Based on Signal Amplification of Hybridization Chain Reaction Combining with Magnetic Separation

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