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

1. 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

1. 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).

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.