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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1.0×10~5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.
Telomerase is a ribonucleoprotein composed of RNA and protein, and it is usually activated in cancer cells.It is closely related to the continuous replication of telomere DNA and the continuous proliferation of cancer cells.Therefore, the detection of telomerase activity The early diagnosis of cancer and the development of anti-cancer drugs targeting telomerase are of great significance.Using hybridization chain reaction (HCR) without amplification of the detection signal, a simple and rapid detection method of telomerase activity The telomerase extension is a piece of DNA that has a (ggttag) _n repeat at its end, and the telomerase extension is attached to the magnetic microspheres through the specific interaction of streptavidin with biotin during the experiment. Design a telomerase extension product specific DNA probe I as a hybridization chain reaction initiation probe DNA probe I 3’-end and telomerase extension product of the repeated sequence matching, by hybridization, DNA probes I was immobilized on the magnetic ball; the 5’-end of DNA probe I initiates a hybridization chain reaction between DNA probe II and probe III.Fluorophores are labeled on both DNA probe II and probe III and can be utilized Fluorescence direct signal detection in the reaction process , The magnetic separation to remove excess unreacted three DNA probes under the optimal conditions, can detect 1.0 × 10 ~ 5 Hela cells in the telomerase activity of the method is simple, rapid, low detection costs, analysis of the whole No enzyme involved in the clinical diagnosis of cancer or cancer and telomerase as a target molecule screening of anticancer drugs has broad application prospects.