有限延伸法检测端粒酶活性

人端粒酶是一种核蛋白体，通过其内含的RNA模板与端粒末端配对把重复端粒片段添加在端粒3＇末端|因此,端粒酶活性与细胞凋亡、衰老、永生化有密切关系，是癌症临床预测诊断的一个生物标签．现有的端粒酶活性检测方法，存在灵敏度低和不易定量等问题．本研究采用错配有限延伸法检测端粒酶活性：在人端粒酶延伸人工合成的游离端粒酶底物时，只加入dATP和dGTP，端粒酶只能把底物延伸4个脱氧核糖核苷酸AGGG．然后加入dNTP，让端粒酶延伸的产物和一条长的引物配对从而延伸出PCR模板|再加入引物进行热启动PCR．PCR后进行非变性PAGE (polyacrylamide gel electrophoresis)，得到希望的唯一1条目标带．同时,用不同的端粒酶浓度梯度进行优化，发现有限延伸法检测端粒酶活性的下限达到250个HeLa细胞．

Detecting Telomerase Activity by Pre-mature Termination

Human telomerase is the ribonucleoprotein complex that adds telomere repeats to the telomere 3＇end, as directed by an RNA template in the RNA component of the enzyme, which pairs with the telomere 3＇end during elongation. Telomerase is tightly correlated with cell apoptosis, senescence and immortalization, and thus telomerase is a target for anticancer therapy and a prognostic biomarker for cancers. Previous methods to detect telomerase activity had low sensitivity and were difficult to quantify. Mismatched pre mature termination was employed to detect telomerase activity. While human telomerase elongates a free and nontelomeric substrate, the product was only elongated to four oligo nucleotides AGGG, through adding only dATP and dGTP, then adding dNTPs. The PCR template would be formed by hybridization of the telomerase product and longer primer|at last, adding primers and dNTP, an unique targeted band was visualized through native PAGE (polyacrylamide gel electrophoresis) via heated PCR. Meanwhile, telomerase concentration gradient experiments were carried out and the lowest detecting telomerase activity was tested by only 250 HeLa cells.