有限延伸法检测端粒酶活性 - researchgate.doc

技术与方法 有限延伸法检测端粒酶活性 赵秀举( （武汉轻工大学生与制药工程学院，武汉 430023） 摘 要 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断添加在端粒3末端端粒酶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．端粒酶活性检测方法，．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四个脱氧核糖核苷酸．然后加入，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PAGEolyacrylamide gel electrophoresis)，得到希望的唯一一条目标带．用不同的端粒酶浓度梯度进行，发现检测端粒酶活性的下限达到250个HeLa细胞． 关键词人端粒酶活性有限延伸法错配 中图分类号Q61 Detecting Telomerase Activity by Pre-mature Termination ZHAO Xiu-Ju* (School of Biology and Pharmaceutical Engineering, Collaborative Innovation Center for Processing of Agricultural Products, Wuhan Polytechnic University, Wuhan 430023, China) Abstract 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 low limit detecting telomerase activity was 250 HeLa cells. Key words human telomerase activity; premature termination; mismatch  人端粒酶是核蛋白体，包括RNA和蛋白部分，通过其内含的RNA模板与端粒末端配对，可以把端粒重复片断加在端粒末端，从而可以消除端粒末端复制问题导致的端粒缩短．端粒酶活性与细胞永生化癌变衰老有密切的关系，从而吸引了大批的科研工作者对端粒酶研究1-4]． 端粒酶由于在细胞内的低丰度，所以一直成为研究工作