Modified telomeric repeat amplification protocol:: A quantitative radioactive assay for telomerase without using electrophoresis
Modified telomeric repeat amplification protocol:: A quantitative radioactive assay for telomerase without using electrophoresis
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DOI：
10.1006/abio.2000.4589
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发表时间：
2000-06-15
影响因子：
2.9
通讯作者：
Aradi, J
中科院分区：
文献类型：
--
作者：
Szatmari, I;Tokés, S;Aradi, J
我们实验室开发了一种基于聚合酶链反应（PCR）的放射性端粒酶检测方法，该方法是定量的，不需要电泳评价（称为TP-TRAP;它利用两个反向引物）。该方法的主要步骤包括：（1）用端粒酶延伸20-mer寡核苷酸底物（MTS），（2）在[H-3]dTTP存在下，使用底物寡核苷酸和两个反向引物扩增端粒酶产物（RPC 3，38聚体; RP，20 mer），（3）通过沉淀和过滤分离扩增的放射性dsDNA，（4）酸不溶性DNA的放射性测定。端粒酶产物的长度在扩增时不增加。通过利用两种反向引物和高度特异性的PCR方案实现了该测定的这一有价值的特征。该检测方法是线性的，准确的，并适用于细胞生物学研究，在端粒酶活性的轻微定量差异必须检测。该试验也适用于筛选和表征端粒酶抑制剂，如化学修饰的寡核苷酸逆转录酶抑制剂[（s（4）dU）（35）]所示。(C)北京大学出版社.
A polymerase chain reaction (PCR)-based radioactive telomerase assay was developed in our laboratory which is quantitative and does not require electrophoretic evaluation (designated as TP-TRAP; it utilizes two reverse primers). The main steps of the assay include (1) extension of a 20-mer oligonucleotide substrate (MTS) by telomerase, (2) amplification of the telomerase products in the presence of [H-3]dTTP using the substrate oligonucleotide and two reverse primers (RPC3, 38 mer; RP, 20 mer), (3) isolation of the amplified radioactive dsDNA by precipitation and filtration, (4) determination of the radioactivity of the acid-insoluble DNA. The length of the telomerase products does not increase on amplification. This valuable feature of the assay is achieved by utilization of the two reverse primers and a highly specific PCR protocol. The assay is linear, accurate, and suitable for cell-biological studies where slight quantitative differences in telomerase activity must be detected. The assay is also suitable for screening and characterization of telomerase inhibitors, as shown with a chemically modified oligonucleotide reverse transcriptase inhibitor [(s(4)dU)(35)]. (C) 2000 Academic Press.