Modified Telomeric Repeat Amplification Protocol: A Quantitative Radioactive Assay for Telomerase without Using Electrophoresis
✍ Scribed by Istvan Szatmari; Szilvia Tőkés; Christopher B. Dunn; Thomas J. Bardos; Janos Aradi
- Publisher
- Elsevier Science
- Year
- 2000
- Tongue
- English
- Weight
- 145 KB
- Volume
- 282
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
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 [ 3 H]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 ].