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PubMed · 10860502

Modified telomeric repeat amplification protocol: a quantitative radioactive assay for telomerase without using electrophoresis.

I Szatmari·S Tókés·C B Dunn·T J Bardos·J Aradi
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Abstract

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)].

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Cell ExtractsCholic AcidsDNADNA PrimersDetergentsEthanolaminesHL-60 CellsHeLa CellsHumansKineticsOligonucleotidesPolymerase Chain ReactionReverse Transcriptase InhibitorsSensitivity and SpecificityTelomeraseTelomereTemperatureJournal ArticleResearch Support, Non-U.S. Gov't

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I Szatmari, S Tókés, C B Dunn, T J Bardos, J Aradi. 2000-06-15. Modified telomeric repeat amplification protocol: a quantitative radioactive assay for telomerase without using electrophoresis.. https://doi.org/10.1006/abio.2000.4589

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