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

Istvan Szatmari, Szilvia Tokés, Christopher B. Dunn, Thomas J. Bardos, Janos Aradi

Research output: Contribution to journalArticle

24 Citations (Scopus)

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 [3H]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 [(s4dU)35]. (C) 2000 Academic Press.

Original languageEnglish
Pages (from-to)80-88
Number of pages9
JournalAnalytical Biochemistry
Volume282
Issue number1
DOIs
Publication statusPublished - Jun 15 2000

Keywords

  • PCR
  • TRAP assay
  • Telomerase
  • Telomerase inhibitor

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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