Real-Time Detection and Quantification of Telomerase Activity Utilizing Energy Transfer Primers

  • Hiroshi Uehara
Part of the Methods in Molecular Biology™ book series (MIMB, volume 335)


A novel closed-tube format telomeric repeat amplification protocol specifically adapted to real-time detection and quantification of telomerase activity was developed. The assay utilizes energy transfer primers, which emit fluorescence only upon incorporation into polymerase chain reaction (PCR) amplification products. The assay, performed on a real-time detection instrument, is highly reproducible, sensitive, and specific. Telomerase activity in as few as 10 cultured cells can be quantified with a linear dynamic range more than 2.5 logs. In addition, the presence of potential PCR inhibitor(s) is readily detectable by inclusion of an internal PCR control labeled with a second color fluorescence.

Key Words

Telomerase TRAP assay energy transfer primer PCR real time 


  1. 1.
    Blackburn, E. H. (1991) Structure and function of telomeres. Nature 350, 569–563.PubMedCrossRefGoogle Scholar
  2. 2.
    Zakitan, V. A. (1989) Structure and function of telomeres. Ann. Rev. Genet. 23, 579–604.CrossRefGoogle Scholar
  3. 3.
    Watson, J. D. (1972) Origin of concatemeric T7 DNA. Nature New Biol. 239, 197–201.PubMedCrossRefGoogle Scholar
  4. 4.
    Olovnikov, A. M. (1973) A theory of marginotomy: the incomplete copying template margin in enzymic synthesis of pronucleotides and biological significance of the phenomenon. J. Theor. Biol. 41, 181–190.PubMedCrossRefGoogle Scholar
  5. 5.
    Greider, C. W. and Blackburn, E. H. (1989) A telomeric sequence in the RNA of Tetrahymena telomerase required for telomere repeats synthesis. Nature 337, 331–337.PubMedCrossRefGoogle Scholar
  6. 6.
    Morin, G. B. (1989) The human telomere terminal transferase enzyme is a ribonucleoprotein that synthesizes TTAGGG repeats. Cell 59, 521–529.PubMedCrossRefGoogle Scholar
  7. 7.
    Kim, N. W., Piatyszek, M. A., Prowse, K. R., et al. (1994) Specific association of human telomerase activity with immortal cells and cancer. Science 266, 2011–2014.PubMedCrossRefGoogle Scholar
  8. 8.
    Shay, J. W. and Bacchetti, S. (1997) A survey of telomerase activity in human cancer. Eur. J. Cancer 33, 787–791.PubMedCrossRefGoogle Scholar
  9. 9.
    Bodnar, A. G., Ouellette, M., Frolkis, M., et al. (1998) Extension of life-span by introduction of telomerase into normal human cell. Science 279, 349–352.PubMedCrossRefGoogle Scholar
  10. 10.
    Bacchetti, S. and Counter, C. M. (1995) Telomeres and telomerase in human cancer. Int. J. Oncology 7, 423–432Google Scholar
  11. 11.
    Counter, C. M., Avilion, A. A., LeFeuvre, C. E., et al. (1992) Telomerase shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity. EMBO J. 11, 1921–1929.PubMedGoogle Scholar
  12. 12.
    Nazarenko, I. A., Bhatnagar, S., and Hohman, R. J. (1997) A closed tube format for amplification and detection of DNA based energy transfer. Nucleic Acids Res. 25, 2516–2521.PubMedCrossRefGoogle Scholar
  13. 13.
    Uehara, H., Nardone, G., Nazarenko, I. A., and Hohman, R. J. (1999) Detection of telomerase activity utilizing energy transfer primer: comparison with gel-and ELISA-based detection. Biotechniques 26, 552–558.PubMedGoogle Scholar
  14. 14.
    Myakishev, M., Khripin, Y., Hu, S., and Hamer, D. (2001) High throughput SNP genotyping by allele-specific PCR with universal energy transfer-labeled primers. Genome Res. 1, 163–169.CrossRefGoogle Scholar
  15. 15.
    Stryer, L. (1978) Fluorescence energy transfer as a spectroscopic ruler. Ann. Rev. Biochem. 47, 819–846.PubMedCrossRefGoogle Scholar
  16. 16.
    Wu, P. and Brand, L. (1994) Resonance energy transfer: methods and applications. Anal. Biochem. 218, 1–13.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2006

Authors and Affiliations

  • Hiroshi Uehara

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