Advertisement

Using PCR Coupled to PAGE for Detection and Semiquantitative Evaluation of Telomerase Activity

  • Laura Gardano
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1054)

Abstract

Telomerase is the enzyme that extends the chromosome ends, thereby contributing to eukaryotic cell genome stability. Telomerase is expressed in the majority of cells that have an unlimited proliferation such as stem cells and cancer cells. The increased interest in telomerase in cancer research, challenged by the low cellular abundance of the enzyme, has led to the development of a reliable and at the same time very sensitive approach to detect telomerase activity. The telomeric repeat amplification protocol (TRAP) represents an easy and rapid method for detection of telomerase activity in cells. A non-telomeric TS primer is extended by telomerase in the first step followed by the PCR amplification of the products. The PCR step renders this protocol very sensitive to detect telomerase activity at the single cell level making it compatible with the analysis of tumor samples. When run on a polyacrylamide gel, the PCR product is a characteristic ladder of bands due to the repetitive nature of telomeric DNA sequence. The densitometric analysis of the ladder allows the TRAP assay to be used for comparative quantification of telomerase activity in different samples.

Key words

Telomerase activity Telomeres Telomerase detection 

References

  1. 1.
    Blackburn EH (1991) Structure and function of telomeres. Nature 350(6319):569–573PubMedCrossRefGoogle Scholar
  2. 2.
    Blackburn EH (1991) Telomeres. Trends Biochem Sci 16(10):378–381PubMedCrossRefGoogle Scholar
  3. 3.
    Greider CW, Blackburn EH (1985) Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell 43(2 Pt 1):405–413PubMedCrossRefGoogle Scholar
  4. 4.
    Olovnikov AM (1973) A theory of marginotomy. The incomplete copying of template margin in enzymic synthesis of polynucleotides and biological significance of the phenomenon. J Theor Biol 41(1):181–190PubMedCrossRefGoogle Scholar
  5. 5.
    Hayflick L, Moorhead PS (1961) The serial cultivation of human diploid cell strains. Exp Cell Res 25:585–621PubMedCrossRefGoogle Scholar
  6. 6.
    Shay JW, Bacchetti S (1997) A survey of telomerase activity in human cancer. Eur J Cancer 33(5):787–791PubMedCrossRefGoogle Scholar
  7. 7.
    Buseman CM, Wright WE, Shay JW (2012) Is telomerase a viable target in cancer? Mutat Res 730(1–2):90–97PubMedGoogle Scholar
  8. 8.
    Kim NW et al (1994) Specific association of human telomerase activity with immortal cells and cancer. Science 266(5193):2011–2015PubMedCrossRefGoogle Scholar
  9. 9.
    Morin GB (1991) Recognition of a chromosome truncation site associated with alpha-thalassaemia by human telomerase. Nature 353(6343):454–456PubMedCrossRefGoogle Scholar
  10. 10.
    Holt SE, Norton JC, Wright WE, Shay JW (1996) Comparison of the telomeric repeat amplification protocol (TRAP) to the new TRAP-eze telomerase detection kit. Methods Cell Sci 18:237–248CrossRefGoogle Scholar
  11. 11.
    Kim NW, Wu F (1997) Advances in quantification and characterization of telomerase activity by the telomeric repeat amplification protocol (TRAP). Nucleic Acids Res 25(13):2595–2597PubMedCrossRefGoogle Scholar
  12. 12.
    Saldanha SN, Andrews LG, Tollefsbol TO (2003) Analysis of telomerase activity and detection of its catalytic subunit, hTERT. Anal Biochem 315(1):1–21PubMedCrossRefGoogle Scholar
  13. 13.
    Gardano L et al (2011) Native gel electrophoresis of human telomerase distinguishes active complexes with or without dyskerin. Nucleic Acids Res 40(5):e36PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, New York 2013

Authors and Affiliations

  • Laura Gardano
    • 1
  1. 1.UFR SMBH/University Paris 13ParisFrance

Personalised recommendations