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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Blackburn EH (1991) Structure and function of telomeres. Nature 350(6319):569–573
Blackburn EH (1991) Telomeres. Trends Biochem Sci 16(10):378–381
Greider CW, Blackburn EH (1985) Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell 43(2 Pt 1):405–413
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):181–190
Hayflick L, Moorhead PS (1961) The serial cultivation of human diploid cell strains. Exp Cell Res 25:585–621
Shay JW, Bacchetti S (1997) A survey of telomerase activity in human cancer. Eur J Cancer 33(5):787–791
Buseman CM, Wright WE, Shay JW (2012) Is telomerase a viable target in cancer? Mutat Res 730(1–2):90–97
Kim NW et al (1994) Specific association of human telomerase activity with immortal cells and cancer. Science 266(5193):2011–2015
Morin GB (1991) Recognition of a chromosome truncation site associated with alpha-thalassaemia by human telomerase. Nature 353(6343):454–456
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 18:237–248
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):2595–2597
Saldanha SN, Andrews LG, Tollefsbol TO (2003) Analysis of telomerase activity and detection of its catalytic subunit, hTERT. Anal Biochem 315(1):1–21
Gardano L et al (2011) Native gel electrophoresis of human telomerase distinguishes active complexes with or without dyskerin. Nucleic Acids Res 40(5):e36
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media, New York
About this protocol
Cite this protocol
Gardano, L. (2013). Using PCR Coupled to PAGE for Detection and Semiquantitative Evaluation of Telomerase Activity. In: Makovets, S. (eds) DNA Electrophoresis. Methods in Molecular Biology, vol 1054. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-565-1_19
Download citation
DOI: https://doi.org/10.1007/978-1-62703-565-1_19
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-564-4
Online ISBN: 978-1-62703-565-1
eBook Packages: Springer Protocols