Assessment and quantification of telomerase enzyme activity

Abstract

The enzyme telomerase is activated in 80-90% of all human malignancies and immortal cell lines, where it functions to maintain the integrity of chromosomal-end structures called telomeres. Telomerase enzyme activity can be detected in whole cell lysates by a polymerase chain reaction (PCR)-based method referred to as the telomeric repeat amplification protocol (TRAP). The TRAP assay involves extension of an oligonucleotide through telomerase-mediated enzymatic addition of telomeric DNA repeats and subsequent PCR amplification of the extension products. While the TRAP assay as originally developed utilizes radioactively labelled nucleotides, protocols are provided herein for nonradioactive versions of the TRAP assay, with options for either qualitative assessment of TRAP products by polyacrylamide gel electrophoresis (standard TRAP), or quantitative analysis by real-time PCR (Q-TRAP). The Q-TRAP method poses the additional advantages of exquisite sensitivity, rapidity, and potential for adoption to a high-throughput format. © Springer Science+Business Media New York 2013.