A sensitive direct human telomerase activity assay

Expression of telomerase, the specialized reverse transcriptase that adds 5'-TTAGGG-3' repeats to the ends of human chromosomes, is upregulated in >= 85% of human cancers and tumor cell lines. We describe a direct primer-extension activity assay(1,2) for human telomerase that displays sensitivity to similar to 10(6) telomerase-positive cells, making the method suitable for use with standard cell culture-based research (Fig. 1). Telomerase is first immunoaffinity purified from cell lysate using an antibody to telomerase and captured using protein G-agarose beads (Steps 14-17). Then telomerase is dissociated from the beads using excess peptide antigen (Step 19). A second affinity purification exploits the stable binding interaction between human telomerase and the telomeric DNA substrate 5'-(TTAGGG)(3)-3' (dissociation half-life >= 10 h at 23 degrees C)(3). Modifying neutravidin beads with 5'-biotin-CTAGACCTGTCATCA(TTAGGG)(3)-3' (Step 5) generates an affinity reagent that captures >90% of immunopurified telomerase(4) (Step 22), providing highly enriched telomerase bound to its DNA substrate in a volume of 20 mu l of beads. Addition of assay buffer results in extension of the bead-immobilized DNA substrate (Step 24). Telomerase extension products are released by heating in denaturing formamide buffer to disrupt the avidin-biotin interaction, and then separated and visualized by standard techniques.