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 > or =85% of human cancers and tumor cell lines. We describe a direct primer-extension activity assay for human telomerase that displays sensitivity to approximately 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 > or = 10 h at 23 degrees C). Modifying neutravidin beads with 5'-biotin-CTAGACCTGTCATCA(TTAGGG)3-3' (Step 5) generates an affinity reagent that captures >90% of immunopurified telomerase (Step 22), providing highly enriched telomerase bound to its DNA substrate in a volume of 20 mul 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.
Cohen
Scott B
SB
Cancer Research Unit, Children's Medical Research Institute, 214 Hawkesbury Road, Westmead, NSW 2145, Australia.
Reddel
Roger R
RR
D001681:Biological Assay / Q000379:methods; D014408:Biomarkers, Tumor / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004273:DNA, Neoplasm / Q000235:genetics; D006801:Humans; D016014:Linear Models; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019098:Telomerase / Q000032:analysis / Q000235:genetics / Q000378:metabolism*
D014408:Biomarkers, Tumor; D004273:DNA, Neoplasm; D019098:Telomerase