Nonradioactive direct telomerase activity detection using biotin-labeled primers.

BACKGROUND

Telomerase is a ribonucleoprotein enzyme responsible for maintenance of telomere length which expressed in more than 85% of cancer cells but undetectable in most normal tissue cells. Therefore, telomerase serves as a diagnostic marker of cancers. Two commonly used telomerase activity detection methods, the telomerase repeated amplification protocol (TRAP) and the direct telomerase assay (DTA), have disadvantages that mainly arise from reliance on PCR amplification or the use of an isotope. A safe, low-cost and reliable telomerase activity detection method is still lacking.

METHOD

We modified DTA method using biotin-labeled primers (Biotin-DTA) and optimized the method by adjusting cell culture temperature and KCl concentration. The sensitivity of the method was confirmed to detect endogenous telomerase activity. The reliability was verified by detection of telomerase activity of published telomerase regulators. The stability was confirmed by comparing the method with TRAP method.

RESULTS

Cells cultured in 32°C and KCl concentration at 200 mM or 250 mM resulted in robust Biotin-DTA signal. Endogenous telomerase activity can be detected, which suggested an similar sensitivity as DTA using radioactive isotope markers. Knockdown of telomerase assembly regulator PES1 and DKC1 efficiently reduced telomerase activity. Compared with TRAP method, Biotin-DTA assay offers greater signal stability over a range of analyte protein amounts.

CONCLUSION

Biotin-labeled, PCR-free, and nonradioactive direct telomerase assay is a promising new method for the easy, low-cost, and quantitative detection of telomerase activity.