Application of DNA sequencing in detection of telomerase activity

The telomeric repeat amplification protocol (TRAP) is the most commonly used method for the detection of telomerase activity. However, this method and other improved immunoassay methods based on TRAP present problems owing to tedious quantification and radioisotope handling. In order to alleviate these inconveniences, a novel method combining DNA sequence analysis with TRAP to detect telomerase activity was developed. TRAP products were refined by hydroxybenzene/chloroform, and deposited by ethanol to eliminate the PCR inhibitors. Then through image analysis of PCR products, telomerase activity was quantitatively determined. We used this new method to detect telomerase enzyme activity in HLF, MCF, K-562, SMMC-7721 cell lines, leukocytes and RNase-pretreated or heat-treated samples as controls. The telomerase activity linearly depended on the number of K-562 cells used in the assay. Telomerase activity in controls was negative. Using this novel methodology for the detection of TRAP products, results were available within a few hours, with high sensitivity, and without the use of radioisotopes. Further studies are aimed at streamlining this approach for large-scale evaluations of clinical samples.