High-Throughput Analysis of Telomerase by Capillary Electrophoresis
Donald H. Atha, K Miller, A D. Sanow, J Xu, J L. Hess, O C. Wu, Wenhua Wang, S Srivastava, W E. Highsmith
The enzyme telomerase is expressed in 85-90% of all human cancers, but not in normal, non-stem cell somatic tissues. Clinical assays for telomerase in easily obtained body fluids would have great utility as non-invasive, cost-effective methods for the early detection of cancer. The most commonly used method for the detection and quantification of telomerase enzyme activity is the PCR based assay known as the telomerase repeat amplification protocol or TRAP assay Most of the TRAP assay systems use a slab-gel based electrophoresis system to size and quantify the PCR-amplified extension products. We are developing high-throughput capillary electrophoresis (CE) methods for the analysis of TRAP/PCR products. The TRAP assay was conducted on lysates of the human lung cancer cell line A-549 in reactions containing 5 to 100 cells. TRAP/PCR products were generated using fluorescent (5-carboxyfluorescein) - labeled CX and TS primers and analyzed on the Applied Biosystems Model 310 capillary electrophoresis system using POP4TM polymer. After analysis with GeneScanTM and GenotyperTM software, the total peak area of the TRAP ladder extension products were computed using Microsoft ExcelTM. Results were compared with unlabeled TRAP/PCR products analyzed on the Bio-Rad BioFocus 3000 capillary electrophoresis system using 6% HMW PVP (high molecular weight polyvinylpyrrolidone) polymer and SYBRTM Green I dye. Both CE systems were able to resolve the TRAP ladder products with high reproducibility and sensitivity (5 cells). With the appropriate robotic sample handling system, these CE methods would streamline validation of the telomerase TRAP assay with increased sensitivity, reproducibility and automation over slab-gel methods.
San Diego, CA
16th International Symposium on Microscale Separation and Analysis
, Miller, K.
, Sanow, A.
, Xu, J.
, Hess, J.
, Wu, O.
, Wang, W.
, Srivastava, S.
and Highsmith, W.
High-Throughput Analysis of Telomerase by Capillary Electrophoresis, Electrophoresis, San Diego, CA
(Accessed June 9, 2023)