High-throughput analysis of telomerase by capillary electrophoresis.
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 noninvasive, 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 polymerase chain reaction (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 5-100 cells. TRAP/PCR products were generated using a fluorescent 4,7,2'4'5'7',-hexachloro-6-carboxyfluorescein(HEX)-labeled TS primer and analyzed on the Applied Biosystems Model 310 CE system using POP4 polymer. After analysis with GeneScan and Genotyper software, the total peak areas of the TRAP ladder extension products were computed using Microsoft Excel. Results were compared with unlabeled TRAP/PCR products analyzed on the Bio-Rad BioFocus 3000 CE system using 6% high molecular weight polyvinylpyrrolidone (HMW PVP) polymer and SYBR Green I dye. Both CE systems were able to resolve the TRAP ladder products with high reproducibility and sensitivity (5-15 cells). With the appropriate robotic sample handling system, these CE methods would enable performing the telomerase TRAP assay with increased sensitivity, reproducibility and automation over slab-gel methods.
Atha
Donald H
DH
Biotechnology Division, National Institute of Standards and Technology, Gaithersburg, MD 20899-8311, USA. donald.atha@nist.gov
Miller
Karen
K
Sanow
Anita D
AD
Xu
Jingfan
J
Hess
Jennifer L
JL
Wu
Osmond C
OC
Wang
Wendy
W
Srivastava
Sudhir
S
Highsmith
W Edward
WE
D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D019075:Electrophoresis, Capillary / Q000379:methods* / Q000706:statistics & numerical data; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D008175:Lung Neoplasms / Q000201:enzymology; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019098:Telomerase / Q000032:analysis*; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D019098:Telomerase