A fluorescent method for detection of telomerase activity.
The telomere repeat amplification protocol (TRAP) was recently developed to detect telomerase activity in cellular protein extracts. Teleomerase synthesizes a specific repeating nucleotide sequence onto the ends of telomeres, which stabilize eukaryotic chromosomes. Telomeric repeats are identified in the TRAP method by polymerase chain reaction amplification and incorporation of radionucleotides detected by autoradiography. Several drawbacks to this method have been recognized, including the time required to complete the assay, the resolution of the results, and the hazards of radioactive material. We have developed a new fluorescent method of detecting telomerase to alleviate these problems. Telomeric repeats are identified in the fluorescent TRAP (F-TRAP) assay by incorporation of fluorescein-labeled primers during amplification and subsequent detection with an automated DNA sequencer. This new method appears to be as sensitive as the standard TRAP assay and offers advantages in speed, resolution, cost, and safety.
Aldous
W K
WK
Department of Clinical Investigation, Madigan Army Medical Center, Tacoma, WA 98431, USA.
Grabill
N R
NR
D017122:Bacteriophage T4 / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D005456:Fluorescent Dyes; D006801:Humans; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D012091:Repetitive Sequences, Nucleic Acid; D015203:Reproducibility of Results; D013095:Spermidine / Q000032:analysis; D019098:Telomerase / Q000032:analysis*; D016615:Telomere / Q000201:enzymology / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics
D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D014764:Viral Proteins; C060035:gp32 protein, Enterobacteria phage T4; D019098:Telomerase; D013095:Spermidine