TRAP-LIG, a modified telomere repeat amplification protocol assay to quantitate telomerase inhibition by small molecules.
The telomerase enzyme is implicated in a large proportion of human cancers. Its major function is to maintain the length of telomeric DNA by synthesizing telomeric DNA repeats, and its enzymatic activity is assayed by means of the telomere repeat amplification protocol (TRAP) assay. We show here that this assay is unable to reliably determine the ability of small molecules to inhibit the enzyme because they interfere with the PCR step of the assay, resulting in a major overestimation of their activity. We report a modified TRAP assay that incorporates the addition of an intermediate step, enabling ligand to be removed from the final PCR process using a commercially available oligonucleotide purification kit, so that more reliable estimates of telomerase inhibition can be made.
Reed
Julie
J
CRUK Biomolecular Structure Group, School of Pharmacy, University of London, London WC1N 1AX, UK.
Gunaratnam
Mekala
M
Beltran
Monica
M
Reszka
Anthony P
AP
Vilar
Ramon
R
Neidle
Stephen
S
D000818:Animals; D002417:Cattle; D045744:Cell Line, Tumor; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D054856:G-Quadruplexes; D006801:Humans; D008024:Ligands; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D015203:Reproducibility of Results; D019098:Telomerase / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D016615:Telomere / Q000378:metabolism*
D004791:Enzyme Inhibitors; D008024:Ligands; D019098:Telomerase; D004259:DNA-Directed DNA Polymerase