Telomerase assay using biotinylated-primer extension and magnetic separation of the products.
Human telomerase, a ribonucleoprotein enzyme is known to be associated with immortalized cancer cells but is absent in most normal tissues. Thus, telomerase appears to be an attractive new target for anticancer agents and an important diagnostic marker of human cancers. Here, we describe an improved telomerase assay method based on the Dynabead biomagnetic separation theory. In this method, 5'-biotinylated (TTAGGG)3 was used as a primer for the telomerase reaction. Telomerase reaction products were then immobilized on streptavidin-coated Dynabeads and washed intensively to eliminate excess [alpha-32P]dGTP. Using this method, without the amplification of telomerase reaction products by the PCR, we were able to quantitatively detect telomerase activity in human HeLa cell extracts equivalent to between 200-500 cells. This method is anticipated to be useful for the measurement of telomerase activity in various tumor cells, for assessing potential telomerase and for understanding the biochemical aspects of the telomerase reaction.
Sun
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Institute for Drug Development, Cancer Therapy and Research Center, San Antonio, TX 78245, USA.
Hurley
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LH
Von Hoff
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D014408:Biomarkers, Tumor; D001710:Biotin; D004796:Clinical Enzyme Tests / Q000379:methods / Q000592:standards; D017931:DNA Primers; D004273:DNA, Neoplasm / Q000302:isolation & purification; D006160:Guanosine Triphosphate; D006801:Humans; D008280:Magnetics; D008863:Microspheres; D012680:Sensitivity and Specificity; D019098:Telomerase / Q000032:analysis / Q000235:genetics*; D016615:Telomere / Q000201:enzymology / Q000235:genetics*; D013696:Temperature
D014408:Biomarkers, Tumor; D017931:DNA Primers; D004273:DNA, Neoplasm; D001710:Biotin; D006160:Guanosine Triphosphate; D019098:Telomerase