Abstract
I have designed an unamplified assay for human telomerase based on the hybridization protection assay. This assay measuresin vitrosynthesis of the human telomeric repeat DNA sequence (TTAGGG)Nby solution hybridization with a chemiluminescent acridinium ester oligonucleotide probe. The assay is capable of detecting less than 0.1 fmol of the telomerase repeat DNA sequence, and has a linear range extending to 3.5 fmol/reaction. Using this assay, telomerase activity can be measured in less than 60 min. The hybridization protection assay can be used to determine the amount of telomerase activity in a cell-free extract obtained from as few as 105cells, and is three to four orders of magnitude less sensitive than PCR-based telomerase assays. However, the precision, accuracy, and rapidity of this telomerase assay make it suitable for enzyme isolation, enzyme characterization, and high-throughput screening applications.
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