A homogeneous chemiluminescent assay for telomerase.
I have designed an unamplified assay for human telomerase based on the hybridization protection assay. This assay measures in vitro synthesis of the human telomeric repeat DNA sequence (TTAGGG)N by 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 10(5) cells, 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.
Lackey
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DB
Chugai Biopharmaceuticals, Inc., 6275 Nancy Ridge Road, San Diego, California 92121, USA.
D000166:Acridines / Q000737:chemistry; D017931:DNA Primers; D015342:DNA Probes; D019286:Endopeptidase K / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods*; D008163:Luminescent Measurements; D012091:Repetitive Sequences, Nucleic Acid; D012260:Ribonucleases / Q000737:chemistry / Q000378:metabolism; D019098:Telomerase / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism
D000166:Acridines; D017931:DNA Primers; D015342:DNA Probes; D019098:Telomerase; D012260:Ribonucleases; D019286:Endopeptidase K