A telomerase enzymatic assay that does not use polymerase chain reaction, radioactivity, or electrophoresis
- Resource Type
- Authors
- Wen Zhou; Murray O. Robinson; Hue Kha; Robert C. Wahl; Brian Rasnow; Karen Kearns; Tisha San Miguel; Alex Mladenovic; Barbara Karan-Tamir; Lisa Zeni; Kui Chen
- Source
- Analytical Biochemistry. 331:230-234
- Subject
- Electrophoresis
Radioisotopes
Detection limit
Telomerase
Lysis
Base Sequence
Biophysics
Enzyme-Linked Immunosorbent Assay
Cell Biology
Biology
Polymerase Chain Reaction
Biochemistry
Molecular biology
Cell Line
law.invention
Telomere
law
Biotinylation
Humans
Primer (molecular biology)
Molecular Biology
Polymerase chain reaction
DNA Primers
Chemiluminescence
- Language
- ISSN
- 0003-2697
A telomerase assay has been developed for high-throughput screening in 96-well microtiter plates. A crude cell lysate which adds telomere repeats to a biotinylated DNA primer is the source of telomerase. The telomerase-extended primer is hybridized to a digoxigenin-labeled telomere antisense DNA probe. The hybrid is further processed by enzyme-linked immunosorbent assay (ELISA) as follows. The biotinylated hybrid is captured on streptavidin-coated microtiter plates. The immobilized hybrid is probed with alkaline phosphatase-antidigoxigenin and detected via chemiluminescent readout. The limit of detection of a chemically synthesized tetra-telomere repeat was about 10 attomoles. Apparent telomerase activity was detected in lysates of 293T cells. The signal to background for the assay (ratio of signal for the complete assay mixture divided by the signal for the assay mixture without primer) was around 10. An automated system that performed unattended runs of up to 17 96-well microtiter plates in 8h was constructed.