Researchers from the University of California, San Francisco, and Bio-Rad Laboratories, Inc. are using Bio-Rad’s ddPCR technology with intercalating dye chemistry to provide absolute quantification of telomerase activity.
Telomeres, the protective structures at the ends of chromosomes, naturally degrade with each cell division. Once they are below a critical length, the cells arrest and become senescent before dying or passing through crisis, incurring additional genomic mutations, and becoming immortalized cancer cells.
One of the mechanisms of immortality is the activation of the telomerase enzyme. This enzyme adds a specific sequence of nucleotide repeats to telomeres, thus rewinding the clock and enabling a cell to divide continuously. Researchers are investigating telomerase activity as a biomarker for cancer diagnosis and as a target for anticancer drugs. Measuring telomerase activity more sensitively may enhance our understanding of its role in oncogenesis and other cellular and physiological phenotypes.
ddPCR Technology Enhances the Telomerase Activity Assay
Traditionally, the telomerase repeat amplification protocol (TRAP) assay measures the presence of active telomerase by measuring the activity of the enzyme on a starting DNA template, which is then amplified by PCR. For samples with abundant telomerase activity, SYBR® Green qPCR assays deliver high throughput and sufficient sensitivity. However, the most sensitive detection method still requires radioactive labeling, laborious polyacrylamide sequencing gels, and densitometry.
In the cited study, single-molecule counting of telomerase-extended templates was accomplished by partitioning the sample into droplets followed by PCR amplification and detection by fluorescence droplet reading using Bio-Rad’s ddPCR technology. The study results indicate that ddPCR is significantly more sensitive than traditional TRAP radiography and is also more amenable to high-throughput analysis.
“This new (ddPCR TRAP) telomerase activity assay demonstrates the ability to quantify telomerase enzymatic activity in droplets,” said George Karlin-Neumann, director of scientific affairs at Bio-Rad’s Digital Biology Center. “The sensitivity of ddPCR technology enables the measurement of telomerase activity in samples below the detection limit of traditional radiographic methods, while also providing a quick and facile method of measurement in cells where telomerase is more highly expressed.