Abstract LB-253: TRAPing telomerase activity using droplet digital PCR.

The aim of this work was to develop a more sensitive and high throughput assay for measuring telomerase activity. Telomeres are the protective structures at the ends of chromosomes consisting of 6 bp repeat sequences. In young cells, these regions can be as long as 15kb and act as caps which protect the DNA ends. These ends naturally degrade with each passing cell division, usually losing 25-200 base pairs per division. Once they are shortened below a critical length (estimated to be 200-300 bp) the cells arrest and become senescent, or “old”. Telomeres can be thought of as a cellular or mitotic clock. Once the clock has wound down, the cells either die or pass through crisis and become immortal. One of the mechanisms of immortality is the activation of the enzyme, telomerase. Telomerase is the endogenous reverse transcriptase responsible for adding repeats to telomeres, rewinding the clock and enabling a cell to continuously divide. Abundant telomerase activity is found in fetal and adult stem cells, germ cells, and cancer. It is also present at much lower levels in non-stem cells, such as immune cells, but these levels can be difficult to measure reliably. The telomerase repeat amplification protocol (TRAP) 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 qPCR assays provide high throughput. However, the current most sensitive method of detection still uses radioactivity, laborious PAGE sequencing gels, and densitometry to quantify telomerase. Here we explore using droplet digital™ PCR (ddPCR™) to provide absolute quantification of telomerase activity. This was achieved through single-molecule counting of telomerase-extended templates that were partitioned into droplets, amplified by PCR and detected by fluorescence droplet flow cytometry. Analysis of control samples suggests ddPCR is at least an order of magnitude more sensitive than TRAP radiography and is more amenable to higher throughput analysis. We extend throughput, sensitivity, and the range of biological samples that can be analyzed for telomerase activity. Citation Format: Dawne Shelton, Jack Regan, George Karlin-Neumann, Jue Lin, Elizabeth Blackburn. TRAPing telomerase activity using droplet digital PCR. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-253. doi:10.1158/1538-7445.AM2013-LB-253