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Droplet Digital TRAP ddTRAP: Adaptation of the Telomere Repeat Amplification Protocol to Droplet Digital Polymerase Chain Reaction
Published on: May 3, 2019
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Quantitative telomerase enzyme activity determination using droplet digital PCR with single cell resolution
Andrew T Ludlow1, Jerome D Robin1, Mohammed Sayed1
1Department of Cell Biology, UT Southwestern Medical Center, Dallas, TX 75390-9039, USA.
Nucleic Acids Research
|May 28, 2014
Summary
A new droplet digital telomere repeat amplification protocol (ddTRAP) assay offers accurate, reproducible measurement of telomerase activity. This digital method enables single-cell analysis, advancing cancer research and therapeutic development.
Area of Science:
- Molecular Biology
- Biochemistry
- Cancer Research
Background:
- The telomere repeat amplification protocol (TRAP) is a long-standing PCR-based method for assessing telomerase activity.
- Traditional TRAP assays have limitations in sensitivity, quantitative accuracy, and throughput, often requiring radioactive gel electrophoresis.
- Droplet digital PCR (ddPCR) offers absolute quantification of nucleic acid molecules, presenting an opportunity to improve TRAP assay performance.
Purpose of the Study:
- To develop and validate a droplet digital TRAP (ddTRAP) assay for precise and reproducible quantification of telomerase activity.
- To demonstrate the utility of ddTRAP for analyzing telomerase activity in various biological contexts, including single cells and primary cells.
Main Methods:
- Adaptation of the conventional TRAP assay to a droplet digital PCR (ddPCR) platform.
- Validation of the ddTRAP assay's reproducibility and accuracy across different cell types.
- Application of ddTRAP for quantifying telomerase activity in single HeLa cells, cancer cell lines, and mitogen-stimulated peripheral blood mononuclear cells.
Main Results:
- The ddTRAP assay accurately and reproducibly quantifies telomerase activity, measuring 57.8 ± 7.5 telomerase-extended products per single HeLa cell.
- Demonstrated successful application in quantifying telomerase activity in diverse cancer cell lines and human primary cells.
- Established a sensitive method for monitoring telomerase enzyme activity at the single-cell level.
Conclusions:
- The droplet digital TRAP (ddTRAP) assay provides a significant advancement over traditional TRAP methods, offering enhanced accuracy and reproducibility.
- This digital approach enables precise monitoring of telomerase activity on a single-cell basis.
- The ddTRAP assay holds potential for applications in cancer diagnostics and the development of novel telomerase-targeted therapies.
