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In vitro Reconstitution of the Active T. castaneum Telomerase
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ddTRAP: A Method for Sensitive and Precise Quantification of Telomerase Activity.

Andrew T Ludlow1, Dawne Shelton2, Woodring E Wright1

  • 1Department of Cell Biology, UT Southwestern Medical Center, Dallas, TX, USA.

Methods in Molecular Biology (Clifton, N.J.)
|May 3, 2018
PubMed
Summary
This summary is machine-generated.

Telomerase, an enzyme crucial for cancer cell replication, can be detected using the droplet digital Telomere Repeat Amplification Protocol (ddTRAP). This sensitive method enhances the analysis of telomerase activity in tumor cells.

Keywords:
Droplet digital PCRTelomerase activity

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Area of Science:

  • Molecular Biology
  • Oncology
  • Biochemistry

Background:

  • Telomerase is a reverse transcriptase essential for maintaining telomere length.
  • Universal expression in over 85% of tumor cells contributes to unlimited replication and avoidance of senescence.
  • Telomere length maintenance is a hallmark of cancer progression.

Purpose of the Study:

  • To detail the procedures for the droplet digital Telomere Repeat Amplification Protocol (ddTRAP).
  • To present an optimized method for detecting telomerase activity.
  • To highlight the advantages of ddTRAP over traditional assays.

Main Methods:

  • The ddTRAP assay involves a two-step process using whole cell lysates.
  • Telomerase-mediated primer extension is followed by droplet digital PCR (ddPCR) detection.
  • This method quantifies the extension of telomere repeat products.

Main Results:

  • Adaptation of the TRAP assay to ddPCR significantly improves throughput.
  • The ddTRAP method offers increased sensitivity for telomerase detection.
  • Enhanced repeatability of the assay is achieved through ddPCR.

Conclusions:

  • ddTRAP provides a highly sensitive and repeatable method for assessing telomerase activity.
  • This protocol facilitates robust detection of telomerase in biological samples.
  • The improved ddTRAP assay is valuable for cancer research and diagnostics.