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Telomeres and Telomerase02:41

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In eukaryotic DNA replication, a single-stranded DNA fragment remains at the end of a chromosome after the removal of the final primer. This section of DNA cannot be replicated in the same manner as the rest of the strand because there is no 3’ end to which the newly synthesized DNA can attach. This non-replicated fragment results in gradual loss of the chromosomal DNA during each cell duplication. Additionally, it can induce a DNA damage response by enzymes that recognize single-stranded...
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Translesion (TLS) polymerases rescue stalled DNA polymerases at sites of damaged bases by replacing the replicative polymerase and installing a nucleotide across the damaged site. Doing so, TLS allows additional time for the cell to repair the damage before resuming regular DNA replication.
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In eukaryotic cells, DNA replication is highly conserved and tightly regulated. Multiple linear chromosomes must be duplicated with high fidelity before cell division, so there are many proteins that fulfill specialized roles in the replication process. Replication occurs in three phases: initiation, elongation, and termination, and ends with two complete sets of chromosomes in the nucleus.
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Droplet Digital TRAP ddTRAP: Adaptation of the Telomere Repeat Amplification Protocol to Droplet Digital Polymerase Chain Reaction
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Droplet Digital TRAP ddTRAP: Adaptation of the Telomere Repeat Amplification Protocol to Droplet Digital Polymerase Chain Reaction

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Telomerase Repeated Amplification Protocol (TRAP).

Ilgen Mender1, Jerry W Shay2

  • 1Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, USA;

Bio-Protocol
|May 17, 2016
PubMed
Summary
This summary is machine-generated.

Telomere Repeat Amplification Protocol (TRAP) measures telomerase activity, crucial for cancer and aging research. This assay detects telomerase in cells and tissues, aiding in understanding cell proliferation and developing targeted therapies.

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Area of Science:

  • Molecular Biology
  • Cell Biology
  • Genetics

Background:

  • Telomeres protect chromosome ends but shorten due to cellular processes, leading to aging and disease.
  • Telomerase, a ribonucleoprotein enzyme, counteracts telomere shortening by adding repeats, enabling continuous cell proliferation.
  • Dysregulated telomerase activity is a hallmark of cancer, making it a target for cancer therapies.

Purpose of the Study:

  • To provide a detailed protocol for the Telomere Repeat Amplification Protocol (TRAP) assay.
  • To explain the TRAP assay's utility in determining telomerase activity in mammalian cells and tissues.
  • To highlight the role of telomere biology in aging, cancer, and therapeutic strategies.

Main Methods:

  • The TRAP assay involves three key steps: extension, amplification, and detection.
  • During extension, telomerase adds repeats to a substrate oligonucleotide (TS).
  • Amplification uses PCR with specific primers, and detection is performed via electrophoresis, including internal controls (TSNT) to identify false negatives.

Main Results:

  • The TRAP assay effectively quantifies telomerase activity by analyzing amplified telomeric repeat products.
  • The inclusion of TSNT controls ensures the reliability of results by detecting potential false negatives.
  • Successful implementation of the TRAP assay allows for the assessment of telomerase presence in various biological samples.

Conclusions:

  • The TRAP assay is a valuable and widely used method for assessing telomerase activity.
  • Understanding telomerase function through assays like TRAP is critical for research in aging and oncology.
  • This protocol facilitates reproducible measurement of telomerase activity, supporting advancements in cancer diagnostics and therapeutics.