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Related Concept Videos

Telomeres and Telomerase02:41

Telomeres and Telomerase

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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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Author Spotlight: Advanced Single-Molecule Techniques for Investigating Telomeric Protein-DNA Interactions
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Author Spotlight: Advanced Single-Molecule Techniques for Investigating Telomeric Protein-DNA Interactions

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High-throughput single-molecule telomere characterization.

Jennifer McCaffrey1, Eleanor Young1, Katy Lassahn2

  • 1School of Biomedical Engineering, Drexel University, Philadelphia, Pennsylvania 19104, USA.

Genome Research
|October 14, 2017
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Summary
This summary is machine-generated.

We developed a novel CRISPR/Cas9 method for single-molecule telomere length analysis. This technique allows global, haplotype-resolved mapping of subtelomeres and telomere lengths, revealing insights into their variation and function.

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Area of Science:

  • Genomics
  • Molecular Biology
  • Epigenetics

Background:

  • Telomeres protect chromosome ends but shorten with cell division.
  • Subtelomeric regions (SREs) are complex and influence telomere length regulation.
  • Current methods lack single-molecule resolution for global telomere analysis.

Purpose of the Study:

  • To develop a novel method for global, haplotype-resolved, single-molecule telomere length analysis.
  • To enable detailed characterization of subtelomere organization and variation.
  • To investigate telomere attrition and subtelomere biases in different cell types.

Main Methods:

  • Utilized an in vitro CRISPR/Cas9 RNA-directed nickase system for specific DNA labeling.
  • Employed genome-wide barcoding with a separate nickase enzyme.
  • Integrated high-throughput imaging and nanochannel array analysis of single DNA molecules.
  • Mapped subtelomere repeat element (SRE) regions to unique chromosomal DNA.

Main Results:

  • Achieved simultaneous and accurate analysis of 30-35 discrete telomeres.
  • Documented telomere attrition in human fibroblasts from early to senescent stages.
  • Identified subtelomere-specific biases for critically short telomeres.
  • Presented the first global, single-telomere-resolved analyses of cancer cell lines.

Conclusions:

  • The novel method provides unprecedented resolution for telomere and subtelomere analysis.
  • Offers insights into the population dynamics and functional roles of subtelomeric regions.
  • Enables detailed study of telomere length dynamics in various biological contexts, including aging and cancer.