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Related Experiment Videos

Mapping DNA Breaks by Next-Generation Sequencing.

Laura Baranello1,2, Fedor Kouzine3, Damian Wojtowicz3

  • 1Laboratory of Pathology, NCI/NIH, Bethesda, MD, 20892, USA. baranellolf@mail.nih.gov.

Methods in Molecular Biology (Clifton, N.J.)
|October 19, 2017
PubMed
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This summary is machine-generated.

We developed two new methods, DSB-Seq and SSB-Seq, to map DNA breaks in human cells. These powerful tools detect both double-strand and single-strand breaks, revealing the genome's "breakome".

Area of Science:

  • Genomics
  • Molecular Biology
  • Cell Biology

Background:

  • DNA damage, including double-strand breaks (DSBs) and single-strand breaks (SSBs), is crucial in cellular processes and disease.
  • Accurate mapping of these breaks is essential for understanding genome stability and disease mechanisms.
  • Existing methods may have limitations in comprehensively detecting the full spectrum of DNA breaks.

Purpose of the Study:

  • To introduce two novel sequencing-based methods, DSB-Seq and SSB-Seq, for high-resolution mapping of DSBs and SSBs in the human genome.
  • To demonstrate the utility of these methods in a relevant cellular context, such as human colon cancer cells.
  • To validate the accuracy and reliability of DSB-Seq and SSB-Seq using a known DNA-damaging agent.

Main Methods:

Keywords:
DNA damageDouble-strand breaks (DSBs)Etoposide (ETO)Single-strand breaks (SSBs)Topoisomerase 2 (Top2)

Related Experiment Videos

  • DSB-Seq: A method specifically designed to capture and sequence DNA fragments resulting from double-strand breaks.
  • SSB-Seq: A complementary method developed for the detection and sequencing of single-strand breaks.
  • Application in HCT1116 cells: Both methods were applied to human colon cancer cells (HCT1116) to assess their performance in a cancer cell line.
  • Validation with etoposide (ETO): The results obtained from DSB-Seq and SSB-Seq were validated using etoposide, a known topoisomerase 2 poisoning agent that induces DSBs.

    • Main Results:

    • Successful implementation of DSB-Seq and SSB-Seq in HCT1116 human colon cancer cells.
    • Demonstration of the methods' ability to detect and map both DSBs and SSBs within the genome.
    • Validation of the detected break sites using etoposide, confirming the accuracy of the developed sequencing approaches.

    Conclusions:

    • DSB-Seq and SSB-Seq provide powerful and direct tools for mapping DNA breaks in human cells.
    • These methods enable the comprehensive analysis of the DNA "breakome", encompassing both physiological and pathological DSBs and SSBs.
    • The developed techniques offer significant potential for advancing research in genome stability, DNA repair, and cancer biology.