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DNA sequencing is a fundamental technique that is routinely used in the biological sciences. This method can be applied to a range of questions at different scales - from the sequencing of a cloned DNA fragment or the study of a mutation in a gene up to whole-genome sequencing. However, despite the widespread use of sequencing today, it was not until 1977 that Fredrick Sanger and his collaborators developed the chain-termination method to decode DNA sequences. It relies on the separation of a...
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In Vivo Genomic Supercoiling Mapping Using Psora-seq.

Bryan J Visser1, David Bates2

  • 1Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA.

Methods in Molecular Biology (Clifton, N.J.)
|July 19, 2024
PubMed
Summary
This summary is machine-generated.

We developed Psora-seq, a sensitive and robust DNA supercoiling assay using psoralen and magnetic beads. This method accurately maps DNA supercoiling at 1kb resolution, overcoming limitations of previous techniques.

Keywords:
Chromosome supercoilingDNA supercoilingDNA topologyPsoralenPsoralen ChIP-seq

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Area of Science:

  • Molecular Biology
  • Genomics
  • Biochemistry

Background:

  • DNA supercoiling is crucial for DNA replication, transcription, and recombination.
  • Existing genomic supercoiling assays are often difficult to perform and lack sensitivity.

Purpose of the Study:

  • To develop a sensitive, robust, and accessible method for mapping genomic supercoiling.
  • To overcome limitations of previous psoralen-based DNA supercoiling assays.

Main Methods:

  • Developed psoralen affinity purification with genomic sequencing (Psora-seq).
  • Utilized biotinylated psoralen and streptavidin magnetic beads for DNA pull-down.
  • Employed deep sequencing for 1kb resolution supercoiling quantification.

Main Results:

  • Psora-seq provides a sensitive and robust assay for DNA supercoiling.
  • The method achieves 1kb resolution in mapping supercoiling.
  • Psora-seq improves upon previous psoralen pull-down assays by enhancing photo-binding and DNA recovery.

Conclusions:

  • Psora-seq is a powerful new tool for studying DNA supercoiling.
  • This assay facilitates research in DNA replication, transcription, and recombination.
  • The method requires only common laboratory equipment and reagents.