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DNA Isolation01:24

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DNA isolation protocols can be fast and straightforward or complex and time-consuming depending on the type and quality of DNA required for further processing. For example, plasmid DNA extraction is a bit more complicated than genomic DNA extraction because of the need for an appropriate lysis method to separate plasmid DNA from gDNA during isolation. However, for specific applications, such as long-range DNA sequencing that require a good yield of high- quality DNA samples, we need to follow...
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Direct Observation of Enzymes Replicating DNA Using a Single-molecule DNA Stretching Assay
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Single-molecule DNA flow-stretch assays for high-throughput DNA-protein interaction studies.

Ayush Kumar Ganguli1, Mohammad Nour Alsamsam1,2, Ugnė Bagdonaitė1

  • 1Institute of Biotechnology, Life Sciences Center, Vilnius University, Lithuania.

FEBS Open Bio
|February 20, 2026
PubMed
Summary

This study optimizes single-molecule DNA flow-stretch assays for studying DNA-protein interactions. The improved protocol enhances reproducibility and accessibility for complex biological system analysis.

Keywords:
DNADNA flow‐stretchDNA‐protein interactionsflowcellfluorescence microscopysingle‐molecule biophysics

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Area of Science:

  • Molecular Biology
  • Biophysics
  • Biochemistry

Background:

  • DNA-interacting proteins are crucial for fundamental cellular processes like replication and repair.
  • Single-molecule techniques offer detailed insights into protein behavior, overcoming ensemble averaging limitations.
  • Existing single-molecule assays have limitations in DNA substrate length and arrangement.

Purpose of the Study:

  • To present an optimized protocol for single-molecule DNA flow-stretch assays.
  • To enhance the reproducibility and accessibility of these assays for complex studies.
  • To advance the application of DNA flow-stretch assays in studying DNA-protein interactions.

Main Methods:

  • Developed an optimized protocol focusing on surface preparation, tethering chemistries, and fluorescent labeling/imaging.
  • Utilized bacteriophage λ DNA for robust flow-induced extension and phiX DNA for assay validation.
  • Employed single-molecule DNA flow-stretch assays with surface-tethered DNA fragments subjected to buffer flow.

Main Results:

  • The optimized protocol provides a more naturalistic approach to probe real-time DNA-protein interactions.
  • Bacteriophage λ DNA is suitable for studying proteins affecting DNA length or translocation.
  • PhiX DNA is useful for assay testing, optimization, and validation.

Conclusions:

  • The optimized protocol significantly improves the study of DNA-protein interactions using single-molecule flow-stretch assays.
  • This advancement facilitates higher-throughput and more complex mechanistic studies across diverse biological systems.
  • The protocol enhances the accessibility and reproducibility of these powerful single-molecule techniques.