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Related Concept Videos

Labeling DNA Probes03:31

Labeling DNA Probes

DNA probes are fragments of DNA labeled with a reporter tag to enable their detection or purification. The resulting labeled DNA probes can then hybridize to target nucleic acid sequences through complementary base-pairing, and may be used to recover or identify these regions.
Radioisotopes, fluorophores, or small molecule binding partners like biotin or digoxigenin, are the most widely used reporter tags for labeling DNA probes. These labels can be attached to the probe DNA molecule via...

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Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy
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Dual fluorescent labeling method to visualize plasmid DNA degradation.

Charudharshini Srinivasan1, Shafiuddin Siddiqui, Lawrence K Silbart

  • 1Department of Pharmaceutical Sciences, University of Connecticut, 69 North Eagleville Road, Unit 3092, Storrs, Connecticut 06269, USA.

Bioconjugate Chemistry
|December 18, 2008
PubMed
Summary

Researchers developed a dual-labeling method to track plasmid DNA stability within cells. This tool helps understand DNA degradation, a key barrier in nonviral gene delivery, at the molecular level.

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Published on: January 7, 2019

Area of Science:

  • Biotechnology
  • Molecular Biology
  • Cell Biology

Background:

  • Nonviral vectors are crucial for gene delivery, but their efficiency is limited by intracellular barriers.
  • DNA instability in the cytoplasm is a significant challenge hindering successful nuclear translocation.
  • Understanding DNA trafficking and degradation is essential for improving gene therapy strategies.

Purpose of the Study:

  • To develop a novel dual-labeling method for tracking plasmid DNA integrity within the intracellular environment.
  • To investigate DNA instability and degradation during nonviral gene delivery using this new tool.
  • To provide a molecular-level understanding of DNA fate within single cells.

Main Methods:

  • Plasmid DNA was dual-labeled with rhodamine and fluorescein using peptide nucleic acid (PNA) linkers.
  • Gel electrophoresis confirmed that labeling did not affect DNA integrity or electrophoretic mobility.
  • Confocal time-lapse imaging tracked dual-labeled DNA complexes (Lipofectamine2000/DNA) in CHO-K1 cells.

Main Results:

  • Intact DNA appeared as a single yellow fluorescence signal due to fluorophore proximity.
  • Separated red and green fluorescence signals indicated DNA degradation or fragmentation.
  • Observation of yellow voxels confirmed intact DNA in the cytoplasm, while red/green signals suggested degradation.

Conclusions:

  • The developed dual-labeling technique offers a robust tool for monitoring DNA stability in real-time within living cells.
  • This method can elucidate the molecular mechanisms of DNA degradation during nonviral gene delivery.
  • The findings contribute to optimizing gene delivery systems by addressing DNA instability challenges.