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Related Experiment Videos

High resolution mapping DNAs by R-loop atomic force microscopy

D V Klinov1, I V Lagutina, V V Prokhorov

  • 1Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry and Institute of Molecular Genetics,Russian Academy of Sciences, Miklukho-Maklaya 16/10, Moscow 117871, Russia.

Nucleic Acids Research
|October 1, 1998
PubMed
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Atomic force microscopy (AFM) visualizes RNA:DNA hybrids called R-loops, enabling precise mapping of human endogenous retrovirus K10 long terminal repeats (LTRs). This R-loop imaging technique offers high accuracy for DNA sequence analysis.

Area of Science:

  • Molecular Biology
  • Nanotechnology
  • Genomics

Background:

  • R-loops, structures of DNA:RNA hybrids, play roles in various cellular processes.
  • Accurate mapping of DNA sequences is crucial for understanding gene function and genetic diseases.
  • Human endogenous retroviruses (HERVs) are remnants of ancient viral infections integrated into the host genome.

Purpose of the Study:

  • To develop and apply atomic force microscopy (AFM) for imaging R-loops formed by short RNA transcripts.
  • To map the long terminal repeats (LTRs) of the human endogenous retrovirus K10 (HERV-K10) family within plasmid and cosmid DNA.
  • To assess the accuracy and potential applications of R-loop imaging for DNA sequence analysis.

Main Methods:

  • R-loop formation using specific RNA probes targeting U3 and U5 regions of HERV-K10 LTRs.

Related Experiment Videos

  • Stabilization of R-loops with glyoxal treatment and adsorption onto mica surfaces.
  • Imaging of R-loops using constant-force atomic force microscopy (AFM) in height mode.

    • Main Results:

    • AFM successfully imaged R-loops, appearing as looped RNA:DNA hybrids.
    • The RNA:DNA hybrids were observed to be 12-15% shorter than corresponding DNA:DNA duplexes.
    • AFM provided accurate mapping of LTR positions within plasmids and cosmids, with precision up to 0.5% of cosmid length.

    Conclusions:

    • AFM-based R-loop imaging is a viable technique for visualizing and mapping specific DNA sequences.
    • The method demonstrates high accuracy and potential for analyzing DNA structures and variations.
    • This technique could be adapted for mapping gene exons, regulatory regions, and detecting genetic alterations associated with diseases.