Direct visualization of abasic sites on a single DNA molecule using fluorescence microscopy

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• DNA damage is a critical factor in genomic instability and disease.
• Abasic sites are common DNA lesions that can lead to mutations if not repaired.
• Existing methods for detecting abasic sites often lack single-molecule resolution.

Purpose of the Study:

• To develop a novel method for the direct visualization and quantification of abasic sites on individual DNA molecules.
• To adapt fluorescence in situ hybridization (FISH) for high-resolution DNA damage detection.
• To characterize the distribution and number of abasic sites within single DNA molecules.

Main Methods:

• Modification of fluorescence in situ hybridization (FISH) for extended DNA molecules.
• Specific labeling of abasic sites using a biotinylated aldehyde-reactive probe.
• Detection and quantification via fluorochrome-conjugated streptavidin and calibrated fluorescence detection.
• Counting individual fluorochrome-DNA complexes to estimate abasic site numbers.

Main Results:

• Successful direct visualization of abasic sites on single DNA molecules.
• Accurate estimation of the number of abasic sites per DNA molecule.
• Demonstration of a calibrated method for single-fluorochrome detection.
• Characterization of abasic site distribution at the single-molecule level.

Conclusions:

• The developed method enables direct visualization and precise quantification of abasic sites in individual DNA molecules.
• This technique offers a powerful tool for studying DNA damage and repair mechanisms at high resolution.
• The findings advance our understanding of DNA lesion dynamics and their implications for genome integrity.