Fluorescent marker for direct detection of specific dsDNA sequences

Area of Science:

• Molecular Biology
• Biotechnology
• Genomics

Background:

• Direct visualization of specific DNA sequences on large genomic scales remains a challenge.
• Existing methods for DNA sequence detection may lack prolonged imaging capabilities or specificity.
• Restriction enzymes offer sequence-specific binding but typically involve DNA cleavage.

Purpose of the Study:

• To develop a novel fluorescent marker for prolonged, direct visualization of specific double-stranded DNA (dsDNA) sequences.
• To create a tool that leverages a modified restriction enzyme for sequence-specific DNA labeling.
• To enable measurement of marker positions along individual DNA molecules.

Main Methods:

• A mutant EcoRI restriction endonuclease (K249C) was engineered and biotinylated at the mutation site.
• The biotinylated enzyme was complexed with fluorescent nanospheres via NeutrAvidin.
• The enzyme-nanosphere complex was incubated with dsDNA under conditions preventing cleavage, followed by fluorescent staining and single-molecule stretching on glass slides.

Main Results:

• A fluorescent marker capable of prolonged, direct visualization of specific dsDNA sequences was successfully created.
• The marker specifically binds to its target sequence, as confirmed by bulk gel-shift assays and single-molecule measurements.
• The method allows for the measurement of bound marker positions along individual DNA molecules.

Conclusions:

• The developed fluorescent marker provides a new method for long-term, direct visualization of specific dsDNA sequences.
• This tool enables precise localization of target sequences within genomic DNA at the single-molecule level.
• The approach offers potential applications in genomics research, diagnostics, and DNA mapping.