Comprehensive FISH probe design tool applied to imaging human immunoglobulin class switch recombination

Area of Science:

• Genomics
• Molecular Biology
• Immunology

Background:

• Fluorescence in-situ hybridization (FISH) is crucial for visualizing DNA.
• Designing effective FISH probes, especially for repetitive sequences, remains challenging.
• Understanding B cell class switch recombination requires precise molecular tools.

Purpose of the Study:

• To present a novel web-based algorithm (webFISH) for designing target-specific FISH probes.
• To enable simultaneous detection of DNA recombination events in single cells.
• To investigate the asynchrony of class switch recombination in immunoglobulin heavy chain alleles.

Main Methods:

• Developed a web engine boosted fluorescence in-situ hybridization (webFISH) algorithm.
• Utilized genome-wide sequence similarity search for probe design.
• Applied webFISH to design probes for the human immunoglobulin heavy chain locus.
• Analyzed primary B cells to detect class switch recombination.

Main Results:

• Successfully designed and utilized webFISH probes for the human immunoglobulin heavy chain.
• Achieved simultaneous detection of class switch recombination in both alleles within single B cells.
• Demonstrated asynchrony of class switch recombination between the two alleles.
• Enabled parallel assessment of protein expression via immunofluorescence staining.

Conclusions:

• The webFISH algorithm effectively designs single-copy and repetitive DNA FISH probes.
• This technique allows for the direct, simultaneous detection of class switch recombination.
• Revealed asynchrony in class switch recombination, offering new insights into molecular mechanisms.