Plug-and-Play Competitive Hairpin Conversion Module Enables Enzyme-Free High-Resolution Discrimination of Single...

Area of Science:

• Molecular Biology
• Biotechnology
• Genetics

Background:

• Single-nucleotide polymorphism (SNP) detection is crucial for genetic analysis but challenged by low thermodynamic differences in long DNA sequences.
• Existing methods often struggle to differentiate single-base mismatches, with probe binding energy masking critical signals.

Purpose of the Study:

• To develop a novel, modular system for accurate SNP detection in complex genetic backgrounds.
• To overcome the limitations of current SNP detection methods by enhancing single-base discrimination.

Main Methods:

• Development of a plug-and-play competitive hairpin conversion module (CHCM) utilizing strand-competitive hybridization and toehold-mediated strand displacement.
• Design of two hairpin probes (H1 and H2) for specific recognition of wild-type and mutant alleles.
• Leveraging thermodynamic competition to modulate probe-target binding stability and generate differential DNA assemblies.

Main Results:

• CHCM achieved precise discrimination of heterozygous samples with as low as 0.1% mutation abundance in long DNA sequences.
• The system demonstrated high accuracy without enzymatic assistance, using conventional fluorescent probes.
• Successful SNP genotyping was validated using soybean leaf genomic DNA, confirming practical utility.
• CHCM showed seamless integration with PCR amplification and catalytic hairpin assembly (CHA) signal amplification.

Conclusions:

• CHCM offers a high-resolution, cost-effective solution for SNP detection in complex biological samples.
• The developed modular framework supports scalable and dynamic nucleic acid probe design for future applications.