Isothermal Detection of Single-Nucleotide Variants via pH-Mediated Toehold-Controlled Ligation on Double-Stranded DNA

Area of Science:

• Molecular Biology
• Biochemistry
• Diagnostics

Background:

• Single-nucleotide variants (SNVs) are crucial biomarkers in molecular diagnostics.
• Current SNV detection methods often rely on thermal denaturation and complex instrumentation, limiting speed and accessibility.
• There is a need for rapid, cost-effective, and instrument-free SNV detection strategies.

Purpose of the Study:

• To develop a novel, simple, and cost-effective physicochemical strategy for direct SNV detection on double-stranded DNA.
• To eliminate the reliance on thermal denaturation in SNV detection workflows.
• To create a rapid, portable, and instrument-free platform for molecular diagnostics.

Main Methods:

• Exploited the pH sensitivity of DNA duplex stability for strand unwinding.
• Utilized a toehold-gated probe for specific SNV site assembly after pH-mediated unwinding.
• Employed toehold-mediated strand displacement and designed ligation for allele recognition.
• Integrated an in vitro transcription reaction for signal amplification and visual readout.

Main Results:

• Developed a pH-mediated, toehold-controlled SNV detection method.
• Eliminated the need for thermal denaturation, replacing it with rapid acid-base treatment.
• Achieved detection and readout within 45 minutes through a two-step workflow.
• Demonstrated a visually distinguishable signal generation via in vitro transcription.

Conclusions:

• The developed method provides a rapid, low-cost, portable, and instrument-free solution for precise SNV identification.
• This approach significantly simplifies SNV detection workflows.
• The platform holds strong potential for rapid on-site molecular diagnostics and biomarker analysis.