Programmable Double Peptide Nucleic Acid-Locked Nucleic Acid Molecular Switch Enables Extraction-Free Direct Zygosity...

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Single-nucleotide polymorphism (SNP) zygosity determination is crucial for understanding genetic disease associations, but current isothermal amplification methods lack allelic resolution.
• Metabolic dysfunction-associated steatohepatitis (MASH) susceptibility is linked to genetic variants influencing lipid metabolism, highlighting the need for precise genotyping.

Purpose of the Study:

• To develop and validate a novel isothermal amplification strategy for direct and accurate SNP zygosity determination.
• To enable allele-resolved genotyping without the need for nucleic acid extraction, facilitating rapid diagnostics.

Main Methods:

• Integration of a programmable double peptide nucleic acid and locked nucleic acid (PNA-LNA) molecular switch with loop-mediated isothermal amplification (LAMP).
• Utilizing allele-specific PNA-LNA clamps for reciprocal suppression of amplification, converting single-base differences into binary outputs.
• Testing the platform using the *PNPLA3* rs738409 variant in various DNA samples, including crude lysates.

Main Results:

• The PNA-LNA-LAMP assay achieved single-copy sensitivity and complete suppression of mismatched alleles.
• Direct zygosity determination (homozygous wild-type, heterozygous, homozygous mutant) was demonstrated without reliance on amplification kinetics.
• Genotype assignments showed full agreement with Sanger sequencing, with consistent results from fluorescence and colorimetric readouts.

Conclusions:

• The developed PNA-LNA molecular switch provides a generalizable framework for allele-resolved isothermal genotyping, extending LAMP capabilities.
• This method enables rapid, naked-eye zygosity-resolved SNP analysis, suitable for laboratory and resource-limited settings.
• The platform offers a significant advancement for genetic analysis in MASH and other genetically influenced diseases.