Programmable Double Peptide Nucleic Acid-Locked Nucleic Acid Molecular Switch Enables Extraction-Free Direct Zygosity Discrimination of Single Nucleotide Polymorphisms.

Accurate discrimination of single-nucleotide polymorphism (SNP) zygosity remains challenging for isothermal amplification platforms, which typically report mutation presence without resolving allelic composition. This limitation is particularly relevant for metabolic dysfunction-associated steatohepatitis (MASH) in which disease susceptibility and progression are strongly influenced by genetic variants that regulate hepatic lipid metabolism. Here, we report a programmable double peptide nucleic acid and locked nucleic acid (PNA-LNA) molecular switch integrated with loop-mediated isothermal amplification (LAMP) for direct zygosity determination without nucleic acid extraction. The strategy employs two complementary reactions in which allele-specific PNA-LNA clamps selectively inhibit amplification of an allele by PNA while permitting amplification of the alternative allele by LNA. This reciprocal suppression architecture converts single-base differences into binary amplification outputs, enabling direct identification of homozygous wild-type, heterozygous, and homozygous mutant genotypes without reliance on amplification kinetics or post hoc curve analysis. Using the clinically relevant PNPLA3 rs738409 variant as a model target, we systematically characterized the analytical performance of the platform. The assay achieves single-copy sensitivity, complete suppression of mismatched alleles under optimized conditions, and clear genotype resolution across synthetic DNA templates, genomic DNA from cultured cell lines, and extraction-free crude lysates spiked in human plasma. Genotype assignments showed full agreement with Sanger sequencing. Both fluorescence and colorimetric readouts produced consistent zygosity calls, supporting implementation of this method as a rapid, naked-eye test in laboratory and resource-limited settings for zygosity-resolved SNP analysis in MASH and other genetically associated diseases. Additionally, the programmable double PNA-LNA molecular switch offers a generalizable framework for allele-resolved isothermal genotyping and extends LAMP from mutation detection to quantitative allelic discrimination. To the best of our knowledge, this work may represent the first isothermal amplification-based strategy employing dual PNA-LNA probe pairs for direct determination of zygosity in human genomic DNA.