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

Authors

Milkiyas Toru Tantu, Md Akeruzzaman Shaon, Farjana Haque, Omar Hamza Bin Manjur, Kiran Shrestha, Md Sajedul Islam, Sharmin Aktar, Romario Nguyen, Liang Qiao, Nidhish Francis, Doaa M Hanafy, Allen G Ross, Muhammad J A Shiddiky, Fatema Zerin Farhana

Published in

Analytical chemistry. Jun 21, 2026. Epub Jun 21, 2026.

Abstract

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.

PMID:
42324653
Bibliographic data and abstract were imported from PubMed on 22 Jun 2026.

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