Accurate determination of allelic zygosity, whether a genetic variant is present in zero, one, or
two copies, is essential for understanding how a gene contributes to disease. Despite this, most
existing single nucleotide polymorphism (SNP) detection methods are primarily designed to
indicate whether a mutation is present or absent and do not reliably distinguish zygosity. Many
of these methods require complicated workflows, long processing times, or careful
interpretation of amplification curves, and their accuracy often decreases when applied to
mixed or crude biological samples. Here, we report for the first time a dual peptide nucleic
acid–locked nucleic acid (PNA-LNA) molecular-switch method combined with loop-mediated
isothermal amplification (LAMP) that enables direct and clear determination of zygosity for
the rs734809 SNP in PNPLA3. For this SNP, disease risk strongly depends on the allelic
composition at this site. Individuals carrying one mutant allele (G) and one wild-type allele (C)
(heterozygous, C/G) usually exhibit moderate disease features, while individuals carrying two
mutant alleles (homozygous, G/G) develop more severe disease and faster progression.
The assay uses two complementary amplification reactions, where each reaction blocks one
allele and allows amplification of the other. This design enables straightforward discrimination
between homozygous wild-type, heterozygous, and homozygous mutant conditions, without
relying on amplification speed or complex signal analysis. The method was validated using
metabolic-dysfunction-associated steatohepatitis (MASH)–specific synthetic DNA targets,
cultured cell lines, and extraction-free crude cell lysates spiked into human plasma. It
demonstrated high specificity, single-copy sensitivity, and full agreement with Sanger
sequencing. This method offers a rapid, low-cost, and extraction-free workflow with both
fluorescence and colorimetric readouts. Importantly, the underlying principle is not target- or
gene-specific and can be readily extended to a wide range of biomedical, environmental,
agricultural, and biosecurity applications.