Dual single nucleotide polymorphisms typing of apolipoprotein E gene based on double restriction endonuclease with lambda exonuclease and triple helix molecular switch assistance.

Single nucleotide polymorphisms (SNPs) are critical determinants of disease susceptibility, pathogenesis, and drug response, underscoring the need for their accurate monitoring in clinical practice. In this study, we propose a novel apolipoprotein E (APOE) genotyping method for the rapid and precise identification of six genotypes (ε2/ε2, ε3/ε3, ε4/ε4, ε2/ε3, ε2/ε4, and ε3/ε4). The method utilizes restriction endonucleases AflIII and HaeII to selectively cleave the rs429358 and rs7412 sites, thereby generating distinct double-stranded DNA fragments. These fragments are subsequently processed by Lambda exonuclease to produce single-stranded DNA, which binds to a triple-helix molecular switch (THMS) and induces its conformational transition into a hairpin structure, resulting in a fluorescence change. The optimized assay exhibits a linear detection range of 5-1000 copies with a minimum detection limit of 2 copies for the rs429358 site, and a range of 10-1000 copies with a minimum detection limit of 6 copies for the rs7412 site. Furthermore, the method was validated using clinical samples from 10 Alzheimer's disease patients, achieving complete concordance with sequencing results, which underlines the high specificity and sensitivity of the method and demonstrates its potential as a valuable tool for the early diagnosis and personalized treatment of Alzheimer's disease.