A stem-loop (two-tailed) primer system with flanking forward/reverse primers enables highly selective PCR-based detection of mutant DNA sequences against a wild-type background.

Patentradars sammanfattning

This disclosure covers methods, compositions, kits, and reaction mixtures for detecting single-nucleotide variants in DNA using a stem-loop primer whose 3' hemiprobe terminus is complementary to the mutant base but mismatched with wild-type sequence, providing allele-discriminating extension selectivity. The stem-loop primer acts as a two-tailed bridge: its 5' hemiprobe and stem-loop structure serve as hybridization targets for a dedicated forward primer, while a conventional reverse primer binds genomically downstream of the variant site, enabling exponential amplification exclusively from mutant-derived extension products. Selectivity for mutant over wild-type can reach 10- to 1,000,000-fold, and the platform is compatible with qPCR, digital PCR, ddPCR, NGS library prep, NIPT, and transplant rejection monitoring. An optional fluorophore/quencher probe targeting the stem-loop region enables real-time detection.

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