Hairpin-Empowered Invasive Reaction Combined with Catalytic Hairpin Assembly Cascade Amplification for the Specific Detection of Single-Nucleotide Polymorphisms

Single-nucleotide polymorphism (SNP) is widely used in the study of disease-related genes and in the genetic study of animal and plant strains. Therefore, SNP detection is crucial for biomedical diagnosis and treatment as well as for molecular design breeding of animals and plants. In this regard, this article describes a novel technique for detecting SNP using flap endonuclease 1 (FEN 1) as a specific recognition element and catalytic hairpin assembly (CHA) cascade reaction as a signal amplification strategy. The mutant target (MT) was hybridized with a biotin-modified upstream probe and hairpin-type downstream probe (DP) to form a specific three-base overlapping structure. Then, FEN 1 was employed for three-base overlapping structure-specific recognition, namely, the precise SNP site identification and the 5' flap of DP dissociation. After dissociation, the hybridized probes were magnetically separated by a streptavidin-biotin complex. Especially, the ability to establish such a hairpin-type DP provided a powerful tool that could be used to hide the cut sequence (CS) and avoid false-positive signals. The cleaved CS initiated the CHA reaction and allowed superior fluorescence signal generation. Owing to the high specificity of FEN 1 for single base recognition, only the MT could be distinguished from the wild-type target and mismatched DNA. Owing to the dual signal amplification, as low as 0.36 fM MT and 1% mutation abundance from the mixtures could be detected, respectively. Furthermore, it could accurately identify SNPs from human cancer cells, as well as soybean leaf genome extracts. This strategy paves the way for the development of more precise and sensitive tools for diagnosing early onset diseases as well as molecular design breeding tools.