Single nucleotide polymorphism discrimination and genotyping based on cascade strand displacement reaction mediated label-free Cas12a system

Single nucleotide polymorphism (SNP) discrimination plays an important role in precision medicine by providing insights into an individual’s genetic information. The low abundance of the mutant target and the minimal impact caused by SNP on the whole nucleic acid sequence remain a challenge during d...

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Bibliographic Details
Published in:Sensors and actuators. B, Chemical Chemical, 2025-01, Vol.423, p.136832, Article 136832
Main Authors: Li, Yilin, Li, Feng, Li, Ruoxuan, Zhu, Yuedong, Lin, Yanan, Zhang, Yan, Sun, Dan, Yu, Yanyan
Format: Article
Language:English
Subjects:
Cas12a
Cascade strand displacement reaction
G-quadruplex
Genotyping
Single nucleotide polymorphism
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Summary:Single nucleotide polymorphism (SNP) discrimination plays an important role in precision medicine by providing insights into an individual’s genetic information. The low abundance of the mutant target and the minimal impact caused by SNP on the whole nucleic acid sequence remain a challenge during discrimination. Herein, a cascade strand displacement reaction mediated label-free Cas12a sensing platform is established for SNP analysis. Split G-quadruplex (G4) motif is recruited as label-free signal output for Cas12a sensing system, and hence overcomes relying of fluorescence labeled substrates like conventional method. The established platform exhibits excellent single base difference discrimination ability attributing to the cascade strand displacement process, during which, single-base mismatch will affect the strand-exchange rate significantly. The limit of detection reaches 1 copy / test after integration with isothermal preamplification. The proposed method can discriminate as low as 0.1 % single base variation and perform robustly in biological matrixes. Human buccal swab samples are successfully genotyped with high accuracy. •Split G4 was utilized as the label-free output method for Cas12a system.•Cascade strand displacement reaction rendered the platform great specificity.•The detection sensitivity reached 1 copy per test with the help of RPA.•Human buccal swab samples were tested with high accuracy.•The platform could serve as a universal platform for SNP discrimination.
ISSN:0925-4005
DOI:10.1016/j.snb.2024.136832