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Ultra-specific genotyping of single nucleotide variants by ligase-based loop-mediated isothermal amplification coupled with a modified ligation probe

Specific and accurate detection of single nucleotide variants (SNVs) plays significant roles in pathogenic gene research and clinical applications. However, the sensitive but ultra-specific detection of rare variants in biological samples still remains challenging. Herein, we report a novel, robust...

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Detalles Bibliográficos
Autores principales: Sun, Yuanyuan, Han, Bingjie, Sun, Fangfang
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The Royal Society of Chemistry 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9032167/
https://www.ncbi.nlm.nih.gov/pubmed/35479710
http://dx.doi.org/10.1039/d1ra00851j
Descripción
Sumario:Specific and accurate detection of single nucleotide variants (SNVs) plays significant roles in pathogenic gene research and clinical applications. However, the sensitive but ultra-specific detection of rare variants in biological samples still remains challenging. Herein, we report a novel, robust and practical SNV assay by integrating the outstanding features of high selectivity of an artificial mismatched probe, and the powerful loop-mediated isothermal amplification. In this strategy, we rationally introduce artificial mismatched bases into the 3′-terminal regions of the probe located in the ligation region to reduce the risk of nonspecific ligation, which can dramatically improve the specificity for the SNV assay. The proposed method can discern as little as 0.01% mutant DNA in the high background of wild-type DNA with high sensitivity (10 aM). In virtue of its outstanding performance, the artificial mismatched probe may also be employed and expanded in various DNA and RNA genetic analyses with ligase-assisted approaches, showing great potential in biomedical research, clinical diagnostics, and bioanalysis.