High-throughput ultra-sensitive discrimination of single nucleotide polymorphism via click chemical ligation

• Published in: Analyst (London) 2020-01, Vol.145 (1), p.172-176
• Main Authors: Zhou, Qian-Yu, Zhong, Xin-Ying, Zhao, Ling-Li, Wang, Li-Juan, Zhou, Ying-Lin, Zhang, Xin-Xiang
• Format: Article
• Language: English
• Subjects: Alkynes; Alkynes - chemistry; Armoracia - enzymology; Azides - chemistry; Benzidines - chemistry; Biomarkers; Chemical reactions; Click Chemistry; Colorimetry; Colorimetry - methods; Coloring Agents - chemistry; Cycloaddition; Cycloaddition Reaction; Deoxyribonucleic acid; Discrimination; DNA; DNA - analysis; DNA - genetics; DNA Probes - chemistry; DNA Probes - genetics; Horseradish Peroxidase - chemistry; Immobilized Nucleic Acids - chemistry; Immobilized Nucleic Acids - genetics; Nucleic Acid Hybridization; Nucleotides; Oligodeoxyribonucleotides - chemistry; Oligodeoxyribonucleotides - genetics; Organic chemistry; Oxidation-Reduction; Pathogenesis; Plates (structural members); Polymorphism; Polymorphism, Single Nucleotide; Sandwich structures; Self-assembly; Steric effects
• ISSN: 0003-2654; 1364-5528
• DOI: 10.1039/c9an01672d

Single nucleotide polymorphisms (SNPs) have been proven to be important biomarkers for disease diagnosis, prognosis and disease pathogenesis. Here, taking the advantages of a self-assembled oligonucleotide sandwich structure and robust chemical reactions, we have developed a simple, high-throughput and effective colorimetric analytical technique termed CuAAC-based ligation-assisted assays (CuAAC-LA) for SNP detection using a DNA-BIND 96-well plate. With the 5'-azide and 3'-alkyne groups labelled on two oligonucleotide probes, the target DNA can direct a Cu(i)-catalyzed alkyne-azide cycloaddition (CuAAC) click reaction. Since the small difference in duplex stability caused by a single-nucleotide mismatch was amplified by the steric effects of these reactive groups for the ligation reaction of an unstable duplex, CuAAC-LA exhibited an ultra-sensitive discrimination ability for a mutant type target in the presence of large amounts of wild type targets. As low as 0.05% SNP could be clearly detected, which was better than most previously reported methods by various DNA ligases, indicating that a simple and rapid synthetic method i.e., the DNA template-directed click reaction held the potential to replace the ligase for SNP detection.