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

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Abstract

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.

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

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Publication details

The article was received on 29 Aug 2019, accepted on 31 Oct 2019 and first published on 01 Nov 2019


Article type: Paper
DOI: 10.1039/C9AN01672D
Analyst, 2020, Advance Article

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

    Q. Zhou, X. Zhong, L. Zhao, L. Wang, Y. Zhou and X. Zhang, Analyst, 2020, Advance Article , DOI: 10.1039/C9AN01672D

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