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Simultaneous multiple single nucleotide polymorphism detection based on click chemistry combined with DNA-encoded probes.

ABSTRACT: Single nucleotide polymorphisms (SNPs) are emerging as important biomarkers for disease diagnosis, prognostics and disease pathogenesis. As one type of disease is always connected to several SNP sites, there is great demand for a reliable multiple SNP detection method. Herein, we mimicked a ligation reaction based on DNA ligase and originally utilized an enzyme-free DNA template-directed click reaction for SNP detection. With 5'-alkyne and 3'-azide groups labelled on two oligonucleotide probes, the target DNA-directed Cu(i)-catalyzed alkyne-azide cycloaddition (CuAAC) click reaction produced a new DNA strand with a triazole backbone, as a mimic of a DNA phosphodiester linkage. Trace amounts of the target (as low as 25 fmol in 50 ?L) could be sensitively detected using capillary gel electrophoresis with laser-induced fluorescence (CGE-LIF). Meanwhile, SNP caused an obvious difference in the efficiency of the click reaction, and 0.5% SNP could be easily detected. More importantly, multiplexed SNP detection in a one tube reaction was successfully achieved only by encoding different lengths of the DNA probes for the different SNP sites.

SUBMITTER: Zhou QY

PROVIDER: S-EPMC5932596 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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Simultaneous multiple single nucleotide polymorphism detection based on click chemistry combined with DNA-encoded probes.

Zhou Qian-Yu QY Yuan Fang F Zhang Xiao-Hui XH Zhou Ying-Lin YL Zhang Xin-Xiang XX

Chemical science 20180222 13

Single nucleotide polymorphisms (SNPs) are emerging as important biomarkers for disease diagnosis, prognostics and disease pathogenesis. As one type of disease is always connected to several SNP sites, there is great demand for a reliable multiple SNP detection method. Herein, we mimicked a ligation reaction based on DNA ligase and originally utilized an enzyme-free DNA template-directed click reaction for SNP detection. With 5'-alkyne and 3'-azide groups labelled on two oligonucleotide probes, ...[more]

PMID: 29780463

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Project description:Summary In this work, we developed a simple and robust assay to rapidly detect SNPs in nucleic acid samples. Our approach combines loop-mediated isothermal amplification (LAMP)-based target amplification with fluorescent probes to detect SNPs with high specificity. A competitive “sink” strand preferentially binds to non-SNP amplicons and shifts the free energy landscape to favor specific activation by SNP products. We demonstrated the broad utility and reliability of our SNP-LAMP method by detecting three distinct SNPs across the human genome. We also designed an assay to rapidly detect highly transmissible severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants from crude biological samples. This work demonstrates that competitive SNP-LAMP is a powerful and universal method that could be applied in point-of-care settings to detect any target SNP with high specificity and sensitivity. We additionally developed a publicly available web application for researchers to design SNP-LAMP probes for any target sequence of interest. Graphical abstract Highlights • A simple and rapid assay to detect specific SNPs in complex nucleic acid samples• Approach leverages competitive probes to specifically detect SNP over non-SNP sequences• Demonstrated utility across diverse targets and sample types• Web application for researchers to design SNP-LAMP probes for any target of interest Motivation Single-nucleotide polymorphisms (SNPs) are the most common source of genetic variation between individuals and have implications in human disease, pathogen drug resistance, and agriculture. SNPs are often detected using DNA sequencing or PCR, both of which can be difficult to deploy for on-site testing or in low-resource settings. There is a need for new SNP detection methods that can be performed rapidly without advanced laboratory equipment or a cold supply chain. Detecting single-nucleotide polymorphisms (SNPs) is important for understanding human disease, the emergence of pathogen variants, and the application of genetic agricultural breeding programs. Hyman et al. develop a simple and rapid assay to detect SNPs in complex nucleic acid samples. A web-based tool allows researchers to design custom SNP probes.

Dataset Information

Simultaneous multiple single nucleotide polymorphism detection based on click chemistry combined with DNA-encoded probes.

Publications

Simultaneous multiple single nucleotide polymorphism detection based on click chemistry combined with DNA-encoded probes.

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