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

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 μL) 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.