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

A novel strategy utilizing a DNA template-directed CuAAC click reaction to mimic a ligation reaction based on DNA ligase was successfully established for multiple SNP detection with high sensitivity and specificity.

(Guangdong, China). Sodium ascorbate was bought from Aladdin (Shanghai, China). Tris(3hydroxypropyltriazolylmethyl)amine (THPTA) was obtained from Sigma-Aldrich (St. Louis, MO, USA) and stored at 4℃. HPLC purified oligonucleotides were obtained from Shanghai Sangon Biotech (Shanghai, China). The sequences and modifications used in this study are listed in Table S1 and S2. Deionized water was used in all experiments. All chemical reagents in the experiment were of analytical grade. Beckman coulter ssDNA 100-R Kit was acquired from Beckman (USA). The CuAAC reaction was performed on a C1000 Touch TM PCR (USA).
HPLC-ESI-MS was carried out on Dionex Ultimate 3000 UHPLC and Thermo Q-Exactive mass spectromer (Thermo Fisher, MA, USA). CuAAC products were separated and detected with a Beckman coulter PA800 plus (USA) quipped with an LIF detector (excitation at 488 nm)

The CuAAC reaction for target ssDNA detection
Probe P M (50 nM) and probe P N (50 nM) were mixed with different concentration of target DNA in 10 mM potassium phosphate solution (PBS, pH 7.4). The resulting solutions were heated to 85℃ for 5 min and then gradually cooled down to room temperature. Then a mixture of the catalyst solutions (2 mM THPTA, 100 µM CuSO 4 , 1 mM sodium ascorbate) was added to the above solutions and the CuAAC reaction was carried out at room temperature for 2 h.

The CuAAC reaction for SNP detection
A certain ratio of probe P M and probe P N was mixed with an appropriate amount of target DNA or mutant DNA in PBS. The resulting solutions were heated to 85℃ for 5 min and then gradually cooled down to 30℃. Then a mixture of the catalyst solutions (2 mM THPTA, 100 µM CuSO 4 , 1 mM sodium ascorbate) was added to the above solutions and the CuAAC reaction was carried out at 30℃ for 0.5 h.

The detection of the CuAAC products
The CuAAC products were mixed with 1 µL FAM labeled internal standard chain (IS) to correct the fluorescence intensity caused by aberration of sample injection, and subsequently injected into capillaries with application of a voltage of 9.3 kV for 2 s. The separation and detection of the CuAAC products were performed on the Beckman coulter PA800 plus with an electrophoresis voltage of 9.3 kV at 30℃ for an hour of running time. The positions and areas of the peaks were determined by the 32 Karat Software (Beckman). The relative peak area (RPA) which was used for the quantification of the CuAAC product was defined as the ratio of the peak area of CuAAC product Electronic Supplementary Material (ESI) for Chemical Science. This journal is © The Royal Society of Chemistry 2018 to the peak area of IS.
For HPLC-ESI-MS detection, elution was performed using 8 mM dimethylhexylamine and acetate acid (160 µL in 500 mL solution) as mobile phase A, and acetonitrile as mobile phase B for HPLC-MS. The injection volume was 20 µl. Scan range: 350 m/z to 4000 m/z. Mass resolution: 70 000.     The effect of ratios (P1:P2) on SNP detection. F 1 and F 0 represent the peak area produced by abundance 0.5%, and 100%, respectively.

Fig. S4
The effect of reaction time on SNP detection. F 1 and F 0 represent the peak area produced by abundance 0.5%, and 100%, respectively.