Method for detecting 5-methylcytosines in nucleic acid
A technology of methylcytosine, nucleic acid, applied in the field of nucleic acid chemistry
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Embodiment 1
[0023] Example 1: Sodium bisulfite treatment of nucleic acid strands containing 5-methylcytosine
[0024] Pipette 2 μL of 5-methylcytosine-containing DNA (10 μM) labeled with the fluorescent substance HEX (Chinese name is 5-hexachlorofluorescein phosphoramidate) at the 3’ end, add 48 μL of water, and then add 5.5 μL of freshly prepared NaOH solution (3M), incubate in a water bath at 42°C for 0.5 hours, add 30 μL of hydroquinone (10 mM) after the completion of the water bath incubation, and then add 520 μL of NaHSO 3 solution (3.6M, pH=5.0) and 200 μL of paraffin oil, and incubated in a water bath at 50° C. for 16 hours in a dark place, to change cytosine on DNA to uracil. After the reaction, use a desalting column to desalt and purify the DNA.
Embodiment 2
[0025] Example 2: DNA treated with sodium bisulfite was treated with compound 1 and volume fraction of 10% piperidine
[0026] Take 4 μL of 5-methylcytosine-containing DNA (10 μM) treated with sodium bisulfite, add 10 μL of acetonitrile, 2 μL of Tris-HCl solution (1M, pH=5.0), 2 μL of water and 2 μL of compound 1 ( 20mM), heat treatment at 50°C for 10 minutes, 5-methylcytosine on the DNA chain specifically binds to compound 1; then precipitated with ice ethanol, centrifuged, spin-dried the solvent, and then added piperidine with a volume fraction of 10% at 90°C for 0.5 h, the position-specific cleavage of the 5-methylcytosine bound to compound 1; then ice-ethanol precipitation, centrifugation, and spin-drying of the solvent. Load the sample to the electrophoresis tank, and observe the gel results after 2 hours. By comparing with the control of the A+G sequence and the control of the G sequence, the position and number of the DNA bands in the polyacrylamide denaturing gel can b...
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