Nucleic acid aptamer specifically combined with Sudan Red and application of nucleic acid aptamer
A technology of nucleic acid aptamer and Sudan red, applied in the field of biomedicine
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Embodiment 1
[0024] Example 1: Screening of Sudan Red Nucleic Aptamer (C07)
[0025] 1. Coupling of Ligand (Sudan Red) and Matrix (Sepharose 6B)
[0026] 1. Suspend 1 g of lyophilized powder Sepharose 6B in 3 mL of distilled water (1 g of lyophilized powder can produce a final matrix volume of about 3.0 mL);
[0027] 2. Immediately rinse with 200 mL of distilled water per gram of powder for 1 hour in a sintered glass filter (multi-void G3);
[0028] 3. Dissolve 6g of ligand with 6mL of coupling buffer solution (0.05M, carbonate buffer at pH 9.6) to make the final concentration 1mg / mL, or transfer the dissolved ligand to the coupling with a desalting column. In the joint buffer solution, adjust the pH value of the aqueous phase;
[0029] 4. Adjust the ratio of the matrix to the buffer solution with a concentration of 1 mg / mL in the above step 3 to dissolve the ligand to a volume ratio of 1:2, mix for 16 hours in a water bath from 25°C to 40°C, and shake at 37°C bed overnight;
[0030]...
Embodiment 2
[0076] Example 2: Cloning and sequencing of nucleic acid aptamers and prediction of single-stranded DNA secondary structure
[0077] 1. Preparation of Escherichia coli DH5α Competent Cells
[0078] 1. Pick a single DH5α colony, inoculate it in 3 mL of LB medium without ampicillin, and cultivate overnight at 37°C. The next day, take the above-mentioned bacterial solution and inoculate it in 50mL of liquid LB medium at a ratio of 1:100, shake at 37°C 2 hours; when the OD600 value reached 0.35, the bacterial culture was harvested;
[0079] 2. Transfer the bacterial culture to a 50mL pre-cooled sterile polypropylene tube and place it on ice for 10 minutes to cool the culture;
[0080] 3. Centrifuge at 4000 rpm for 10 minutes at 4°C, discard the culture medium, and invert the tube for 1 minute to drain the remaining culture medium;
[0081] 4. Add 150 μL ice-cooled 0.1 mM CaCl each 2 Solution, combine two tubes, ice bath for 10min;
[0082] 5. Centrifuge at 4000rpm for 10min ...
Embodiment 3
[0096] Embodiment 3: circular dichroism to K + Research on the relationship between concentration and nucleic acid aptamer C07
[0097] 1. After diluting the aptamer to 20 μM with 20 mM Tris-HCl buffer (pH 7.2), denature at 94 ° C for 0.5 min and cool to 25 ° C at a speed of 0.5 ° C / min.
[0098] 2. The nucleic acid aptamer C07 was diluted to 2.5 μM with 20 mM Tris-HCl buffer (pH 7.2) containing different concentrations (0, 5, 10, 20, 50 mM) of KCl.
[0099] 3. Detect with a circular dichroism spectrometer at 25°C at a wavelength of 220–340nm (see the results in figure 1 ), the results showed that the nucleic acid aptamer C07 had a peak at 275 nm in different solutions, indicating that the nucleic acid aptamer had a stem-loop and G-quadruplex structure.
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