SsDNA aptamer for specifically recognizing 6 '-sialyllactose as well as screening method and application of ssDNA aptamer
A sialyllactose and screening method technology, which is applied in the field of ssDNA aptamers that specifically recognize 6'-sialyllactose, can solve the problem of poor affinity and specificity between molecularly imprinted polymers and boronate, and inability to analyze sugar differences , the problem of high cost of antibody preparation, to achieve the effect of high sensitivity, short operation process and strong specificity
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Embodiment 1
[0053] (1) Construction of random ssDNA library and its primers:
[0054] a. Construct a random ssDNA library with a length of 79 bases
[0055] 5'-TAGGGAATTCGTCGACGGATCC-N35-CTGCAGGTCGACGCATGCGC CG-3', wherein N represents any one of bases A, T, C, and G.
[0056] b. Synthesis of upstream primers
[0057] Upstream primer 1: 5'-TAGGGAATTCGTCGACGGAT-3';
[0058] Upstream primer 2: 5′-FAM-TAGGGAATTCGTCGACGGAT-3′;
[0059] c. Synthesize downstream primers
[0060] Downstream primer 1: 5'-CGGCGCATGCGTCGACCTG-3';
[0061] Downstream primer 2: 5'-biotin-CGGCGCATGCGTCGACCTG-3'.
[0062] (2) In vitro screening of nucleic acid aptamers:
[0063] S1. Using upstream primers containing fluorescent groups and downstream primers modified with biotin, PCR amplifies the 79nt-length single-stranded DNA library to construct double-stranded DNA for 6′-sialyllactose-specific aptamer screening Library, in which 25 μL of the PCR amplification system is shown in Table 1.
[0064] Table 1 PCR...
Embodiment 2
[0084] S1. Denature the aptamer and the signal probe at 95°C for 5 minutes, then incubate at 37°C for 10 minutes, then add streptavidin-modified magnetic beads and shake slightly for a period of time to form a For the nucleic acid molecular hybridization system of aptamers and signal probes, use a magnet to magnetically separate the solution, discard the supernatant, wash the pellet once with PBS buffer, wash the pellet three times with Tris-HCl buffer, and resuspend In Tris-HCl buffer;
[0085] S2. Add 50 μL of 6′-sialyllactose containing different concentrations to the above Tris-HCl buffer and mix well, and incubate at room temperature for 1 hour; perform magnetic separation with a magnet, and obtain the supernatant, which contains the signal probe. The signal probe sequence is: 5'-CCGTAGTGCGCTCTCGCTCGTGGCT-3';
[0086] S3. Take 200 μL of 1 μmol·L respectively -1 Hairpin probe Hp1 with 200 μL 1 μmol L -1 The hairpin probe Hp2 was denatured at 95°C for 5 minutes, and then...
Embodiment 3
[0095] In order to further verify the accuracy of this method in determining the content of 6′-sialyllactose in actual samples, milk products processed by centrifugal supernatant were selected.
[0096] Take 10mL milk sample and centrifuge at room temperature for 5min (5,000×g), then remove the upper fat layer. Then the milk product was diluted 20 times with Tris-HCl buffer. Then add different concentrations of 6′-sialyllactose and mix well. 5μmol·L -1 The aptamer sequence and the complementary signal probe were denatured at high temperature for 5 minutes, then incubated in ice bath for 10 minutes, and then incubated at 37°C for 1 hour. After that, the supernatant was removed by magnetic separation, and the precipitate was washed once with PBS buffer and Tris-HCl buffer. After washing 3 times, resuspend, mix with 50 μL of milk products containing different concentrations of 6′-sialyllactose, and incubate at room temperature for 30 min with slight shaking. The supernatant wa...
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