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ssDNA aptamer specifically recognizing 6'-sialyllactose and its screening method and application

A sialyllactose, aptamer technology, applied in DNA preparation, recombinant DNA technology, DNA/RNA fragments, etc., can solve the problem of poor affinity and specificity between molecularly imprinted polymer and boronate, high cost of antibody preparation, It can not analyze problems such as sugar difference, and achieve the effect of high sensitivity, short operation process and reduced impact.

Active Publication Date: 2022-05-03
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the aforementioned biorecognition elements are limited in the construction and application of biosensors due to the complexity of sugars
For example, lectins can only recognize specific types of sugars, and further differential analysis of sugars cannot be performed; the high cost of antibody preparation limits its application in sugar detection; the affinity of synthetic molecularly imprinted polymers with borate and less specific

Method used

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  • ssDNA aptamer specifically recognizing 6'-sialyllactose and its screening method and application
  • ssDNA aptamer specifically recognizing 6'-sialyllactose and its screening method and application
  • ssDNA aptamer specifically recognizing 6'-sialyllactose and its screening method and application

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Experimental program
Comparison scheme
Effect test

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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Abstract

The present invention uses streptavidin-modified magnetic beads-SELEX technology to screen through the immobilized DNA library method, and obtains 4 ssDNA adaptations with high affinity and specificity to 6'-sialyllactose from 3187 sequencing results Using ssDNA aptamer as 6'-sialyllactose biorecognition element, catalytic hairpin self-assembly as signal amplification unit, and quantum dots as signal label, a fluorescent biosensor based on fluorescence resonance energy transfer was constructed to detect6 '‑sialyllactose. The invention provides a recognition element and a detection method with high affinity, high specificity, easy modification and labeling for the detection of 6'-sialyllactose.

Description

technical field [0001] The invention relates to the fields of biochemistry and molecular biology, analytical chemistry and combinatorial chemistry, in particular to a ssDNA aptamer specifically recognizing 6'-sialyllactose and its screening method and application. Background technique [0002] Sialyllactose is a kind of human milk oligosaccharides (HMOs). As a prebiotic, it plays an important role in promoting infant brain development and improving infant immunity. 6'-sialyllactose and 3'-sialyllactose are the main forms of sialyllactose, both of which have antibacterial activity and immunomodulatory effects. The content of 6′-sialyllactose is an important nutritional index in infant formula milk powder. So far, high performance liquid chromatography, high pH anion exchange chromatography, proton nuclear magnetic resonance spectroscopy and mass spectrometry have been widely used in the qualitative and quantitative detection of 6′-sialyllactose. However, although these meth...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/115C12N15/10G01N33/53C12Q1/6825
CPCC12N15/115C12N15/1048G01N33/5308C12Q1/6825C12N2310/16C12N2330/31C12Q2531/113C12Q2523/308C12Q2563/107C12Q2563/131C12Q2541/101C12Q2525/205C12Q2525/301
Inventor 周楠迪陈金日张雨婷王晓丽
Owner JIANGNAN UNIV
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