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Aptamer screening method

An aptamer and random sequence technology, applied in biochemical equipment and methods, microbial measurement/testing, DNA preparation, etc., can solve the problems of time-consuming, expensive, cumbersome SELEX process, etc., and achieve the effect of simplifying experimental design

Pending Publication Date: 2022-05-27
HUAQIAO UNIVERSITY
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AI Technical Summary

Problems solved by technology

The traditional SELEX process is cumbersome, expensive and time-consuming, so a new SELEX technology is proposed to simplify the procedure and improve the selection efficiency

Method used

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Embodiment 1

[0031] Insert a random sequence into the non-cutting functional part of the self-cleaving DNA self-cleaving enzyme, destroy its original shearing secondary structure, and make the random sequence form a specific secondary structure under the action of the target molecule, making the DNA self-cleaving. Cleavage occurs after lyase restores the cleavage structure. The random library is:

[0032] I-R3-random: 5’-GTAACGTAGTTGAGCTG-N60-TGACGTTGAAGCGTTACGCAGCTGTGGGTTGATTCC-3’

[0033] II-R1-ramdom: 5'biotin-CATGACCACTAGGAGCATCTTTGGCG-N47-TAGGGGAATAAATCTTTGGCACCTAGTGGTCATG-3'

[0034] Among them, N60 and N47 represent 60 or 47 random bases, respectively.

[0035] The specific steps include the following:

[0036] 1. Screening of R1-random random library

[0037] 1. Preparation of I-R3 DNA random library. The ordered random library was scaled to 100 μM and then bound to the 5’ end biotin-modified oligonucleotide sequence (5’ oligo) sequence. Make the library fold correctly.

[0...

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Abstract

The invention discloses a method for screening an aptamer, which comprises the following steps: 1) inserting a random sequence into self-cleavage enzyme to enable the self-cleavage enzyme to lose a self-cleavage function; (2) the random sequence is combined with the target molecule, the self-cleavage function of the self-cleavage enzyme is recovered, and self-cleavage occurs; and 3) carrying out repeated separation and screening on the cut sequence to enrich the aptamer capable of sensing the target molecule. According to the present invention, the self-shearing characteristic of the enzyme is utilized to achieve the precise separation of the sequence combined with the small molecule and subjected to the shearing, such that the complex experiment design is simplified, and the convenient separation screening method is provided.

Description

technical field [0001] The present invention relates to a method for screening aptamers based on DNA self-cleaving enzyme SELEX. Background technique [0002] SELEX technology is actually a screening process that starts with a huge library of random nucleic acid sequences. In theory, the library contains two fixed ends that can be PCR-amplified, with a random region in the middle, usually 40 nucleotides, which yields a theoretical library of 440 different sequences, and this contains A random library of approximately 1015 different molecules is incubated with the target, then the nucleic acid molecules bound to the target are isolated and the DNA bound to the target is amplified by PCR. This process is repeated until a DNA pool with high affinity for the target substance is isolated. This pool was then sequenced and characterized to identify aptamers with the highest affinity. SELEX strategies have been used to identify aptamers for a variety of targets ranging from small...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1048C12Q2531/113C12Q2565/125C12Q2521/345C12Q2563/143C12Q2563/149
Inventor 李三暑敖雅琪
Owner HUAQIAO UNIVERSITY
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