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In-vitro screening method of nucleic acid aptamer, nucleic acid aptamer and kit for detecting target molecule

A nucleic acid aptamer, in vitro screening technology, applied in the biological field, can solve the problems of the existence of PCR product fidelity, nucleic acid aptamers need to be studied, monomer synthesis difficulties, etc., to achieve easy screening, simple synthesis method, and applicability wide effect

Active Publication Date: 2021-02-26
TSINGHUA UNIV
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Problems solved by technology

However, this method is difficult to synthesize monomers on the one hand, and on the other hand, when using DNA containing unnatural bases as a template and unnatural base monomers for PCR, the polymerases that can be selected during PCR amplification are limited, and the PCR products fidelity challenges
[0005] Therefore, the in vitro screening method of nucleic acid aptamers still needs to be studied.

Method used

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  • In-vitro screening method of nucleic acid aptamer, nucleic acid aptamer and kit for detecting target molecule
  • In-vitro screening method of nucleic acid aptamer, nucleic acid aptamer and kit for detecting target molecule
  • In-vitro screening method of nucleic acid aptamer, nucleic acid aptamer and kit for detecting target molecule

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Embodiment 1

[0042] 1. Construction of PS-random library

[0043] First obtain a random library through solid-phase synthesis, the sequence is ATACCAGCTTTCTGTTCT-N30-AGATAGTAAGTGCAATCT, and synthesize B-DNA, its sequence is: biotin-AAAAAAAAAAAAAGAACAGAAAGACC, the random library can be hybridized with the B-DNA through the middle GGTCTTTCTGTTCT sequence, Then connect to the avidin-modified magnetic ball through the biotin on the B-DNA. At the same time, synthesize primers for PCR, the forward primer is AP1: 5'-ATACCAGCTTATTCAATT-3', the reverse primer is TER-AP2: 5'-A20-Spacer18-AGATTGCACTTACTATCT-3', and the thio monomer is selected from thio dATP, other monomers are natural monomers dTTP, dGTP, dCTP. Add 2.5 μl AP1, 2.5 μl TER-AP2, 1 μl 10 mM sulfo-dATP, 0.5 μl dTTP, 0.5 μl dGTP, 0.5 μl dCTP, 41.5 μl HO to a 200 μl PCR tube 2 O. 0.5 μl Phusion enzyme and 0.5 μl random library template. Place the above PCR system on a PCR machine, denature at 98°C for 3min, denature at 98°C for 20s, ann...

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Abstract

The present invention provides an in-vitro screening method of a nucleic acid aptamer, which comprises: providing a random library having a thiophosphate group modification and a binding group modification, the binding group being suitable for binding to a target molecule, the thiophosphate group being bound to the binding group; and contacting the random library with a target, separating the random library combined with the target molecule, and taking the random library as a target nucleic acid aptamer obtained by screening. The random library is modified by the thiophosphate group and the binding group, the binding group generates affinity with a target, and the distance between the random library and the target is shortened, so that the random library is endowed with initial binding activity with the target, the random library and the target are easier to bind, and the nucleic acid aptamer is easier to successfully screen.

Description

technical field [0001] The present invention relates to the field of biology. Specifically, the present invention relates to an in vitro screening method for nucleic acid aptamers, nucleic acid aptamers and kits for detecting target molecules. Background technique [0002] Nucleic acid aptamer (aptamer) refers to RNA or single-stranded DNA that can form a certain spatial structure and specifically bind to target substances. Its target substances range widely, including proteins, small molecules, metal ions and even whole cells. The binding force between aptamer and target substance is mainly various weak forces, including hydrogen bond, intermolecular force, electrostatic adsorption or base stacking force, etc. Compared with antibodies (a protein), aptamer has several advantages: a. It can be synthesized artificially, with low production cost and stable performance; b. It can tolerate the transition from "high temperature to low temperature" without harsh storage conditions...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12N15/115G01N33/53
CPCC12N15/1048C12N15/115G01N33/5308C12N2320/13C12N2310/16C12N2310/315C12Q2525/205C12Q2525/113
Inventor 向宇顾春梅徐潇
Owner TSINGHUA UNIV
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