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Fluorescence chemical sensor for simultaneously detecting multiple DNA glycosylase and detection method and application thereof

A technology of chemical sensor and glycosylase, which is applied in the field of fluorescent chemical sensor and its detection, can solve the problems of low sensitivity and limited application, and achieve high sensitivity, prevent non-specific amplification, and good specificity

Active Publication Date: 2021-07-09
SHANDONG NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The colorimetric method is simple and easy to implement, but it is only suitable for systems with relatively simple components and color development that are not easily disturbed
Luminescence, electrochemical and fluorescence methods have fast analysis speed, good selectivity, and can be used for accurate quantification, but their low sensitivity limits their application in low-concentration complex samples

Method used

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  • Fluorescence chemical sensor for simultaneously detecting multiple DNA glycosylase and detection method and application thereof
  • Fluorescence chemical sensor for simultaneously detecting multiple DNA glycosylase and detection method and application thereof
  • Fluorescence chemical sensor for simultaneously detecting multiple DNA glycosylase and detection method and application thereof

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preparation example Construction

[0074] The second aspect of the present invention provides a method for preparing a fluorescent chemical sensor for simultaneous detection of multiple DNA glycosylases, comprising: combining multiple DNA glycosylase recognition sequences with a fluorescent group-modified reporter probe in a buffer The reaction is carried out by heating in the liquid, and after cooling, a fluorescent chemical sensor for simultaneously detecting multiple DNA glycosylases is formed.

[0075] Preferably, the concentration of each DNA glycosylase recognition sequence and the reporter probe modified by the fluorescent group is 10 μM / L;

[0076] Preferably, the heating temperature is 80-100°C, and the temperature is kept for 5-10 minutes, preferably 95°C for 5 minutes.

[0077] The third aspect of the present invention provides an application of a fluorescent chemical sensor for simultaneous detection of multiple DNA glycosylases in DNA glycosylases.

[0078] Preferably, there are at least two DNA g...

Embodiment 1

[0095] 1. Preparation of bifunctional double-stranded DNA probes.

[0096] 10 μmol hAAG probe, 10 μmol UDG probe, 10 μmol Cy3-labeled reporter probe and 10 μmol Cy5-labeled reporter probe were dissolved in 10×NEB buffer 4 (500 mmol potassium acetate, 200 mM Tris(hydroxymethyl)aminomethane-acetic acid, 100 mmol magnesium acetate, 10 mmol dithiothreitol, pH 7.9) were incubated at 95°C for 5 min, then slowly cooled to room temperature to form bifunctional double-stranded DNA probe. The obtained bifunctional double-stranded DNA probes were stored at 4°C for future use.

[0097] 2. DNA glycosylase-induced base excision reaction and strand displacement amplification reaction.

[0098] The base excision reaction induced by DNA glycosylase was carried out in a 10 microliter reaction system containing 1 micromole of bifunctional double-stranded DNA probe, 1×NEB buffer 4, 1×UDG reaction buffer, 2U APE1 and different concentrations of hAAG and UDG were incubated at 37°C for 1 hour. T...

Embodiment 2

[0114] 1. Feasibility verification of the experimental method

[0115] Using non-denaturing polyacrylamide gel electrophoresis (PAGE, figure 2 A) and fluorescence measurements ( figure 2 B-D), to verify the feasibility of the proposed method. In the presence of hAAG, a distinct 12-base band was observed for the Cy3-labeled reporter probe ( figure 2 A, Lane 3, the region indicated by the arrow at the bottom), showing the release of Cy3 reporter probe induced by hAAG-mediated base removal and strand displacement amplification reaction. Likewise, a unique 12-base band formed by the Cy5-labeled reporter probe was observed in the presence of UDG ( figure 2 A, lane 4, the region indicated by the middle arrow), showing the release of Cy5 reporter probe induced by UDG-mediated base removal and strand displacement amplification reaction. When both hAAG and UDG are present, bands of Cy3-labeled reporter probe and Cy5-labeled reporter probe can be observed ( figure 2 A, Lane 1)...

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Abstract

The invention belongs to the technical field of molecular detection, and particularly relates to a fluorescent chemical sensor for simultaneously detecting multiple DNA glycosylase as well as a detection method and application of the fluorescent chemical sensor. The fluorescent chemical sensor comprises a double-stranded DNA substrate, two or more than two DNA glycosylase recognition sequences and a reporter probe, wherein the double-stranded DNA substrate comprises a recognition sequence for recognizing one or more DNA glycosylase; the 5' tail ends of the two chains of the double-chain DNA substrate are respectively modified with fluorescence quenching groups; the reporter probe is modified by a fluorophore; the base sequence connected with the fluorescence quenching group is hybridized with the reporter probe to form a base pair. According to the invention, the signal amplification technology based on the fluorescence chemical sensor can realize ultra-sensitive detection of various DNA glycosylase.

Description

technical field [0001] The invention belongs to the technical field of molecular detection, and in particular relates to a fluorescent chemical sensor for simultaneously detecting multiple DNA glycosylases, a detection method and an application thereof. Background technique [0002] The information disclosed in this background section is only intended to increase the understanding of the general background of the present invention, and is not necessarily taken as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to those skilled in the art. [0003] DNA stores the genetic information that organisms rely on for survival and reproduction. The integrity and stability of its molecular structure is of great significance to the survival of cells and the normal physiological activities. However, in daily life, the influence of endogenous and exogenous physical and chemical factors such as ultraviolet rays, ionizing radiation, c...

Claims

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Application Information

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IPC IPC(8): C12Q1/34C12Q1/682
CPCC12Q1/34C12Q1/682C12Q2521/531C12Q2563/107C12Q2521/301C12Q2531/119
Inventor 张春阳张艳胡金萍
Owner SHANDONG NORMAL UNIV
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