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Fluorescent chemical sensor for simultaneous detection of multiple dna glycosylases, 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: 2022-07-29
SHANDONG NORMAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • Fluorescent chemical sensor for simultaneous detection of multiple dna glycosylases, detection method and application thereof
  • Fluorescent chemical sensor for simultaneous detection of multiple dna glycosylases, detection method and application thereof
  • Fluorescent chemical sensor for simultaneous detection of multiple dna glycosylases, detection method and application thereof

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preparation example Construction

[0074] In a second aspect of the present invention, there is provided a preparation method of a fluorescent chemical sensor for simultaneously detecting multiple DNA glycosylases, comprising: adding multiple DNA glycosylase recognition sequences and a reporter probe modified by a fluorescent group in a buffer The reaction is carried out by heating in the liquid, and after cooling, a fluorescent chemical sensor for simultaneously detecting a variety of DNA glycosylases is formed.

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

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

[0077] In a third aspect of the present invention, there is provided an application of a fluorescent chemical sensor for simultaneous detection of multiple DNA glycosylases in DNA glycosylase.

[0078] Prefe...

Embodiment 1

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

[0096] Mix 10 μM hAAG probe, 10 μM UDG probe, 10 μM Cy3-labeled reporter probe, and 10 μM Cy5-labeled reporter probe in 10×NEB buffer 4 (500 mM potassium acetate, 200 mM molar tris(hydroxymethyl)aminomethane-acetic acid, 100 mmol magnesium acetate, 10 mmol dithiothreitol, pH 7.9) incubate at 95°C for 5 min, then slowly cool 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 DNA glycosylase-induced base excision reaction was carried out in a 10 microliter reaction system, which contained 1 micromolar bifunctional double-stranded DNA probe, 1 × NEB buffer 4, 1 × UDG reaction buffer, 2U APE1 and various concentrations of hAAG and UDG were incubated at 37°C for 1 hour. Then 0.5 mM dNTPs, 1...

Embodiment 2

[0114] 1. Feasibility verification of the experimental method

[0115] Using native 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 distinct band was observed with the Cy3-labeled reporter probe ( figure 2 A, Lane 3, the lowermost region indicated by the arrow), showing the release of the Cy3 reporter probe induced by hAAG-mediated base removal and strand displacement amplification reactions. 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 the Cy5 reporter probe induced by UDG-mediated base removal and strand displacement amplification reactions. When both hAAG and UDG are present, bands of Cy3-labeled reporter probe and Cy5-labeled reporter probe can be observed ( figure 2 A, ...

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Abstract

The invention belongs to the technical field of molecular detection, in particular to a fluorescent chemical sensor for simultaneously detecting multiple DNA glycosylases, a detection method and application thereof. The fluorescent chemical sensor includes a double-stranded DNA substrate, two or more DNA glycosylase recognition sequences, and a reporter probe; the double-stranded DNA substrate includes a double-stranded DNA substrate for identifying one or more DNA glycosylation The enzyme recognition sequence, the 5' ends of the two strands of the double-stranded DNA substrate are respectively modified with fluorescent quenching groups, the reporter probe is modified with fluorescent groups, and the base sequence connected to the fluorescent quenching group is the same as that of the reporter probe. The needles hybridize to form base pairs. Signal amplification technology based on fluorescent chemical sensors can realize ultrasensitive detection of various DNA glycosylases.

Description

technical field [0001] The invention belongs to the technical field of molecular detection, in particular to a fluorescent chemical sensor for simultaneously detecting multiple DNA glycosylases, a detection method and application thereof. Background technique [0002] The information disclosed in this Background section is only for enhancement of understanding of the general background of the invention and should not necessarily be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person of ordinary skill in the art. [0003] DNA stores the genetic information that organisms depend on for survival and reproduction, and the integrity and stability of its molecular structure are of great significance for cell survival and normal physiological activities. However, in daily life, the influence of endogenous and exogenous physicochemical factors such as ultraviolet rays, ionizing radiation, chemical mutagens, reacti...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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