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Fluorescent chemical sensor for synchronously detecting multiple DNA (deoxyribonucleic acid) glycosylases and detection method and application of fluorescent chemical sensor

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 no specificity, harmful radiation, low sensitivity, etc., and achieve the effect of increased sensitivity and good specificity

Active Publication Date: 2020-05-15
SHANDONG NORMAL UNIV
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0004] HPLC is an efficient detection method, but it requires tedious DNA lysis and expensive instrumentation
Gold nanoparticles colorimetric method can visually detect DNA glycosylase, but due to the complicated preparation and modification process of gold nanoparticles (AuNP), the detection sensitivity is low
Gel electrophoresis combined with radiolabeling is considered the gold standard method for the analysis of DNA glycosylases such as uracil DNA glycosylase (UDG) and human alkyladenine DNA glycosylase (hAAG), but this method is time-consuming. Long time, low sensitivity, large sample consumption, high requirements for operating skills, not suitable for quantitative analysis and accompanied by harmful radiation
The closest existing implementations to the present invention are glycosylase assays based on rolling circle amplification, which usually involve fluorophore-quencher-labeled probes, and because all starting materials are in a homogeneous In the system, so there is no specificity in the amplification process, only one DNA glycosylase can be detected

Method used

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  • Fluorescent chemical sensor for synchronously detecting multiple DNA (deoxyribonucleic acid) glycosylases and detection method and application of fluorescent chemical sensor
  • Fluorescent chemical sensor for synchronously detecting multiple DNA (deoxyribonucleic acid) glycosylases and detection method and application of fluorescent chemical sensor
  • Fluorescent chemical sensor for synchronously detecting multiple DNA (deoxyribonucleic acid) glycosylases and detection method and application of fluorescent chemical sensor

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Embodiment 1

[0086] 1. Preparation of Reagents

[0087] Preparation of bifunctional dsDNA substrates: 1 μM hAAG probe and 1 μM UDG probe were mixed in 10 mM Tris-HCl (pH 8.0), 50 mM Tris-HCl (pH 8.0) NaCl and 1 mM EDTA in annealing buffer, incubate at 95°C for 5 min, and then slowly cool to room temperature to form a bifunctional dsDNA substrate. The obtained bifunctional dsDNA substrate was stored at 4°C for further use.

[0088] DNA glycosylase-induced excision reaction and RCA reaction: DNA glycosylase-induced excision reaction was performed in 10 μl reaction solution containing 100 nanomoles per liter of bifunctional dsDNA substrate, 1x NEB buffer 4. 1x UDG reaction buffer, 2U APE1 and different concentrations of hAAG and UDG, react at 37°C for 1 hour. Then combine 50 nmoles per liter of hAAG cyclic template, 50 nmoles per liter of UDG cyclic template, 0.1 mg per ml of BSA, 0.25 mmoles per liter of dATP, 0.25 mmoles per liter of dTTP, 10 Micromoles per liter of Cy3-labeled dCTP (Cy3...

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Abstract

The invention belongs to the technical field of DNA (deoxyribonucleic acid) glycosylase detection and in particular relates to a fluorescent chemical sensor for synchronously detecting multiple DNA glycosylases and a detection method and application of the fluorescent chemical sensor. The invention provides a method for simultaneously detecting multiple DNA glycosylases, the detection precision can be improved, the analysis and detection cost can be reduced, and the clinical application practicability can be improved. The invention provides a method for simultaneously detecting human alkyl adenine DNA glycosylase (hAAG) and uracil DNA glycosylase (UDG), monomolecular detection and rolling circle amplification driven fluorescent molecules of different codes are combined, multiple DNA glycosylases in cancer cells can be simultaneously detected at a monomolecular level, and normal cells and cancer cells can be also distinguished. The fluorescent chemical sensor can be further applied to analysis on enzyme kinetic parameters and screening on DNA glycosylase inhibitors, and has great potential in biomedical research, clinical diagnosis and medicine development.

Description

technical field [0001] The invention belongs to the technical field of DNA glycosylase detection, in particular to a fluorescent chemical sensor for simultaneously detecting the quantity and activity of human alkyladenine DNA glycosylase (hAAG) and uracil DNA glycosylase (UDG) and the Its detection method and application. 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 damage is a hazard produced spontaneously by an organism, and to counteract the deleterious effects of DNA damage, cells engage in multiple repair mechanisms, such as base excision repair (BER). The BER pathway is initiated by DNA glycosylase, which recognizes damaged and mismatched bases and excises ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6844C12Q1/34
CPCC12Q1/6844C12Q1/34C12Q2531/125C12Q2563/107C12Q2521/301C12Q2521/319C12Q2563/143C12Q2563/149
Inventor 张春阳李琛琛陈慧燕胡娟
Owner SHANDONG NORMAL UNIV
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