Fluorescence chemical sensor and method for simultaneously detecting diversified DNA (deoxyribonucleic acid) glycosylases on single-molecular levels and application of fluorescence chemical sensor
A chemical sensor and detection method technology, applied in the field of biological analysis, can solve the problems of increased detection complexity and cost, high requirements for the reaction system, and generation of false positive signals, etc., to prevent non-specific reactions, simple preparation and detection methods, The effect of reducing inspection costs
Active Publication Date: 2018-02-23
SHANDONG NORMAL UNIV
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These methods improve the detection sensitivity through signal amplification through amplification, but exonuclease-assisted signal amplification requires high reaction system and poor reproducibility
However, target-mediated autocatalytic DNAzyme-generated rolling circle amplification is prone to non-specific amplification, resulting in false positiv
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[0062] Experimental method steps
[0063] 1. Molecular beacon preparation: 6 micromoles per liter of hOGG1 probe, 6 micromoles per liter of hAAG probe or 6 micromoles per liter of hOGG1 and 6 micromoles per liter of hAAG probe were added containing 50 millimoles per liter of chloride Sodium, 10 mmol per L Tris-HCl, 10 mmol MgCl, 1 mmol dithiothreitol, pH 7.9 buffer, heat at 95 °C for 5 min, then slowly cool to room temperature, and place the product on ice spare.
[0064] 2. Enzyme digestion reaction: 20 microliters containing 50 mmol per liter of sodium chloride, 50 mmol per liter of potassium acetate, 20 mmol per liter of Tris-Ac, 10 mmol of per liter of magnesium acetate, 10 mmol of potassium chloride, 10 mmol ammonium sulfate, 2 mmol magnesium sulfate, 30 mmol per liter Tris-HCl, 10 mmol magnesium chloride, 1 mmol dithiothreitol, and 0.1% Triton X-100 were added with different concentrations of hOGG1 and hAAG, 0.1 units per microliter of human apurinic / apyrimidinic endon...
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The invention discloses a fluorescence chemical sensor and a method for simultaneously detecting diversified DNA (deoxyribonucleic acid) glycosylases on single-molecular levels and application of thefluorescence chemical sensor. The fluorescence chemical sensor, the method and the application have the advantages that the fluorescence chemical sensor is based on two different molecular beacons including a molecular beacon modified by 8-hydroxyl guanine and a molecular beacon modified by deoxygenated hypoxanthine, the tail end of the molecular beacon modified by the 8-hydroxyl guanine is labeled by cyanine 3 (Cy3) and quenching groups, the tail end of the molecular beacon modified by the deoxygenated hypoxanthine is labeled by cyanine 5 (Cy5) and quenching groups, the fluorescence chemicalsensor is used for detecting 8-hydroxyl guanine DNA glycosylases and N-methylpurine DNA glycosylases and is different from the traditional molecular beacons which can be severely affected by dynamicsand thermodynamics, signal restoration of the Cy3 and the Cy5 depends on molecular beacon splitting with the DNA glycosylases used as media, the DNA glycosylases can be simultaneously sensitively detected by the aid of the method without optional signal amplification, the activity of hOGG1 and hAAG can be detected by the aid of the method in an ultra-sensitive manner without optional signal amplification, the fluorescence chemical sensor can be easily, conveniently and quickly operated, and accurate and reliable test results can be obtained.
Description
technical field [0001] The invention belongs to the technical field of biological analysis, and in particular relates to a fluorescent chemical sensor based on a single-molecule level for simultaneously detecting multiple DNA glycosylases, a detection method and an application thereof. Background technique [0002] DNA glycosylase is an initial enzyme in the base excision repair pathway. This enzyme can recognize and excise mutated DNA bases and generate apurine or apyrimidine (AP) sites. The normal DNA glycosylase The activity ensures the stability of base excision repair and thus the stability of the genome. The base excision repair pathway involves at least 11 different mammalian glycosylases that operate by relying on different mutation types. In addition, abnormal DNA glycosylase is closely related to a series of diseases such as cancer, neuropathy, cardiovascular disease and inflammation. [0003] Traditional methods for detection of glycosylases include radiolabelin...
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Login to view more IPC IPC(8): C12Q1/44C12Q1/34C12N15/11G01N21/64
CPCC12Q1/34C12Q1/44G01N21/6428G01N2021/6432
Inventor 张春阳胡娟刘明昊
Owner SHANDONG NORMAL UNIV
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