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Oxidative damage DNA detection method

A technology of oxidative damage and buffer solution, which is applied in the fields of biochemical equipment and methods, and the determination/inspection of microorganisms. Achieve the effect of low detection limit, high sensitivity and easy promotion

Inactive Publication Date: 2017-07-11
CHINA INST OF SPORT SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0015] The abasic site formed after the base falls off makes the local hydrogen bond force and base stacking force unbalanced, resulting in the instability of the local structure and even the overall structure of DNA

Method used

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  • Oxidative damage DNA detection method

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preparation example Construction

[0100] Preferably, the oxidative damage DNA standard sample is prepared by Fenton reaction, and its preparation method comprises the following steps:

[0101] (a) Dilute the extracted genomic DNA samples to different concentrations, and perform ultraviolet measurement to obtain the relationship between absorbance and DNA concentration;

[0102] (b) Take 10 μg of DNA samples with different concentrations and add 50 μL of 80 μM FeCl 2 , 40 μL 0.5 mM H 2 o 2 , 100 μL Tris-acetate buffer solution, reacted at 37°C for 4 hours in the dark; after the reaction, take it out quickly, add 20 μL 3M sodium acetate solution and 500 μL absolute ethanol, and react at -20°C for 10 minutes; centrifuge the solution to precipitate Wash the DNA pellet with 70% ethanol; dry the DNA pellet and redissolve it with 50 μL of water;

[0103] (c) performing ultraviolet measurement on the solution obtained in step (b), obtaining the DNA concentration in the solution according to the relationship between...

Embodiment approach

[0122] As a preferred embodiment, replace step (a') with the following steps:

[0123] Take 5 μg of oxidative damage DNA standard sample or 25 μL of detection sample, add 0.5 μL 10 U / μL formamide pyrimidine-DNA glycosylase, 5 μL NEB buffer 1 and 20 μL water, react at 37 °C for 90 minutes, and stop the reaction at 75 °C for 10 minutes minute;

[0124]The NEB buffer 1 comprises 10mM Bis-Tris-Propane-HCl, 10mM MgCl 2 and 1 mM DTT, pH 7.0 ± 0.5.

[0125] In this preferred technical scheme, step (a') can be performed not only using endonuclease VIII, but also using FPG protein. Endonuclease VIII mainly recognizes oxidized pyrimidine bases, and FPG protein mainly recognizes oxidized purine bases. Oxidative damaged DNA is transformed into DNA containing 3',5' phosphate group nucleotide gaps.

[0126] In order to further understand the present invention, the present invention will be further explained and described below in conjunction with specific examples.

[0127] The instrume...

Embodiment 1

[0129] Example 1 Effect of different concentrations of oxidatively damaged DNA on fluorescence detection signal

[0130] (a) The preparation method of oxidatively damaged DNA standard samples at different concentrations is as follows:

[0131] Dilute the extracted genomic DNA samples to different concentrations, and use a NanoDrop 2000 ultra-micro spectrophotometer to measure at the maximum absorption wavelength of 260nm to obtain the relationship between absorbance and DNA concentration;

[0132] Take 10 μg of DNA samples with different concentrations and add 50 μL of 80 μM FeCl 2 , 40 μL 0.5 mM H 2 o 2 , 100 μL Tris-acetate buffer solution (Tris-acetate buffer solution is a 2-fold concentrated buffer solution, which contains 20 mM Tris and 13 mM acetic acid, pH=7.3), reacted at 37 ° C for 4 hours in the dark; Take it out, add 20 μL of 3M sodium acetate solution (pH=5.2) and 500 μL of absolute ethanol (4°C), react at -20°C for 10 minutes, then take out the solution and cen...

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Abstract

The invention discloses an oxidative damage DNA detection method, and relates to the technical field of DNA oxidative damage detection. The method comprises (a) obtaining of DNA containing 3 ', 5'-phosphate group nucleotide voi, to be more specific, adding a DNA repair enzyme for recognition and cutting of oxidized DNA bases; (b) introduction of hydroxyl into 3 ', 5' site, to be more specific, adding alkaline phosphatase for dephosphorylation to obtain the DNA containing 3 ', 5'-hydroxyl group nucleotide voids; (c) introduction of a fluorescent marker, to be more specific, adding DNA polymerase I and the fluorescent marker to obtain fluorescence-labeled DNA; (d) formation of a PFP-DNA complex, to be more specific, adding PFP to obtain the PFP-DNA complex; and (e) oxidative damage DNA detection, to be more specific, detecting damage DNA by FRET. The method alleviates the disadvantages of tedious steps, low sensitivity and low selectivity of DNA damage detection in the prior art. The method has the advantages of high sensitivity, strong specificity and high accuracy.

Description

technical field [0001] The invention relates to the technical field of DNA oxidative damage detection, in particular to a method for detecting oxidative damage to DNA. Background technique [0002] DNA (deoxyribonucleic acid, deoxyribonucleic acid) is the carrier of eukaryotic genetic information, and its integrity is crucial to the survival and normal reproduction of organisms. DNA oxidative damage plays an important role in the formation of diseases and human aging. Studies have found that DNA oxidative damage is related to Alzheimer's disease, Parkinson's disease, diabetes, metabolic syndrome, atherosclerosis and other aging diseases. The mechanism of self-initiated DNA damage is mainly due to the oxidative phosphorylation during the generation of ATP by the aerobic respiration of the cell mitochondria. Oxygen is reduced to water as the final electron acceptor. In the process of electron transfer, electrons combine with oxygen molecules to generate superoxide free base, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6851C12Q2521/331C12Q2521/301C12Q2545/113C12Q2563/107C12Q2565/401
Inventor 洪平张栋成永强
Owner CHINA INST OF SPORT SCI
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