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Method for quantitatively detecting deoxyribonucleic acid (DNA) demethylation capability of pollutant

A quantitative detection method and demethylation technology, applied in the direction of biochemical equipment and methods, microbial determination/inspection, etc., can solve the problem of inability to quantify the demethylation ability of pollutants, quantitative detection, no detection technology, etc. problems, to achieve the effect of easy popularization and application, low requirements for hardware facilities, and simple steps

Inactive Publication Date: 2010-11-17
CHINESE RES ACAD OF ENVIRONMENTAL SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the demethylation ability of pollutants cannot be quantified, and there is no report of practical application (Okochi-Takada E, Ichimura S, Kaneda A, et al.Establishment of a detection system fordemethylating agents using an endogenous promoter CpG island.Mutat Res, 2004; 568(2): 187-94.)
Some scholars in our country have proposed that the methylation level of DNA can be further analyzed in conventional animal experiments; however, due to the difficulty in operation, there is still no relatively mature detection technology (Yang Jianping, Zhu Zhiliang, Yuan Jianhui, etc. DNA methylation Application prospects in toxicology. Environmental and Occupational Medicine, 2007; 24(5): 546-9.)
[0005] Generally speaking, the current detection methods only focus on one aspect of the ability of pollutants to cause damage to human health; as for the ability of pollutants to cause human DNA demethylation, it is currently impossible to perform quantitative detection due to the lack of corresponding detection methods

Method used

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  • Method for quantitatively detecting deoxyribonucleic acid (DNA) demethylation capability of pollutant
  • Method for quantitatively detecting deoxyribonucleic acid (DNA) demethylation capability of pollutant
  • Method for quantitatively detecting deoxyribonucleic acid (DNA) demethylation capability of pollutant

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Embodiment 1

[0030] The present invention will be further described in detail by taking the quantitative detection of the demethylation ability of edible aquatic products polluted by the coast as an example. Specific steps are as follows:

[0031] (1) Methylation treatment of fluorescent plasmid

[0032] Purchase the EGFP-C3 plasmid (hereinafter referred to as the C3 plasmid) from Clontech Company of the United States. Purchase DNA methylase Msss.I from NEB Company of the United States. Mix the C3 plasmid with DNA methylase Msss.I, etc., and incubate at 37°C for 3 hours, so that the CMV promoter region of the EGFP gene in the C3 plasmid is in a hypermethylated state. Specific reaction system composition: 4 μl buffer stock solution (10×NE Buffer2), 4 μl S-adenosylmethionine stock solution (100×SAM), 8 μl C3 plasmid DNA (1.7 μM), 6 μl methylase M.SssI, Supplement 18 μl of double distilled water to 40 μl (10×NE Buffer2 buffer stock solution, 100×SAM adenosylmethionine stock solution are pr...

Embodiment 2

[0048] Taking the quantitative detection of demethylation ability of groundwater samples as an example, the present invention will be further described in detail below. Specific steps are as follows:

[0049] (1) Methylation treatment of fluorescent plasmid

[0050] Purchase the EGFP-C3 plasmid (hereinafter referred to as the C3 plasmid) from Clontech Company of the United States. Purchase DNA methylase Msss.I from NEB Company of the United States. Mix the C3 plasmid with DNA methylase Msss.I, etc., and incubate at 37°C for 3 hours, so that the CMV promoter region of the EGFP gene in the C3 plasmid is in a hypermethylated state. Specific reaction system composition: 4 μl buffer stock solution (10×NE Buffer2), 4 μl S-adenosylmethionine stock solution (100×SAM), 8 μl C3 plasmid DNA (1.7 μM), 6 μl methylase M.SssI, Supplement 18 μl of double distilled water to 40 μl (10×NE Buffer2 buffer stock solution, 100×SAM adenosylmethionine stock solution are provided free of charge with...

Embodiment 3

[0066] Taking the quantitative detection of demethylation ability of surface water samples as an example, the present invention will be further described in detail below. Specific steps are as follows:

[0067] (1) Methylation treatment of fluorescent plasmid

[0068] Purchase the EGFP-C3 plasmid (hereinafter referred to as the C3 plasmid) from Clontech Company of the United States. Purchase DNA methylase Msss.I from NEB Company of the United States. Mix the C3 plasmid with DNA methylase Msss.I, etc., and incubate at 37°C for 3 hours, so that the CMV promoter region of the EGFP gene in the C3 plasmid is in a hypermethylated state. Specific reaction system composition: 4 μl buffer stock solution (10×NE Buffer2), 4 μl S-adenosylmethionine stock solution (100×SAM), 8 μl C3 plasmid DNA (1.7 μM), 6 μl methylase M.SssI, Supplement 18 μl of double distilled water to 40 μl (10×NE Buffer2 buffer stock solution, 100×SAM adenosylmethionine stock solution are provided free of charge wi...

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Abstract

The invention relates to a method for quantitatively detecting the deoxyribonucleic acid (DNA) demethylation capability of pollutant, and belongs to the field of the methods for detecting the health damaging capability of the pollutant. At present, no mature detection technology is provided for evaluating the DNA demethylation capability of the pollutant. The method comprises the following steps of: manually methylating fluorescent plasmids; transferring Hela cells from highly-methylated fluorescent plasmids to prepare reconstitution cell strains; performing pretreatment on a pollutant sample; co-culturing 5-methylcystein standard series and the reconstitution cell strains and synchronously co-culturing the tested sample and the reconstitution cell strains; performing fluorescent photographing and fluorescence intensity treatment on the reconstitution cell strains; drawing a pollutant demethylation capability detection standard curve; and quantitatively detecting the demethylation capability of the tested sample pollutant. If green fluorescent light emitted from tested cells is brighter, the demethylation capability of the pollutant is higher and the risk of health damage to a human body is higher. A rapid, simple and quantitative detection method is provided for evaluating the human DNA demethylation capability of the pollutant.

Description

technical field [0001] The invention relates to a quantitative detection method for DNA demethylation ability caused by pollutants. Background technique [0002] Toxic and harmful pollutants widely exist in the human living environment. In order to understand the health hazard ability of various pollutants and provide the public with a relatively safe and healthy production and living environment, the detection of the health damage ability of pollutants in food, drinking water, and air has become a major issue in food safety, drinking water safety, Important content in related fields such as environmental protection and public health. In recent years, studies have found that pollutants can change the methylation level of human DNA, thereby affecting the expression of key genes and causing various serious health damages such as leukemia. The stronger the ability of pollutants to demethylate, the greater the risk of causing health damage to the human body. Quantitative dete...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 王先良
Owner CHINESE RES ACAD OF ENVIRONMENTAL SCI
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