Eureka AIR delivers breakthrough ideas for toughest innovation challenges, trusted by R&D personnel around the world.

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
View PDF2 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • 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

Examples

Experimental program
Comparison scheme
Effect test

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...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12Q1/68
Inventor 王先良
Owner CHINESE RES ACAD OF ENVIRONMENTAL SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com