Method for detecting 8-hydroxy-2'-deoxyguanosine (8-OHdG) and 2'-deoxyguanosin (dG) in cell DNA

A technology of hydroxydeoxyguanosine and deoxyguanosine, which is applied in the field of genotoxicity assessment of harmful components in cigarette smoke, can solve problems such as instrument damage and complexity, and achieve the effects of low detection limit, accurate results and high sensitivity

Inactive Publication Date: 2014-12-03
ZHENGZHOU TOBACCO RES INST OF CNTC
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  • Abstract
  • Description
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Problems solved by technology

The gas chromatography/mass spectrometry (GC/MS) method needs to derivatize the damaged base, which is relatively complicated; the enzyme-linked immunosorbent (ELISA) method has cross-reactivity; the high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method has high sensitiv

Method used

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  • Method for detecting 8-hydroxy-2'-deoxyguanosine (8-OHdG) and 2'-deoxyguanosin (dG) in cell DNA
  • Method for detecting 8-hydroxy-2'-deoxyguanosine (8-OHdG) and 2'-deoxyguanosin (dG) in cell DNA
  • Method for detecting 8-hydroxy-2'-deoxyguanosine (8-OHdG) and 2'-deoxyguanosin (dG) in cell DNA

Examples

Experimental program
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Effect test

example 1

[0022] Prepare 8-OHdG and dG into a mixed series of standard working solutions with different concentrations, the concentration gradient of 8-OHdG is 0.01, 0.02, 0.05, 0.10, 0.2, 0.5, 1 and 2ng / mL, and the concentration gradient of dG is 1, 2, 5 , 10, 20, 50, 100 and 200ng / mL, the internal standards of the two targets are [ C 13 N 15 ]-8-OH-G and D 1 -dG at concentrations of 10 and 20 ng / mL, respectively. Determination was performed by HPLC-MS / MS (see Table 1 and Table 2 for specific conditions). Perform linear regression analysis on the ratio of the chromatographic peak area of ​​the target substance to the peak area of ​​the internal standard to its concentration ratio, and obtain the standard curve regression equation and correlation coefficient of the two kinds of standards for the target substance, and mix the standard working solution with the lowest concentration for 10 times Determination and calculation of the standard deviation of the two target objects, with 3 ...

example 2

[0025] Collect the cells and do 6 parallel determinations at the same time, extract DNA from the cells according to the steps (1) (2) in the content of the invention above, and measure the purity and concentration of DNA with a nucleic acid concentration analyzer, take 6 μg of DNA and add deferoxamine methanesulfonate solution , the DNA was digested with deoxyribonuclease I and alkaline phosphatase. Take 10 μL of the solution after enzymatic hydrolysis and add 20ng D 1 -dG internal standard, diluted to 1mL with ultrapure water, filtered through a 0.22μm filter membrane and determined by HPLC-MS / MS; take 150μL of the solution after enzymatic hydrolysis, add 1ng[ C 13 N 15 ]-8-OH-G internal standard, flowed through a C18 extraction column pretreated with methanol and water, rinsed with 2 mL of water, then eluted with 1 mL of methanol, dried with nitrogen at room temperature, and reconstituted with 100 μL of water , into HPLC-MS / MS determination, respectively measured the con...

example 3

[0028] Collect the cells and do a parallel measurement at the same time, extract DNA from the cells according to the steps (1) (2) of the previous invention, and measure the purity and concentration of the DNA with a nucleic acid concentration analyzer. Take 6 μg of DNA and add it to the deferoxamine methanesulfonate solution. Deoxyribonuclease I and alkaline phosphatase hydrolyze the DNA, and a certain amount of mixed standard solution of 8-OHdG and dG is added to the solution. Take 10 μL of the enzymolysis solution and add 20ng D 1 -dG internal standard, diluted to 1 mL with ultrapure water, filtered through a 0.22 μm filter membrane, and determined by HPLC-MS / MS. Take 150μL of the solution after enzymatic hydrolysis, add 1ng[ C 13 N15 ]-8-OH-G internal standard, purified by methanol and water-activated C18 extraction column, rinsed with 2 mL of water, eluted with 1 mL of methanol, dried with nitrogen at room temperature, reconstituted in 100 μL of water, and sent to HPLC...

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Abstract

The invention belongs to a cell DNA detection technology and in particular relates to a method for detecting 8-hydroxy-2'-deoxyguanosine (8-OHdG) and 2'-deoxyguanosin (dG) in cell DNA. The method can be used for quantitatively detecting the change of the content of 8-OHdG and dG in cell DNA, caused by exposure of harmful ingredients in cigarette smoke, so that the purpose of evaluating DNA oxidation damage caused by induction of the harmful ingredients can be achieved. According to the method, deoxyribonuclease I and alkaline phosphatase are used for hydrolyzing DNA in a cell, and deferoxamine mesylate is added into enzymatic hydrolysate, so that the oxidation of dG in the enzymolysis process is avoided; enzymatic hydrolysate is detected by HPLC-MS/MS; the cell DNA oxidation damage degree is evaluated according to the value of 8-OHdG/10<6>dG. By virtue of the method, the interference of high-concentration metal ions and artificial dG oxidation is removed, and thus the result is relatively accurate; the method is low in detection limit, high in sensitivity, good in repeatability and high in recovery rate.

Description

[0001] technical field [0002] The invention relates to the detection technology of cell DNA oxidative damage marker 8-hydroxydeoxyguanosine and the DNA component deoxyguanosine, which is used for genotoxicity evaluation of harmful components of cigarette smoke. Specifically, it is an analytical method for determining the content of 8-hydroxydeoxyguanosine and deoxyguanosine in cell DNA by HPLC-MS / MS combined technology. Background technique [0003] DNA oxidative damage is an important form of DNA damage. When the body is exposed to various physical and chemical inducers or metabolic processes, a large number of reactive oxygen species (reactive oxygen species, ROS) will be generated, resulting in oxidative damage to DNA molecules. Since deoxyguanosine (2'-deoxyguanosin, dG) is the component with the lowest ionization potential among the various components that make up DNA molecules, it is easy to become the target of ROS attack and generate unstable free radical catio...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/06
Inventor 赵阁张婷婷余晶晶王昇谢复炜
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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