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Method for using biomarker to detect compound induced DNA interstrand cross-linking damage, and use thereof

A technology of biomarkers and DNA strands, which is applied in biochemical equipment and methods, microbiological determination/inspection, etc., can solve the problems of complicated operation, difficult operation, and high experimental cost, and achieve stable gene expression, easy operation, and sensitive sex high effect

Inactive Publication Date: 2013-10-16
BEIJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] 1. The experimental procedure of gel electrophoresis method is relatively simple, but the quantitative effect is poor: the degree of cross-linking can be compared according to the band intensity, but a clearer quantitative relationship cannot be obtained, so electrophoresis is often used for simple qualitative analysis of DNA cross-linking ;
[0007] 2. Fluorescence spectroscopy detection method and alkali elution method require special fluorescent dyes, which are easily interfered by sample impurities, the reproducibility of the experimental results is low, the alkylation site cannot be determined, and the experimental process is not easy to operate;
[0008] 3. The single-cell gel electrophoresis method to detect cross-linking damage between DNA strands requires the addition of a breaking agent, which is easily interfered by external factors, and the reproducibility of the experimental results is poor;
[0009] 4. Chromatography-mass spectrometry detects cross-linking damage between DNA strands. The sample needs to go through complex pretreatment processes such as desalting and concentration. Moreover, mass spectrometry instruments are expensive, complicated to operate, high in requirements for the analysis environment, and high in experimental costs.

Method used

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  • Method for using biomarker to detect compound induced DNA interstrand cross-linking damage, and use thereof
  • Method for using biomarker to detect compound induced DNA interstrand cross-linking damage, and use thereof
  • Method for using biomarker to detect compound induced DNA interstrand cross-linking damage, and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Using real-time quantitative PCR chip technology to find effective markers for evaluating cross-link damage between DNA strands

[0031] 1) In the research of exploring molecular biomarkers, chip technology has played a role that cannot be underestimated. Real-time quantitative PCR chip technology was used to determine gene mRNA expression and Western Blot to detect protein expression levels, and finally the effector molecular biomarkers that caused cross-linking damage between DNA strands could be determined. We selected two known compounds, methylnitrosourea (MNU) and nimustine (ACNU), which have similar chemical structures and similar mechanisms of action but differ in the extent of inducing DNA interstrand cross-link damage, and used a dose of 150 μg / ml was used to collect samples after 2 hours of infecting mouse embryonic fibroblast NIH / 3T3 cells; the sample was mouse fibroblast NIH / 3T3;

[0032] 2) Real-time quantitative PCR chip technology was used to ...

Embodiment 2

[0035] Verification of the effectiveness of the markers in Example 2

[0036]1) In order to verify whether the gene can be used as a marker for evaluating DNA interstrand crosslink damage, we use compounds that can induce different degrees of DNA interstrand crosslink damage and compounds that cannot induce DNA interstrand crosslink damage to investigate this gene. Changes in gene expression.

[0037] 2) Mouse embryonic fibroblast NIH / 3T3 cells were selected for experiments. Cells were cultured in DMEM medium (GIBCO) containing 10% (v / v) fetal bovine serum (HyClone) in 5% (v / v) CO 2 (i.e. introducing CO into the air 2 gas makes CO 2 The volume percentage of gas is 5%) in a 37°C incubator. Subculture once every 3 to 4 days.

[0038] 3) Select 5 compounds that can induce different degrees of cross-linking damage between DNA strands and 2 compounds that cannot induce cross-linking damage between DNA strands. The compounds capable of inducing different degrees of cross-linki...

Embodiment 3

[0040] Example 3 The biomarker Erbb2 gene was used to detect compound-induced DNA interstrand cross-linking damage.

[0041] 2 hours after compound exposure to NIH / 3T3 cells, the total RNA was extracted and reverse-transcribed to obtain cDNA, which was used as a template to detect the expression of Erbb2 gene in the cells. The specific implementation method is as follows:

[0042] 1) Extract total RNA from cells

[0043] Total RNA in cells was extracted with QIAGEN's RNeasy Plus Mini Kit.

[0044] 2) Quantitative and quality detection of total RNA

[0045] Take 2 μl of RNA solution and dilute it by 1:200 times, and measure the absorbance value (A260) at 260nm with a UV spectrophotometer, and calculate the RNA concentration according to the following publicity.

[0046] RNA concentration = A260 × dilution factor × 40 (ng / μl)

[0047] Measure the absorption value (A280) at 260nm, and calculate A260 / A280. Generally, the ratio of A260 / A280 is between 1.8 and 2.0, which can mee...

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Abstract

The present invention relates to the technical field of biology, specifically to a method for using a biomarker to detect compound induced DNA interstrand cross-linking damage, and a use thereof. According to the present invention, research results find that Erbb2 expression is specifically up-regulated after a compound induces DNA interstrand cross-linking damage and an Erbb2 expression up-regulation degree presents regular change according to different DNA interstrand cross-linking damage degrees, and the results prove that positive correlation of the Erbb2 expression and the chemical substance induced DNA interstrand cross-linking damage degree is relatively unique, such that Erbb2 can be adopted as a marker for in vitro detection of chemical substance induced DNA interstrand cross-link damage. The present invention further provides a method for adopting the biomarker Erbb2 to detect chemical substance induced DNA interstrand cross-linking damage.

Description

technical field [0001] The present invention relates to the field of biotechnology. More specifically, the present invention relates to the application of Erbb2 gene as an effect marker in detecting cross-linking damage between DNA strands induced by chemical substances. Background technique [0002] Cross-link damage between DNA strands is one of the important causes of major diseases such as cancer. There are many factors that lead to cross-linking damage between DNA strands, such as free radicals and other active intermediates produced in the metabolic process in the body, ultraviolet rays and ion radiation in the environment, some endogenous and exogenous chemical substances, etc. Among them, electrophilic alkylating agents, as a class of important chemical substances, can be decomposed into active ions in the cell body, and these ions can undergo alkylation reactions with bases on one DNA chain to form active intermediates, and then react with another DNA chain An alk...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 钟儒刚赵琳娜
Owner BEIJING UNIV OF TECH
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