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Integral genome DNA methylation level analysis based on immune capillary electrophoresis-laser induced fluorescence

A laser-induced fluorescence and capillary electrophoresis technology, which is applied in the field of biological detection, can solve the problems of complicated DNA enzymatic hydrolysis operation and limited analysis, and achieves the effect of overcoming the complicated DNA operation, the small amount of DNA, and the wide application prospect.

Inactive Publication Date: 2010-02-03
RES CENT FOR ECO ENVIRONMENTAL SCI THE CHINESE ACAD OF SCI
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among these detection methods, capillary electrophoresis-laser-induced fluorescence (CE-LIF) has the advantages of high sensitivity and less DNA consumption, but the DNA enzymatic hydrolysis operation in the sample preparation process is cumbersome and requires chemical modification of nucleosides, which limits its use. Analysis of large scale and special samples

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  • Integral genome DNA methylation level analysis based on immune capillary electrophoresis-laser induced fluorescence
  • Integral genome DNA methylation level analysis based on immune capillary electrophoresis-laser induced fluorescence
  • Integral genome DNA methylation level analysis based on immune capillary electrophoresis-laser induced fluorescence

Examples

Experimental program
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Effect test

Embodiment 1

[0032] Example 1. Immuno-CE-LIF detection of overall methylation level in lambda DNA

[0033] In this example, the primary antibody is a mouse-derived 5-methylcytosine monoclonal antibody, which can specifically recognize and bind to 5-methylcytosine (5mC) in single-stranded DNA; Alexa Fluor 546 fluorescently labeled Fc is specific The F(ab') fragment of anti-IgG1 is the secondary antibody used in this example. After the λDNA to be tested was treated at 95°C for 5 minutes, the double-stranded DNA was denatured into a single strand, and placed on ice for 10 minutes to prevent the renaturation of the single-stranded DNA, and reacted with the above-mentioned primary antibody and secondary antibody overnight at 4°C to form a DNA-antibody immune complex. The reaction system of DNA and antibody was 20 μL, and the concentrations of primary antibody and secondary antibody were 1 μg / mL and 2 μg / mL, respectively.

[0034] The reaction mixture was separated in a CE-LIF device with an ex...

Embodiment 2、A549

[0039] Example 2. Immuno-CE-LIF detection of the overall methylation level of genomic DNA in A549 cells

[0040] 50 μg / mL of A549 cell genomic DNA was denatured into single-stranded DNA, reacted with 5-methylcytosine monoclonal antibody and fluorescently labeled secondary antibody overnight at 4°C, and carried out immuno-CE-LIF analysis according to the conditions described in Example 1. attached Figure 5 For the detection of the overall methylation level of A549 cells, the electrophoresis peak type is similar to the peak type of mλDNA, indicating that the genomic DNA of A549 cells can react with antibodies to form obvious DNA-antibody immune complexes. attached Figure 6 It shows that when the concentration of genomic DNA in A549 cells is in the range of 0-50 μg / mL, CE-LIFP detection is performed, and the amount of DNA-antibody immune complex has a good linear relationship with the DNA concentration (R 2 =0.99), the example shows that the present invention can be used to d...

Embodiment 3

[0041] Example 3, CE-LIF detection of the effect of 5-Aza-dC on the overall methylation level of cellular genomic DNA

[0042] After exposing A549 cells and HepG2 cells to different concentrations of 5-Aza-dC for 72 hours, genomic DNA was extracted respectively, and 50 μg / mL of cellular genomic DNA was reacted with antibodies overnight, and immuno-CE-LIF detection was carried out according to the above method. attached Figure 7 It shows the effect of different concentrations of 5-Aza-dC on the overall methylation level of genomic DNA in A549 cells and HepG2 cells. As the concentration of 5-Aza-dC increases, the overall DNA methylation level of cells shows a downward trend. At the highest exposure concentration (10 μM), the overall methylation level of genomic DNA in A549 cells and HepG2 cells decreased by 64% and 46%, respectively, and when the exposure concentration of 5-Aza-dC was 0.01 μM, the overall methylation level of DNA was inhibited ( 10%-20%) can be obtained throug...

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Abstract

The invention relates to an efficient and fast detection method of integral DNA methylation level. The invention realizes separation and detection of a DNA-antibody immune compound by an immune capillary electrophoresis-laser induced fluorescence technology to quantitatively analyze the integral methylation level in the DNAs to be detected. Compared with the traditional detection method of the integral DNA methylation level, the method requires no the steps such as bisulfite treatment, DNA enzymolysis, PCR amplification and the like, and has the advantages of simple and fast operation, a smallquantity of the DNAs, high detection sensitivity and the like, thus realizing efficient and fast detection of the integral DNA methylation level; and the method is applicable to detection of variousbiological samples, fast screening of influence of environmental pollutants on DNA methylation and epigenetics evaluation on diseases such as cancer and the like.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to an efficient and rapid detection method for DNA overall methylation level based on immunocapillary electrophoresis-laser induced fluorescence technology. Background technique [0002] DNA methylation is one of the most important epigenetic modifications in vertebrates. It plays an important role in the normal development of mammalian embryos and is closely related to the regulation of gene expression, the inactivation of the X chromosome in females, and genetic imprinting. Aberrant DNA methylation can negatively affect the genome, is associated with many human diseases, and is an important marker of cancer. Among them, the overall hypomethylation state of the genome, as one of the abnormal DNA methylation forms, can be detected in a variety of cancer cells, which can affect the chromosome structure, increase the frequency of chromosomal recombination and mutation, an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N33/561G01N21/64
Inventor 汪海林王晓利
Owner RES CENT FOR ECO ENVIRONMENTAL SCI THE CHINESE ACAD OF SCI
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