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A method for detecting active demethylation of DNA

A demethylation and methylation technology, applied in recombinant DNA technology, biochemical equipment and methods, DNA/RNA fragments, etc.

Active Publication Date: 2015-07-29
南通中科医学检验实验室有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] In summary, so far there is no experimental cell model that can provide functional studies on DNA methylation or DNA demethylation of related genes; and the study of the mechanism of DNA methylation or DNA demethylation is very important for solving the problems caused by DNA methylation. Diseases caused by abnormalities are of great significance

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  • A method for detecting active demethylation of DNA
  • A method for detecting active demethylation of DNA
  • A method for detecting active demethylation of DNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1. Construction and in vitro methylation of artificial CpG island reporter gene carrier PCBS-luc

[0037] 1. Design of PCBS-luc

[0038] Using the method of Escherichia coli recombinant plasmid construction, with PCDNA3.1 as the backbone, luciferase reporter gene, SV40 promoter, BD domain binding site, and a high CpG fragment derived from Streptomyces coelicolor were inserted in sequence to obtain (named PCBS -luc).

[0039] 2. In vitro methylation of PCBS-luc

[0040] PCBS-luc with restriction endonuclease Pst I was linearized by single enzyme digestion and incubated at 37 degrees for 2 hours. Afterwards, the product was purified (using the Axygen Gel Recovery Kit), half of the recovered product was used for methylation treatment, and the other half was not treated as a non-methylated control. In vitro methylation treatment using M.Sss I (NEB Corporation). After the methylation treatment, the product was purified again in the same way as before. rest...

Embodiment 2

[0043] Example 2: Screening of monoclonal stably transfected cell lines

[0044] 1. Determine the optimal drug concentration for screening

[0045] The optimal drug concentration refers to the lowest concentration that can cause all cells to die. The selected drug is hygromycin B (Hygromycin B), its concentration is 50mg / ml. The cells used were HEK293. Specific steps are as follows:

[0046] (1) Resuscitate HEK293 cells into a 6cm dish, passage when they are full, transfer 3ml of 18-20ul per well into a 96-well plate, and make up 100ul / well.

[0047] (2) According to the literature, the optimal concentration of this drug for HEK293 cells is 200ug / ml, and it is also recommended to be 100ug / ml. Therefore, the pre-experimental drug concentrations were selected as 80 ug / ml, 100 ug / ml, 150 ug / ml, and 200 ug / ml to determine the final suitable drug concentration for this experiment.

[0048] (3) After the cells in the 96-well plate are well-passed, pressurize and screen. After 6...

Embodiment 3

[0069] Example 3: Identification of methylated monoclonal stably transfected cell lines

[0070] 1. Identification of Luciferase Activity in Methylated and Unmethylated Monoclonal Stably Transfected Cell Lines

[0071] (1) Transfer the screened stably transfected monoclonal cell lines into 96-well plates, and transfect within 12-16 hours. PM is used as the empty control.

[0072] (2) After 72 hours, the luciferase value was measured and calculated.

[0073] (3) The result is as follows Figure 4 Shown in C and D, C is the transfection response luciferase activity of methylated monoclonal cells to PMVP16, D is the transfection response luciferase activity of non-methylated monoclonal cells to PMVP16 ; Select methylated cell clone 7# with higher luciferase activity, methylated cell clone 9# with lower luciferase activity, and unmethylated cell 8# for further identification.

[0074] 2. Identify the degree of methylation in methylated cell lines and non-methylated cell lines...

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Abstract

The invention belongs to the technical field of biology, relating to a detection method for a gene-induced DNA demethylation. A cell strain in a determined methylation state is screened out. The method uses the cell strain as base and comprises the steps of: collecting a mixed clone of the gene and the cell strain, and determining the DNA methylation state of the clone; and comparing the DNA methylation state of the clone with that of a non-transgenic cell strain to get a detection result. The invention also constructs an artificial CpG island reporter gene vector PCBS-luc and uses methyltransgerase M. SssI to perform methylation treatment to the vector in vitro. The methylated vector and an anti-hygromyc gene expression vector co-transfect HEK293 cells and PCBS stable integrated cell strains which are fully methylated, partly methylated or fully unmethylated are respectively screened out using hygromycin B. The method can be used for observation of a demethylation function of a target protein, detection of DNA methylation or demethylation functions, observation of the relation between a histone modification and a DNA methylation, and study of the influence of environment factors on the cell DNA methylation.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to a method for detecting DNA active demethylation. The method constructs an artificial CpG island reporter gene carrier and carries out methylation in vitro, and transfects cells and uses the drug resistance of cells to screen out Monoclonal stably transfected cell lines, further detection of gene-induced DNA demethylation. Background technique [0002] Epigenetics refers to heritable changes in gene expression that do not change the DNA sequence. A prospective epidemiological study of several large-scale monozygotic twins at the beginning of this century found that genetic variation only played a key role in the occurrence of some rare hereditary tumors, while sporadic tumors accounted for more than 95% of all tumors It is mainly determined by environmental factors. At present, the research scope of epigenetics mainly includes DNA methylation, histone modification, chromatin r...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/02C12Q1/66C12N15/63C12N15/117C12N5/10
Inventor 马端张进王慧君杨璐
Owner 南通中科医学检验实验室有限公司
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