Method for screening methylation sensitive gene of DNA

A methylation and gene technology, applied in the field of genetic engineering, can solve the problems of lack of stem cells, lack of disease models, and high experimental technical requirements, and achieve the effect of easy operation and easy acquisition.

Inactive Publication Date: 2018-05-04
LONGYAN UNIV
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Problems solved by technology

The limitations of MeDIP technology include: the protein MBD2b or 5'-methylcytosine antibody that specifically binds to methylated DNA is required, the amount of cells used is large, the requirements for experimental techniques are high, and the enrichment of DNA fragments with low methylation Low, and still need ...

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  • Method for screening methylation sensitive gene of DNA
  • Method for screening methylation sensitive gene of DNA
  • Method for screening methylation sensitive gene of DNA

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Embodiment Construction

[0020] Below in conjunction with accompanying drawing and embodiment the present invention will be further described:

[0021] Such as Figure 1-4 As shown, in the first embodiment of the present invention, a method for screening DNA methylation-sensitive genes is provided, the method comprising the following steps:

[0022] Construction steps of hypomethylated cell model: thaw animal adult fibroblasts, culture to 1-2 passages until the cells grow well, wait for the cells to grow to 60%-70% enrichment, and transfect the interference group with anti-DNMT1siRNA , and prepare a negative control and / or a blank control group, and perform passage or detection operations when the cells begin to overgrow;

[0023] The detection step of gene expression change: detecting the expression level of the target gene in the interference group, the negative control group and / or the blank group;

[0024] The detection steps of gene-specific region methylation: screen the CpG island of the targ...

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Abstract

The invention discloses a method for screening a methylation sensitive gene of DNA, relating to the genetic engineering field. The method comprises the following steps: a low methylation cell model construction step, a genetic expression change detection step, a gene specific region methylation detection step and a methylation sensitive gene identification step. According to the method, an adult fibroblast cell system is easily obtained by virtue of a common laboratory, the expression of a DNMT1 gene for maintaining the methylation of DNA in the cell is continuously inhibited by virtue of in-vitro-transfected anti-DNMT1 small RNA, and 1-3 generations of the cell are continuously interfered according to the activity and proliferation expression of passage cells; and practices prove that anefficient low methylation cell model can be obtained and can be combined with common PCR or high-throughput sequencing according to different research purposes so as to judge whether a target gene issensitive to the methylation of DNA or screen methylation sensitive transcription sequences of DNA in large scale. The cell model established by the technique is simple, efficient and easy to acquireand operate.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for screening DNA methylation sensitive genes. Background technique [0002] DNA methylation is very important to life activities and is one of the means of gene regulation (mainly through methylation of CpG islands and their adjacent sequences located in the 5'-UTR region, promoter region and the first exon and other functional regulatory regions. It plays a vital role in maintaining the normal function of cells, transmitting genome imprinting, embryonic development, tumorigenesis, etc., and it is a hot spot in epigenetics research. [0003] Current understanding of the methylome remains limited. Like other epigenetic phenomena, the study of methylation levels is complicated by the need for different study modes in different cell types (Laird et al. 2010). At present, the main technical methods for screening DNA methylation-sensitive genes include: DNA methylation c...

Claims

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Application Information

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IPC IPC(8): C12Q1/686C12Q1/6869C12N5/10
CPCC12N5/0656C12N2510/00C12Q1/686C12Q1/6869C12Q2535/122
Inventor 许卫华康丽珠杨小燕
Owner LONGYAN UNIV
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