DNA methylation is one of the earliest discovered forms of gene epigenetic modification. In eukaryotes, methylation occurs only at cytosine, where DNA methyltransferases (DNMTs) convert the 5′ end cytosine of CpG dinucleotides to 5′-methylcytosine. Numerous studies have shown that DNA methylation can induce changes in chromatin structure, DNA conformation, DNA stability, and the interaction patterns between DNA and proteins, thereby controlling gene expression. DNA methylation typically suppresses gene expression, while demethylation induces reactivation and expression of genes. This form of DNA modification regulates gene expression without altering the gene sequence. CpG sites are rare in the genome and are densely located near gene promoters, collectively referred to as CpG islands. CpG islands contain approximately 500 or more bases and are located in the promoter region or the first exon region of genes. The methylation status of vertebrate DNA is closely related to growth and development regulation. For instance, during tumorigenesis, the unmethylated degree of CpG sequences outside of tumor suppressor gene CpG islands increases, while the CpG within CpG islands is highly methylated, leading to reduced expression of tumor suppressor genes.
1. Finding Methylation Sites Using Second-Generation Sequencing
Conduct methylation chip detection on both control and experimental groups to identify relevant methylation sites;
Some related studies show a decrease in expression levels of certain genes, which can predict the presence of CpG islands in those genes;
Search literature for relevant gene methylation sites.
2. Validate Methylation Sites and Analyze Methylation Levels
Use samples from control and experimental groups for methylation validation;
MSP method: Can validate methylation at specific sites, suitable for results after methylation chip analysis, but cannot analyze the degree of methylation;
BSP cloning sequencing method: Validates methylation sites of certain relevant genes and calculates the precise percentage of methylation levels;
HRM method: Suitable for large-scale sample methylation validation and can calculate the range of methylation levels;
Pyrosequencing method: Validates methylation sites of certain relevant genes and calculates the precise percentage of methylation levels.
3. Analyze the Correlation of Methylation Sites with Research
Analyze the correlation with research based on detection results from control and experimental group samples;
Compare whether there are differences in methylation of the same methylation sites between control and experimental groups;
Compare the differences in methylation levels of the same gene promoter regions between control and experimental groups.
4. Validate Changes in Expression Levels of Genes with Methylated Promoters
Use quantitative PCR methods to detect the expression levels of relevant genes. If the expression level decreases, it may be due to methylation occurring in the promoter region, affecting certain pathways of the gene, leading to diseases or phenotypic changes; if there is no change, it may be because the methylated region is not closely related to transcription factors, thus not affecting gene expression.
Sample Requirements:
1. DNA samples: Concentration ≥100ng/ul, volume ≥20ul of total DNA samples;
2. Blood samples: Please provide ≥500ul of anticoagulated blood; use standard sampling tubes, EDTA anticoagulation or sodium citrate anticoagulation;
3. Tissue samples: Please provide sufficient tissue samples (≥300mg);
4. Cells (≥106);
5. Methylation site information: Can provide methylation island analysis.
Experimental Reports
1. MSP: Primer sequences, experimental reports, including electrophoresis images of MSP products;
2. BSP: Primer sequences, sequencing peak charts, positive clone plasmids, and experimental reports.
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