Epigenetic changes can be defined as genetic or acquired changes in a gene, but this change is not related to changes in DNA sequence. DNA methylation is the most common epigenetic change; methylation changes in the promoter or first exon CpG island will result in inactivation of gene expression; chemical modification of histones can also be used as epigenetic changes; Genes in which acetylation changes in proteins are usually turned on.
Abnormal methylation of CpG islands is the leading cause of gene silencing and overexpression, and conventional methods cannot examine methylation at the genome-wide level. Combining epigenetic analysis with gene chip technology enables high-throughput qualitative and quantitative analysis of methylation.
Two chip-based methylation analysis methods have been established: one is the methylation specific oligonucleotide arrays (MSO) method, and the other is the differential methylation hybridization method. DMH). The first method utilizes the principle of direct hybridization, except that the DNA is treated with sodium bisulfite prior to labeling, thereby converting all unmethylated cytosines to uracil, while methylated cytosines are not subject to this treatment. The effect is still cytosine. This method generally studies the regulatory regions of known individual genes and cannot analyze the methylation of a large number of genes, especially unknown genes.
The DMH method is a new method that provides a high-throughput technology platform for large-scale research on CpG island methylation profiles. The DMH method is similar to the expression profile gene chip, except that the target gene and probe preparation methods are more complex than the cDNA expression profile chip. The DMH method was originally based on a CpG island (CGl) library. Cross et al. constructed an affinity matrix containing a methylated CpG binding domain to isolate CpG island sequences from human genomic DNA. The restriction enzyme MseI digests DNA into small fragments, but has no effect on most CpG island sequences. The GC-enriched MseI-treated fragment was isolated using an affinity column and the library was constructed by cloning into a human vector. The CpG island clone was pre-screened to have multiple BstUI cleavage sites, and the amplified fragment was used for chip preparation. Genomic DNA was extracted from the experimental samples and treated with MseI. The digested probe is capable of binding to the Mse engineered target gene in the CGI library. The digested GC-rich fragment was ligated into the adaptor (1 inker) and then treated with methylation-sensitive endonuclease BstUI, BSTUI-treated and untreated control DNA for linker-PCR and fluorescent labeling. Subsequent hybridization, image and data processing procedures are identical to the expression profile chips.
Initially, DMH was applied to nylon membranes to analyze CpG islands in human breast cancer cell lines and was later applied to glass chips to increase detection throughput. In the analysis of epigenetic changes in breast cancer, it was found that hypermethylation of CpG islands can lead to silencing of tumor suppressor genes and downregulation in some cells. Generally, poorly differentiated tumor tissues exhibit a higher degree of methylation than moderately differentiated or well differentiated normal tissues.
DMH has been successfully applied to detect methylation profiles of ovarian cancer. More methylated CpG islands were seen in ovarian cancer tissue samples than ovarian cancer cell lines. Ottaviano et al. identified the feasibility of the MS() strategy by assessing the methylation status of the CpG island located in the first exon of the human ERa gene. The MSO chip experimental results are consistent with the results of traditional Southern blot and sulfite sequence analysis methylation methods. Rober et al. used this technique to study the promoter region of the tumor suppressor gene p16, which is hypermethylated in many tumors. Hypermethylation of this promoter inhibits transcription of p16 and is associated with some tumors; they found that the promoter region of p16 is uniformly methylated at the CpG site of the H1299 cell line.
Different methylation profiles reflect different stages or different types of tumors. The hypermethylation site of CpG islands is associated with tumorigenesis and can therefore be a unique acquired marker for specific tumor subtypes. Therefore, just like gene expression profiling, clustering analysis based on methylation profiles can also be used to analyze tumor subtypes. These basic researches have potential applications, and the direction of applied research includes the development of complementary diagnostic methods based on tumor-associated DNA methylation patterns and by inhibiting DNA methylation or histone deacetylation of tumor cells. Methods and drugs to treat tumors.
Abnormal methylation of CpG islands is the leading cause of gene silencing and overexpression, and conventional methods cannot examine methylation at the genome-wide level. Combining epigenetic analysis with gene chip technology enables high-throughput qualitative and quantitative analysis of methylation.
Two chip-based methylation analysis methods have been established: one is the methylation specific oligonucleotide arrays (MSO) method, and the other is the differential methylation hybridization method. DMH). The first method utilizes the principle of direct hybridization, except that the DNA is treated with sodium bisulfite prior to labeling, thereby converting all unmethylated cytosines to uracil, while methylated cytosines are not subject to this treatment. The effect is still cytosine. This method generally studies the regulatory regions of known individual genes and cannot analyze the methylation of a large number of genes, especially unknown genes.
The DMH method is a new method that provides a high-throughput technology platform for large-scale research on CpG island methylation profiles. The DMH method is similar to the expression profile gene chip, except that the target gene and probe preparation methods are more complex than the cDNA expression profile chip. The DMH method was originally based on a CpG island (CGl) library. Cross et al. constructed an affinity matrix containing a methylated CpG binding domain to isolate CpG island sequences from human genomic DNA. The restriction enzyme MseI digests DNA into small fragments, but has no effect on most CpG island sequences. The GC-enriched MseI-treated fragment was isolated using an affinity column and the library was constructed by cloning into a human vector. The CpG island clone was pre-screened to have multiple BstUI cleavage sites, and the amplified fragment was used for chip preparation. Genomic DNA was extracted from the experimental samples and treated with MseI. The digested probe is capable of binding to the Mse engineered target gene in the CGI library. The digested GC-rich fragment was ligated into the adaptor (1 inker) and then treated with methylation-sensitive endonuclease BstUI, BSTUI-treated and untreated control DNA for linker-PCR and fluorescent labeling. Subsequent hybridization, image and data processing procedures are identical to the expression profile chips.
Initially, DMH was applied to nylon membranes to analyze CpG islands in human breast cancer cell lines and was later applied to glass chips to increase detection throughput. In the analysis of epigenetic changes in breast cancer, it was found that hypermethylation of CpG islands can lead to silencing of tumor suppressor genes and downregulation in some cells. Generally, poorly differentiated tumor tissues exhibit a higher degree of methylation than moderately differentiated or well differentiated normal tissues.
DMH has been successfully applied to detect methylation profiles of ovarian cancer. More methylated CpG islands were seen in ovarian cancer tissue samples than ovarian cancer cell lines. Ottaviano et al. identified the feasibility of the MS() strategy by assessing the methylation status of the CpG island located in the first exon of the human ERa gene. The MSO chip experimental results are consistent with the results of traditional Southern blot and sulfite sequence analysis methylation methods. Rober et al. used this technique to study the promoter region of the tumor suppressor gene p16, which is hypermethylated in many tumors. Hypermethylation of this promoter inhibits transcription of p16 and is associated with some tumors; they found that the promoter region of p16 is uniformly methylated at the CpG site of the H1299 cell line.
Different methylation profiles reflect different stages or different types of tumors. The hypermethylation site of CpG islands is associated with tumorigenesis and can therefore be a unique acquired marker for specific tumor subtypes. Therefore, just like gene expression profiling, clustering analysis based on methylation profiles can also be used to analyze tumor subtypes. These basic researches have potential applications, and the direction of applied research includes the development of complementary diagnostic methods based on tumor-associated DNA methylation patterns and by inhibiting DNA methylation or histone deacetylation of tumor cells. Methods and drugs to treat tumors.
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