DNA methylation is an epigenetic modification. It is a reaction in which DNA methyltransferase (DNMT) uses S- adenosylmethionine as a methyl donor to convert cytosine into 5- methylcytosine (mC). In eukaryotic DNA, 5- methylcytosine is the only chemically modified base. CG dinucleotide is the most important methylation site. It is unevenly distributed in the genome, and there are hypermethylated, hypomethylated and unmethylated regions. In mammals, mC accounts for about 2-7% of total C. Generally speaking, DNA methylation can inhibit gene expression. DNA methylation plays an important role in maintaining chromosome structure, X chromosome inactivation, gene imprinting and tumor development.
The distribution of CpG dinucleotides in human genome is very uneven, but in some segments of the genome, CpG remains or is higher than the normal probability, and these segments are called CpG islands. CpG islands are mainly located in the promoter and the first exon region of the gene. About 60% of gene promoters contain CpG islands. The study of CpG methylation plays a very important role in tumor research. Methylation of cytosine in the promoter region and CpG island near it can regulate the expression of genes at transcription level, thus silencing the corresponding genes, and demethylation can restore their expression. Under physiological conditions, DNA methylation is involved in controlling the temporal and spatial expression of genes. When tumor occurs, the whole genome hypomethylation of tumor cells is an important feature. Compared with normal cells, the degree of genomic methylation of tumor cells is only 20-60%, accompanied by hypermethylation of local genes, including tumor suppressor genes, tumor metastasis and infiltration suppressor genes, cell cycle regulator genes, DNA repair genes, angiogenesis suppressor genes and so on. However, the limitations of current research methods limit the extensive research on DNA methylation.
In recent years, researchers have been exploring qualitative and quantitative methods to detect single or multiple methylation sites. However, due to the high density of methylation polymorphic regions, it is difficult to design primer positions for extension reaction. Pyrophosphate sequencing technology, as a new sequence analysis technology, can quickly detect the frequency of methylation and qualitatively and quantitatively detect methylation sites in samples, which provides a new way for methylation research.
In principle, pyrophosphate sequencing is a method of sequence analysis by synthetic method. Pyrophosphate sequencing starts the enzyme cascade reaction by pyrophosphoric acid released after nucleotide binding to template, so that fluorescein can emit light and be detected. This technique has been used to detect the genotype and haplotype of single nucleotide polymorphism (SNP), and to identify and type bacteria and viruses. One of the main features of this technology is that it is used in Pyrogarm? The peak height displayed on the software comes from the original data of sequence analysis, and the allele frequency in the mixed DNA template can be accurately detected by the peak height.
At present, in the study of methylation, many reports on quantitative analysis of methylation use bisulfite to treat methylated samples and mixed PCR products.
As a correction, the main principle is that bisulfite can convert unmethylated cytosine into uracil, while methylated cytosine remains unchanged under appropriate experimental conditions. Therefore, after the sample was treated with bisulfite and amplified by PCR, the methylation site could be regarded as SNP of C/T, and its gene frequency was 0- 100%. Here, we introduce a researcher who uses pyrophosphate sequencing.
Six methylation sites were detected simultaneously in pyrosequencing reaction. This method can also be used to embed tissues in paraffin with high repeatability and accuracy. The CpG island of the transcription initiation site of glutathione -S- transferase π(GSTP 1) was selected for detection. These sites are unmethylated in normal prostate tissue. However, it is highly methylated in tumor samples. A fragment of 140bp containing 17 methylation polymorphism site was amplified by PCR, and the 15 site was studied with four sequencing primers (table 1). Sequencing primers were designed by online SNP sequencing primer design software (pyrosequencing AB). Some methylation polymorphic sites are replaced by the most probable bases to reduce the number of calculations. Then manually detect the possible mismatch of sequencing primers. In addition, a blank control was run on the PSQ 96MA DNA analyzer to subtract the background caused by sequencing primers, biotin-labeled primers or templates.
The PCR primer design is completely matched with the template and does not cover any methylation polymorphism region. Gstp/kloc-was amplified with 10ng bisulfite transformed DNA sample or 10 fmol purified PCR product, 10 pmol forward (5'-gtgattagtattgg-3') and reverse (5'- biotin -aacttaaaacccatc-3') primers. The reaction system was 60 mM Tris-SO4, pH 8.9 18 mm (NH4) 2SO4, 1 mm MgSO4, 200 micron DNTPS, 3 U platinum Taq DNA high fidelity polymerase, final volume 50μL, PCR cycle setting: denaturation at 95℃ for 4 minutes, and then repeated at 95℃ for 30 seconds, 50. The last extension step was carried out at 72℃ for 4 minutes, and the reaction was stopped. PCR reaction was carried out in Mastercycler 96 of Eppendorf.
In mammalian genome, DNA methylation refers to the fifth carbon source of cytosine in CpG dinucleotide.