Sulfite sequencing process

Bisulfite Sequencing is one of the most commonly used and accurate techniques to study DNA methylation status. By sulfite treatment on DNA samples, this method can effectively distinguish between methylated and non-methylated cytosines, thereby revealing changes in methylation patterns in the genome. The widespread application of this technology provides an important tool for biomedical research, especially in the fields of cancer, gene regulation and developmental biology.

Sulfite Sequencing

1. DNA sulfite treatment purpose: convert cytosine (C) that has not methylated in DNA into uracil (U), while methylated cytosine remains unchanged. Treatment procedure: Treat genomic DNA using sulfites such as sodium bisulfite.

2. Specific primer design Principles: Do not contain CpG sites to avoid differences in methylation and non-methylated DNA. The primer amplified fragment should contain as many CpG sites as possible. Usually, the length of the BSP amplified fragment is 300~500bp. The more CpG sites it contains, the higher the primer score.

3. Purpose of PCR amplification: Amplify the sulfite-treated DNA fragments, at which time uracil (U) will be converted into thymine (T) during PCR. Amplification process: PCR amplification of treated DNA using specific primers.

4. Sequencing library construction purpose: convert PCR amplification products into libraries suitable for high-throughput sequencing. Construction process: Purification of PCR amplification products, end repair, A-tail addition, ligation of sequencing linkers, etc., to build a sequencing library.

5. High-throughput sequencing and sequencing platform: Use a high-throughput sequencing platform (such as Illumina) to sequence the sequencing library. Sequencing results: A large number of sequencing reads were obtained, which contained the sequence information of the DNA fragment and the methylation status.

6. Bioinformatics analysis of methylation site identification: By comparing the sequencing results with the original DNA sequence, the CpG site where methylation occurs was identified. Methylation quantification: count the methylation ratio of each CpG site, that is, the proportion of the readings that occur in methylation to the total reading.

7. Precautions During the entire sequencing process, experimental conditions need to be strictly controlled to avoid DNA contamination and degradation. Primer design is one of the key steps to successful sequencing and requires careful selection and verification. The accuracy and reliability of sequencing results depend on multiple factors, including DNA quality, performance of the sequencing platform, and accuracy of bioinformatics analysis.

The sulfite sequencing process includes multiple steps such as DNA sulfite treatment, specific primer design, PCR amplification, sequencing library construction, high-throughput sequencing and bioinformatics analysis. This process can clarify the methylation status of each CpG site in the target fragment, providing reliable data support for subsequent methylation studies.

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