DNA 5-methylcytosine and 5-hydroxymethylcytosine gene map sequencing method

Abstract

Provided is a sequencing method for the genetic mapping of DNA 5-methylcytosine and 5-hydroxymethylcytosine. The method employs next generation sequencing and library construction technology for trace free DNA and a high-efficiency chemical bioorthogonal reaction, which greatly enhances the selectivity and efficiency of a solid-phase surface binding to a DNA modified base.

Description

technical field [0001] The invention relates to a DNA 5-methylcytosine and 5-hydroxymethylcytosine gene map sequencing method, which belongs to the field of gene sequencing. Background technique [0002] DNA is not only composed of four bases, cytosine, thymine, guanine, and adenine. 5-Methylcytosine and 5-Hydroxymethylcytosine are important modifications in DNA. Modified bases are the fifth and sixth bases on DNA. They are important markers in the regulation of biological pathways, the cell life cycle, and serve multiple biological functions, including transcriptional regulation, transposon silencing, gene imprinting, and X chromosome inactivation. In many diseases, especially major diseases such as cancer, there are specific genome distributions, and there are obvious distribution changes in the early stages of fertilization and development, which are considered to be involved in and mark these processes. Therefore, it is particularly important to use sequencing technolo...

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Problems solved by technology

[0004] According to the above analysis, there are three reasons why the existing enrichment methods have not achieved major breakthroughs: First, the DNA after enrichment is too small, and the loss is too large during the construction of the sequencing library

The traditional process of building a library involves multi-step reaction purification, and the purification efficiency of a small amount of DNA is low. Second, the efficiency of the enrichment method on low-concentration DNA decreases. When using antibody enrichment, the binding efficiency is directly related to the antigen concentration. The reported antigen-antibody binding ability cannot meet the reaction at extremely low DNA concentration; the third is that the enriched DNA cannot be effectively eluted from the solid phase, and the solid phase-loaded DNA only accounts for the original concentration during the enrichment process. Within 5% of DNA

The purification or chemical reaction involved in the elution process will greatly lose the DNA fragments loaded on the solid phase, resulting in failure of subsequent PCR reactions or introducing a large number of sequencing errors

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