DNA5-methylcytosine and 5-hydroxymethylcytosine genome sequencing method

Abstract

The invention provides a DNA5-methylcytosine and 5-hydroxymethylcytosine genome sequencing method, comprising the following steps: (1), purifying and fragmentation pretreating DNA: extracting targeted DNA, breaking it into fragments having an average length of 50 nucleotides to 10000 nucleotides; (2), repairing trace DNA and connecting linker-adaptors: repairing and connecting pretreated DNA fragments to sequencing linker-adaptors required for next-generation sequencing; (3), covalently labeling 5-methylcytosine and 5-hydroxymethylcytosine; (4), enriching in solid phase DNA fragments of labeled 5-position modified cytosine, and in the solid phase, amplifying with PCR (polymerase chain reaction) amplimers of the corresponding linker-adaptors; purifying obtained PCR products to obtain a distribution map of genome 5-position modified cytosine required for next-generation sequencing. The invention greatly enhances bonding selectivity and efficiency of solid phase surface with DNA modified bases.

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...

AI Technical Summary

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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