Reagent for DNA hypermethylation region enrichment, enrichment method and application thereof

Abstract

The present application discloses a reagent for DNA hypermethylation region enrichment, an enrichment method and the application thereof. A DNA hypermethylation region enrich reagent according to thatpresent invention comprise a probe having a single-stranded loop structure, wherein the sequence of the probe comprises an endonuclease cleavage region, a prim binding region, a DNA 3 '-end high-C fragment binding region, a DNA middle high-C fragment binding region, and a DNA 5'-end high-C fragment binding region. The reagent of the present invention effectively enriches the hypermethylated DNA fragment by rolling-loop amplification after cyclization of the hypermethylated DNA fragment, meets the sequencing requirements, ensures the methylation information of DNA to the maximum extent, and avoids the nonspecific amplification and amplification errors caused by the conventional PCR amplification enrichment. The reagent and the enrichment method of the present invention are simple in operation and low in price.

Description

technical field [0001] The present application relates to the field of nucleic acid enrichment in vitro, in particular to a reagent, enrichment method and application for enrichment of hypermethylated regions of DNA. Background technique [0002] Epigenetics is to affect and regulate the expression and function of genes through DNA methylation, histone modification, chromosome remodeling and non-coding RNA regulation without changing the genomic DNA sequence, and this change Stable transmission can occur during cell proliferation and ontogeny. In recent years, with the continuous deepening of life science research, the research methods of epigenetics have also been developed rapidly, and the related research fields have been increasingly broadened, and its research on the occurrence and development of tumors has achieved breakthroughs. progress. DNA methylation is a chemical modification that occurs at the CG site. S-adenosylmethionine is used as a methyl donor. Under the ...

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Problems solved by technology

[0005] 1) For the genome-wide methylation sequencing method, since the total amount of free DNA is extremely small, 8 mL of whole blood only contains about 30-50 ng of free DNA, while the current high-throughput sequencing method requires an initial DNA amount of 100 ng, and Before the sequencing process, bisulfite treatment and library construction will cause DNA loss, which will eventually lead to the scarcity of methylation information contained in the sequencing data; it cannot meet the needs of use

[0006] 2) The high methylation amplification method based on the PCR method, which amplifies and enriches the free DNA through PCR amplification, although it can solve the problem of too little free DNA, and can effectively enrich and detect the high methylation region, However, due to the bias of the sequencing data caused by the amplification of conventional PCR primers, the information of some sites may be lost; the methylation information of free DNA cannot be accurately and comprehensively reflected

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