Bridge-PCR-based method for detecting DNA hydroxymethylation

Abstract

The invention discloses a bridge-PCR-based high-flux chip detection method for DNA hydroxymethylation. . The detection method comprises: firstly fixing different forward primers for amplyfying DNA hydroxymethylation sites to a chip or a microballon; secondly using beta-glucanotransferase to glycosylating all 5hmC on DNA, then using MspI enzyme for enzyme digestion, wherein hydroxymethylated CCGG cannot be cleaved and non-hydroxymethylated CCGG can be cleaved; thirdly hybridizing genome DNA subjected to enzyme digestion with the chip, and performing bridge PCR, wherein the hydroxymethylated CCGG sites can have amplification because the hydroxymethylated CCGG cannot be cleaved, and conversely the non-hydroxymethylated CCGG sites cannot have amplification because the non-hydroxymethylated CCGG can be cleaved; and finally, according to presence or absence of fluorescence at different matrix points of the chip, determining whether CCGC at different positions of the genome DNA is subjected to hydroxymethylization. The beneficial effects comprises: parallelism detection on different DNA hydroxymethylization sites is realized, and the detection results have the characteristics of high flux and high sensitivity.

Description

technical field [0001] The invention belongs to a high-throughput and high-sensitivity detection technology for detecting DNA hydroxymethylation by using a chip. Background technique [0002] 5-Hydroxymethylcytosine (5hmC) is a newly discovered modified base (Kriaucionis and Heintz, 2009; Tahilianie et al., 2009), which exists in a variety of mammalian cell types at low levels. 5hmC is produced by enzymes of the 10-11 translocation (TET) family by oxidation of 5-methylcytosine (5mC). 5hmC can not only reduce the affinity between the methylation-binding domain (MBD) of MeCP protein and methylated DNA, but also has the potential transcriptional regulation function involved in the regulation of gene expression, and participates in the process of DNA demethylation. 5hmC may Become a new molecular marker for the diagnosis of certain diseases. Therefore, the research on 5hmC is increasingly favored by scholars. The research on 5hmC detection method is the premise and important ...

AI Technical Summary

Problems solved by technology

Although these detection methods can detect the fragment sequence of the 5hmC site in the whole genome and the distribution characteristics of 5hmC, they are prone to non-specific binding and cause false positive results due to antibody immunization, and it is difficult to accurately analyze the situation of specific base 5hmC and the cost of detection is high. shortcoming

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