Chromatin conformation capturing method

Abstract

The invention relates to the field of biology, and particularly discloses a chromatin conformation capturing method. The method comprises the steps of carrying out an in-situ proximity ligation reaction in a cell and labeling a ligation position with biotin; identifying an adjacent ligation position by using biotin antibody and secondary antibody specificity, binding Tn5 in pA-Tn5 to DNA near the adjacent ligation position in a targeted manner through binding of pA and antibody, breaking the DNA near the adjacent ligation position by Tn5 enzyme digestion, and simultaneously accessing an N5 linker sequence; and after the DNA is purified, enriching DNA with biotin by using magnetic beads as a template, performing PCR amplification on the template DNA by using a specific primer of an observation site and a P5 primer to construct a sequencing library, sequencing and analyzing data. According to the method, the dependency of the observation site on the restriction enzyme site is greatly reduced, the chromatin three-dimensional conformation in which the observation site participates is detected with higher resolution, the number of required cells is greatly reduced, and the method can be applied to clinical samples.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for capturing chromatin conformation. Background technique [0002] The importance of chromatin interactions in genome function has attracted much attention. At present, there are two main categories of technologies for studying chromatin interactions: molecular probe technology and molecular interaction mapping technology, which can capture the interaction between chromatin points or between multiple points. Molecular interaction mapping technology includes chromatin conformation capture (3C) technology and ChIP-3C, 4C, 5C technology based on 3C technology. [0003] The experimental process of 4C is: after the cross-linked cells are permeabilized through the cell membrane, the chromatin is decomposed by a restriction endonuclease that can cut DNA on the side of the DNA site of interest (target site / observation site). DNA fragmentation; use DNA ligase to join spatially adj...

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Problems solved by technology

[0004] The 4C experiment process is lengthy, the effective information capture rate is low, and the construction of a single library often needs to start experiments with more than ten million cells, and the repeatability is low

Therefore, in the published literature, authors usually use gene editing to introduce variants of interest into cell lines or animal models before conducting 4C studies, and cannot directly use a limited number of clinical samples

In addition, through preliminary attempts, the applicant found that the 4C experiment process is complicated, the operation is difficult, it takes a long time (6-7 days) and consumes a lot of resources and funds

The 4C experiment involves two rounds of enzyme digestion. The detection of chromatin conformation in the target region is highly dependent on the distribution of restriction endonuclease recognition sites and the effectiveness of primers. Any position cannot be used as a detection site for effective detection. detection

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