Method for capturing interacted DNA fragments in tissue nuclear genome

A technology for organizing cells and nuclei, which is applied in the field of molecular biology experiments, can solve the problems of unrealistic culture of cell lines, and achieve the effect of expanding the scope of use

Inactive Publication Date: 2017-04-26
WUHAN FRASERGEN CO LTD
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Problems solved by technology

However, in different stages of individual development, tissue differentiation, and physiological or pathological development, the gene interaction is likely to be different. is unrealistic

Method used

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  • Method for capturing interacted DNA fragments in tissue nuclear genome
  • Method for capturing interacted DNA fragments in tissue nuclear genome
  • Method for capturing interacted DNA fragments in tissue nuclear genome

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Embodiment Construction

[0028] The principles and features of the present invention are described below in conjunction with examples, which are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0029] 1. Process animal tissue to obtain dispersed histiocytes

[0030] 1) Take 1g of fresh animal tissue sample, cut it into pieces as much as possible, put it on a 50ml centrifuge tube with a cell sieve (40μm), add 5mL room temperature 1×PBS to wash, and discard the PBS;

[0031] 2) Slowly and continuously add 10 mL room temperature 1×PBS, and squeeze the ground tissue sample through the cell sieve with a syringe plunger, and collect the filtrate in a 50 mL centrifuge tube;

[0032] 3) Centrifuge at 2000g RT for 5min, and discard the supernatant.

[0033] 2. Formaldehyde crosslinking

[0034] 1) Add 10 mL of formaldehyde with a final concentration of 1% prepared in 1×PBS, gently pipette and resuspend the cells, and shake at room temperature for ...

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Abstract

The invention relates to a method for capturing interacted DNA fragments in tissue nuclear genome. The method comprises the following steps: S1) taking fresh animal tissue, fragmenting the tissue and filtering the tissue with a cell sieve to obtain a filtrate containing dispersed tissue cell, performing centrifugation to collect the tissue cell; S2) resuspending the obtained tissue cell obtained in the S1 in formaldehyde for crosslinking; S3) cracking the crosslinked tissue cell; S4) inactivating endogenous enzyme, adding restrictive enzyme digesting genome; S5) filling a cohesive end generated through restrictive enzyme digesting genome in the S4, adding a marker; S6) connecting the DNA fragments which are mutually closed due to combination with protein; and S7) fragmenting chromatin to capture molecule-labelled fragments, and constructing a library. According to the invention, the animal tissue is subjected to Hi-C operation, a usage scope of a Hi-C technology is enlarged, the Hi-C technology is not limited in cell line, and more species tissue samples can be used for genomics research.

Description

technical field [0001] The invention relates to the technical field of molecular biology experiments, more particularly, to a method for capturing interacting DNA fragments in the genome of tissue cell nuclei. Background technique [0002] The three-dimensional conformation of the genome plays an important role in the regulation of gene expression, but the mechanism of the formation of higher-level chromosome structures is still not thoroughly studied. The regulation of gene expression in higher eukaryotes usually involves the physical interaction of genomic sequence elements on the same chromosome. Chromosome conformation capture (3C) technology can provide detailed information on associations between distant loci, which can be captured from formaldehyde-fixed nuclei and inferred from the three-dimensional folding of chromosomes. The 3C technology is based on formaldehyde-crosslinked chromatin, which captures chromosome conformation through restriction enzyme digestion and...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1051C12Q2521/301C12Q2563/131
Inventor 张骥诚虞诗诚马志远方涛张会君
Owner WUHAN FRASERGEN CO LTD
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