Fusion protein, kit and CHIP-seq detection method

A fusion protein and chip-seq technology, which is applied in the direction of fusion polypeptide, chemical instruments and methods, biochemical equipment and methods, etc., can solve the problems of high probability of experimental failure, high library background, library information loss, etc., and simplify the experimental process , improve the efficiency of database construction, and improve the effect of resolution

Active Publication Date: 2018-07-17
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] 1. The efficiency of library construction is low, resulting in serious loss of library information when making a small number of cells
[0009] When building a library of DNA fragments released after Protein A-MNase cleavage, the traditional TruSeq library building strategy needs to be used, and the adapter and DNA fragment connection efficiency is low when building a library
Especially when a small number of cells are used as the starting material, too many DNA fragments are lost during the library construction process, which eventually leads to the loss of the main information of the library, the high probability of experimental failure, and the inability to obtain a protein-DNA interaction map at the genome-wide level
[0010] 2. Higher library background
[0012] 3. The existing CHIP-seq technology cannot perform in situ detection on tissue sections, etc., destroying its spatial resolution

Method used

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  • Fusion protein, kit and CHIP-seq detection method
  • Fusion protein, kit and CHIP-seq detection method
  • Fusion protein, kit and CHIP-seq detection method

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0061] The present embodiment provides a CHIP-seq detection method (cells are not cross-linked, in a natural state), and the method comprises the following steps:

[0062] 1) Collect about 1,000,000 embryonic stem cells cultured in vitro, wash them twice with PBS, collect the cells by centrifugation, and wash them three times with washing buffer 1;

[0063] 2) Resuspend the cells in binding buffer, add an appropriate amount of antibody, the antibody is H3K4Me3 antibody, and incubate at 4°C for 30 min to fully bind the antibody to the protein;

[0064] 3) Wash the cells 3 times with washing buffer 2 to remove excess unbound antibodies;

[0065] 4) Resuspend the cells with washing buffer 2, then add the fusion protein, and incubate at 4°C for 30 minutes to fully bind the fusion protein to the antibody;

[0066] 5) Wash the cells 3 times with washing buffer 2 to remove excess fusion protein;

[0067] 6) Add the reaction buffer to activate the activity of the fusion protein. In ...

Embodiment 2

[0072] The present embodiment provides a kind of CHIP-seq detection method (cell cross-linking), and described method comprises the following steps:

[0073] 1) Collect 1,000,000 embryonic stem cells, crosslink with 1% FA at room temperature for 3-10min, neutralize glycine and wash with PBS 3 times;

[0074] 2) Resuspend the cells in a hypotonic solution containing 0.3% SDS, and incubate at 37°C for 30 minutes to fully open the chromatin;

[0075] 3) Centrifuge to remove the supernatant;

[0076] 4) Wash cells once with binding buffer, then resuspend cells with binding buffer and add antibody, the antibody is H3K4Me3 antibody, incubate at 4°C for 30 min to fully bind antibody to protein;

[0077] 5) washing the cells with washing buffer 2 for 3 times to remove excess unbound antibodies;

[0078] 6) Resuspend the cells with washing buffer 2, then add the fusion protein, and incubate at 4°C for 30 minutes to fully bind the fusion protein to the antibody;

[0079] 7) washing t...

Embodiment 3

[0085] The present embodiment provides an in situ CHIP-seq detection method, the method comprising the following steps:

[0086] 1) Wash the tissue section 3 times with PBS, and then wash the section 3 times with washing buffer 1;

[0087] 2) Wash the section once with binding buffer, cover the tissue section with binding buffer, add antibody, the antibody is H3K4Me3 antibody, and incubate at 4°C for 1 hour to fully bind the antibody to the protein;

[0088] 3) wash the section with washing buffer 2 for 3 times to remove excess unbound antibody;

[0089] 4) Cover the tissue section with washing buffer 2, then add the fusion protein, and incubate at 4°C for 30 minutes to fully bind the fusion protein to the antibody;

[0090] 5) wash the section with washing buffer 2 for 3 times to remove excess fusion protein;

[0091] 6) Add the reaction buffer to activate the activity of the fusion protein. In order to reduce the reaction background, the reaction is carried out at 4°C for ...

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Abstract

The invention relates to fusion protein, a kit and a CHIP-seq detection method. The fusion protein comprises Tn5 transposase and Fc binding protein, the kit comprises the fusion protein and other auxiliary detection reagents, and the method is the CHIP-seq detection method by using the fusion protein or the kit. According to the fusion protein, the kit and the method, the library establishment efficiency can be improved in the CHIP-seq detection process, the library background is reduced, the accuracy of the CHIP-seq detection method is improved accordingly, and the experimental process of CHIP-seq is simplified.

Description

technical field [0001] The present invention relates to the field of epigenetics, in particular to a fusion protein, a kit and a CHIP-seq detection method. Background technique [0002] With the completion of gene sequencing and the advent of the post-genome era, epigenetics has become a research hotspot in the biological field. Epigenetics mainly studies the heritable modification of DNA and related protein molecules without changing the nucleotide sequence of the gene. These modifications can be "memorized" by cells and retained during subsequent cell divisions Next, its research direction includes: one is the regulation of selective expression of gene transcription level, and the other is the regulation of gene post-transcription. At present, the hotspots of epigenetics mainly focus on the selective expression regulation of gene transcription level, especially the interaction between transcription factors and DNA, DNA methylation and histone modification, etc. [1] . ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N9/12C12Q1/6804C12Q1/6869
CPCC12N9/1241C12Q1/6804C12Q1/6869C07K2319/705C07K2319/70C12Q2525/191C12Q2527/125C12Q2535/122C07K14/31C07K2319/00C12N9/22
Inventor 何爱彬艾珊珊罗颖洁
Owner PEKING UNIV
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