Capacity detection method for assembling chromatin structure on specific site of DNA sequence

A DNA sequence and specific site technology, applied in the field of molecular biology, can solve the problems of cumbersome experimental steps, high experimental operation requirements, and high price, and achieve the effect of high specificity, high accuracy and simple implementation process

Active Publication Date: 2018-01-05
INNER MONGOLIA UNIV OF SCI & TECH
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Problems solved by technology

[0006] (3) The formation of nucleosomes will affect the interaction between trans-acting factors and nucleosomal DNA;
[0010] ChIP-seq method: It is suitable for large-scale detection such as the whole genome. It is expensive and requires high experimental operations. It is not suitable for detecting the nucleosome occupancy rate of a few specific sites.
[0011] Tilling-PCR method: a large number of nested primers need to be designed, the experimental steps are cumbersome and expensive

Method used

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  • Capacity detection method for assembling chromatin structure on specific site of DNA sequence
  • Capacity detection method for assembling chromatin structure on specific site of DNA sequence
  • Capacity detection method for assembling chromatin structure on specific site of DNA sequence

Examples

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example 1

[0060] Example 1: Detection of ability to assemble nucleosomes at specific sites of reconstituted chromatin on circular plasmids (1) Reconstituted chromatin structure on circular plasmids. The purified circular plasmid pUC601 was mixed with the purified histone octamer as shown in Table 1, respectively, and dialyzed in TE solution containing 2 mol / L NaCl. During the dialysis process, TE was pumped into the dialysate to dilute the NaCl concentration in the dialysate to 0.6 mol / L, and the dialysis process was controlled for 16 hours; the dialysate was replaced with TE buffer without NaCl and continued dialysis for 3 hours. Wherein, the reaction temperature of the whole experiment process was controlled at 4°C.

[0061]

[0062] Table 1

[0063] (2) Restriction endonuclease digestion. The nucleosome occupancy near the Nde I single restriction site on the pUC601 plasmid sequence was detected. Take 1 / 5 of the dialyzed sample, add 8U of Nde I, and digest at 37°C for 1 hour.

...

example 2

[0066] Example 2: Detection of the ability to assemble nucleosomes at specific sites in remodeled chromatin on linear DNA sequences

[0067] (1) Reconstruction of chromatin structure on linear DNA sequence. Plasmid pUC19 was digested with restriction endonuclease Ssp I into a linear DNA sequence. Mix the digested and purified linear plasmid pUC601 with the purified histone octamer as shown in the table below, and dialyze in TE solution containing 2mol / L NaCl. During the dialysis process, TE was pumped into the dialysate to dilute the NaCl concentration in the dialysate to 0.6 mol / L, and the dialysis process was controlled for 16 hours; the dialysate was replaced with TE buffer without NaCl and continued dialysis for 3 hours. The whole experimental process was controlled at 4°C.

[0068]

[0069] Table 2

[0070] (2) Restriction endonuclease digestion. The nucleosome occupancy near the EcoR I single restriction site on the pUC601 sequence was detected. Take 1 / 5 of the d...

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Abstract

The invention relates to the field of molecular biology, in particular to a capacity detection method for assembling a chromatin structure on a specific site of a DNA sequence, and aims to solve the problem that an existing technology cannot detect the nucleosome occupying rate at a fixed site to determine the chromatin structure assembling capacity. The method is mainly to assemble the DNA sequence containing a specific restriction enzyme site into the chromatin structure; enzyme digestion is carried out through restriction enzyme to remove various types of protein, then electrophoresis detection is carried out, and the site and the nucleosome occupying rate of the DNA sequence near the site are quantitatively calculated to judge a local chromatin structure near the site. Therefore, the efficiency of assembling the chromatin structure on the specific site on the DNA sequence and the nucleosome assembling capacity are analyzed. The detection method is simple and quick in realizing process, and high in specificity and accuracy.

Description

technical field [0001] The application relates to the field of molecular biology, in particular to a method for detecting the ability to assemble chromatin structure at a specific site in a DNA sequence. Background technique [0002] The nucleosome is the basic structural unit of eukaryotic chromatin. Two copies of each of the four histones H2A, H2B, H3, and H4 form a histone octamer, on which a DNA sequence of about 147 bp is wound in a left-handed helical manner 1.7 circles, constituting nucleosome core particles. The core particles are connected by 20-80bp DNA, which is further compressed and packaged into a 30nm chromatin secondary structure under the action of histone H1. [0003] As a basic structural unit of eukaryotic chromatin, nucleosome and chromatin have dual functions of structure and function. The position of nucleosomes on genomic DNA and the local structure of chromatin regulate many biological processes including DNA replication, transcription, repair, rec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 赵宏宇蔡禄张凤慧赵秀娟刘国庆
Owner INNER MONGOLIA UNIV OF SCI & TECH
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