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MNase digestion optimization method applied to nucleosome loci in tissue samples

A technology for tissue samples and optimization methods, applied in the field of immunoassays, can solve problems that have not been applied clinically, and achieve the effects of improving the research process, optimizing enzyme digestion reaction conditions, and improving enzyme digestion efficiency and recovery rate

Inactive Publication Date: 2018-05-18
上海嘉因生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, MNase-Seq technology is only applied abroad, but has not been promoted in China. It has not been well applied in clinical practice, and there is no optimization method for MNase digestion of clinical samples.

Method used

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Embodiment 1

[0031] Embodiment 1: This embodiment specifically introduces the method for nucleosome positioning, as follows:

[0032] Nucleosome positioning, considered as the probability that a nucleosome originates at a specific base pair within the genome, plays an equally important role in gene and chromatin regulation with known regulatory mechanisms, suggesting that nucleosomes are Genomic DNA is not randomly distributed, but may tend to localize in or away from specific regions of the genome for some purpose. Far from being independent of other transcriptional mechanisms such as histone modifications and chromatin remodeling, nucleosome positioning is tightly entwined with them. However, the positioning of nucleosomes on DNA is very complex, and four simple descriptors can be used to characterize nucleosome positioning:

[0033] (1) Nucleosome repeat length (NRL):

[0034] The nucleosome repeat length is the average length of the DNA associated with one nucleosome, including the c...

Embodiment 2

[0041] Embodiment 2: This embodiment specifically illustrates the processing steps applied to the optimization method for MNase digestion at nucleosome sites in tissue samples, as follows:

[0042] Step 1: Prepare chromatin:

[0043] First, the collected tissue samples are washed with physiological saline pre-cooled to 0°C and added to red blood cell lysate to remove red blood cells, washed with sterile distilled water to obtain white blood cells, red blood cell lysate includes: 30mM KHCO 3 , 250mMNH 4 Cl, 10mM EDTA-Na 2 ;

[0044] Secondly, repeat the following operation twice to obtain precipitate A. The operation is: add TEDP solution, and place the obtained mixed solution in a water bath at 37°C for high-speed centrifugation; TEDP solution includes: 50mM Tris-HCl, 2mM EDTA, 2mM DDT, 10wt% Glycerol, 50 μg / mL PMSF;

[0045] Then, the precipitate A was added to the TEDP solution and spread on the 300mM sucrose solution, and the homogenate was centrifuged at 70000g for 1h ...

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Abstract

The invention relates to an MNase digestion optimization method applied to nucleosome loci in tissue samples. The MNase digestion optimization method includes chromatin preparation, DNA preparation, MNase digestion, DNA fragment recycle, high-throughput sequencing and frequency detection. The MNase digestion optimization method has the advantages that pre-collection methods for tissue cell nucleuses are improved, enzyme digestion reaction conditions are optimized, and enzyme digestion efficiency and recycle rate are increased; correct chromatin fragments can be acquired effectively, binding loci of regulation elements on whole genomes can be positioned and transcribed accurately, and research progresses of clinical samples are improved.

Description

technical field [0001] The invention belongs to the technical field of immunoassay, and in particular relates to an MNase digestion optimization method applied to nucleosome sites in tissue samples. Background technique [0002] In eukaryotic cells, the nucleosome is a complex structure formed by a 147bp DNA fragment tightly wound on the histone octamer, two adjacent nucleosomes are connected by a DNA fragment, and multiple nucleosomes sequential ligation to form chromatin. The interaction between nucleosome and DNA is a dynamic process, and the position of nucleosome is not constant. In most cases, regions of DNA free of nucleosome binding are readily accessible and bound by various regulatory proteins. Therefore, it is suspected that there is some kind of intrinsic link between the positioning of nucleosomes and the transcription of genes. In order to further prove the relationship between nucleosome positioning and gene transcription, more and more researches are devot...

Claims

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Application Information

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IPC IPC(8): C12Q1/6869G06F19/24
CPCC12Q1/6869G16B40/00C12Q2521/345C12Q2565/125C12Q2535/122
Inventor 李旦
Owner 上海嘉因生物科技有限公司
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