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Chromatin hypersensitive-site DNase (deoxyribonuclease) I enzyme digestion method applied to tissue sample

A technology of ultra-sensitive sites and tissue samples, which is applied in the field of immunoassay, can solve the problems of no enzyme digestion method, achieve the effect of optimizing enzyme digestion reaction conditions, improving enzyme digestion efficiency, and reducing false positives

Inactive Publication Date: 2015-06-03
TONGJI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no optimal DNase I digestion method for clinical samples

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Take clinical lung adenocarcinoma tissue samples. The specific method is as follows:

[0027] Under sterile conditions, take about 50mg of lung adenocarcinoma cancer tissue, add pre-cooled 1ml PBS (containing 1× protein inhibitor) to wash 3 times; add NEHB solution (10mM HEPES pH 7.9, 25mM KCl, 0.15mM spermine , 0.5mM spermidine, 1mM EDTA, 2M sucrose, 10% glycerol, 10mM NaF, 1mM orthovanadate, 1mM PMSF, 0.5mM DTT, and 1X protease inhibitor cocktail) and 1.15% KCL, with a 50ml tissue grinder, in an ice bath The homogenate was ground repeatedly in medium, and the cell nuclei were collected by ultracentrifugation at 25,000 rpm for 30 minutes. After washing twice with PBS, suspend in pre-cooled digestion buffer A (15mM Tris-Cl, pH 8.0; 15mM NaCl; 60mM KCl; 1mM EDTA, pH 8.0; 0.5mM EGTA, pH 8.0), 37 Water bath for 2 minutes. Add DNase I enzyme (ranging from 0-60U), and after digesting at 37 degrees for 5 minutes, add an equal amount of digestion stop solution (10mM Tris, p...

Embodiment 2

[0030] The DNase I digestion method applied to the chromatin hypersensitive site in tissue samples comprises the following steps:

[0031] (1) Pretreatment of tissue samples:

[0032] Under sterile conditions, take about 50 mg of mouse liver tissue, add pre-cooled 1ml PBS detergent containing protein inhibitors, and wash 3 times; the content of protease inhibitor cocktail (100X) in PBS detergent is 1X (that is, dilute to double), the protein inhibitor is specifically composed of AEBSF, EDTA, Leupeptin and Pepstatin A.

[0033] (2) Grinding tissue homogenate to collect cell nuclei:

[0034] Add NEHB solution and 1.15wt% KCL to the pretreated tissue sample, use a tissue grinder, grind in an ice bath, and collect cell nuclei by ultracentrifugation at 25,000 rpm for 30 minutes;

[0035] NEHB solution specifically includes: 10mM 4-hydroxyethylpiperazineethanesulfonic acid, 25mM KCl, 0.15mM spermine, 0.5mM spermidine, 1mM EDTA, 2M sucrose, 10v / v% glycerol, 10mM NaF, 1mM orthovanad...

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PUM

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Abstract

The invention relates to a chromatin hypersensitive-site DNase (deoxyribonuclease) I enzyme digestion method applied to a tissue sample, which comprises the following steps: carrying out tissue sample pretreatment, adding an NEHB solution and 1.15% KCl into the tissue sample, grinding with a tissue grinder in an ice bath, and carrying out supercentrifugation to collect the karyons; and carrying out DNase enzyme digestion reaction, purifying to extract the enzyme digestion DNA (deoxyribonucleic acid), and finally, recovering the DNA segment with glue. Compared with the prior art, the method improves the early-stage collection process of the tissue karyons, optimizes the enzyme digestion reaction conditions by the quantity of karyons, and enhances the DNase enzyme digestion efficiency. By combining the DNase gradient enzyme digestion process with the high flux process, the method can effectively acquire the correct chromatin segment, can lower the false positive generated in the early-stage ground sample, can accurately position the combination site of the transcription regulatory element on the genome, and enhances the clinical sample research progress.

Description

technical field [0001] The invention belongs to the technical field of immunodetection, and in particular relates to a DNase I enzyme cutting method applied to chromatin hypersensitive sites in tissue samples. Background technique [0002] The expression of oncogenes is regulated by the combination of multiple functional transcription elements (transcription elements, TEs) binding sites. ), which plays an important role in regulating transcription. DNase I endonuclease can sensitively identify open regions in chromatin of the whole genome, and can predict the position of functional regulatory elements (such as promoters, enhancers, silencers, and insulators, etc.) related to tumor gene expression in tumor cells on the genome. specific binding site. [0003] At present, DNase-seq technology is only applied abroad and has not been promoted in China, and this method is only used in cell samples, and the research on binding sites of regulatory elements is limited to the cell l...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/44
CPCC12Q1/6809C12Q1/44
Inventor 李旦刘小乐
Owner TONGJI UNIV