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Method for enriching and extracting circulating tumor DNA

A tumor and enrichment technology, applied in the biological field, to achieve effective separation and enrichment, high throughput, and high selectivity

Inactive Publication Date: 2019-06-25
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The main issues are extraction efficiency and reproducibility

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  • Method for enriching and extracting circulating tumor DNA
  • Method for enriching and extracting circulating tumor DNA
  • Method for enriching and extracting circulating tumor DNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Two-component polymer functionalized materials are as follows:

[0028]

[0029] Wherein R is carboxyl, nitro, methyl, ethyl formate or hydrogen; X=0.01-0.5.

[0030] In the present invention, the material of the substrate is Si, Cu, Ag, Au, Pt, CuO, Fe 3 o 4 , Porous SiO 2 , Porous Al 2 o 3 , Porous TiO 2 or porous ZrO 2 Any one of them or a mixture of two or more in any proportion.

[0031] Taking X=0.2 as an example, add 0.4mmol of isopropylacrylamide and 0.1mmol of thiourea functional monomer to a 25ml three-necked flask successively, the molar ratio of the two is 4:1, and at the same time add 6mL of deionized water and 6mL of DMF to make Solvent; nitrogen gas was introduced under stirring, after the monomer was fully dissolved, catalyst CuBr 0.032g and bipyridyl ligand 0.16mL were added under the protection of nitrogen gas, and then the reaction system was vacuumed-filled with nitrogen gas to remove the residual oxygen in the reaction system ; Immerse th...

Embodiment 2

[0035] A method for enriching and extracting circulating tumor DNA, using a column solid-phase extraction mode, the circulating tumor DNA enrichment material adopts the two-component polymer functionalized material prepared in Example 1, and the specific steps are as follows:

[0036] 1) Put the circulating tumor DNA enrichment material into a pipette tip or a gel loading tip to form a micro solid phase extraction column, use the eluent and sample solution to activate and balance the micro solid phase extraction column, and put The biological sample is dissolved in the sample solution and loaded onto the micro solid phase extraction column. The mass ratio of the circulating tumor DNA enrichment material to the biological sample is 1000:1;

[0037] 2) Rinse the extraction column with an eluent of 5-200 times the column volume, and the pH of the eluent is 0-7;

[0038] 3) DNA is obtained by eluting with an eluent of 5-100 times column volume, and the pH of the eluent is 10-12; ...

Embodiment 3

[0045] A method for enriching and extracting circulating tumor DNA, using a column solid-phase extraction mode, the circulating tumor DNA enrichment material adopts the two-component polymer functionalized material prepared in Example 1, and the specific steps are as follows:

[0046] 1) Put the circulating tumor DNA enrichment material into a pipette tip or a gel loading tip to form a micro solid phase extraction column, use the eluent and sample solution to activate and balance the micro solid phase extraction column, and put The biological sample is dissolved in the sample solution and loaded onto the micro solid phase extraction column. The mass ratio of the circulating tumor DNA enrichment material to the biological sample is 1000:1;

[0047] 2) Rinse the extraction column with an eluent of 5-200 times the column volume, and the pH of the eluent is 0-7;

[0048] 3) DNA is obtained by eluting with an eluent of 5-100 times column volume, and the pH of the eluent is 10-12;

...

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Abstract

The invention provides a method for enriching and extracting a circulating tumor DNA, which belongs to the field of biotechnology. The method comprises the specific steps that 1) a circulating tumor DNA enrichment material is loaded into a pipette tip or a gel loading tip to form a microsolid phase extraction column; an eluent and a loading solution are used to activate and balance the microsolidphase extraction column; a biological sample is dissolved in the loading solution and loaded to the microsolid phase extraction column; the mass ratio of the circulating tumor DNA enrichment materialto the biological sample is 100 to 1000:1; 2) the extraction column is rinsed with an eluent of 5 to 200 times of column volume; and 3) the DNA is eluted by an eluate of 5 to 100 times of column volume, and the entire process is performed at 10 to 60 DEG C. The circulating tumor DNA enrichment material is a two-component polymer functionalized material. The method provided by the invention has thecharacteristics of high selectivity, high flux and the like in extracting the circulating tumor DNA, and can effectively separate and enrich the circulating tumor DNA.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for enriching and extracting circulating tumor DNA. Background technique [0002] Circulating tumor DNA, ctDNA (circulating tumor DNA), refers to the release of DNA fragments produced by tumor cells through apoptosis or necrosis into the circulatory system. ctDNA exists in body fluids such as blood, synovial fluid, and cerebrospinal fluid. Since the half-life of ctDNA is about 1 hour, which can accurately reflect the current situation of the tumor, ctDNA can be used as a non-invasive alternative to tissue biopsy. In addition, ctDNA plays a role in early cancer diagnosis, prognosis and determination of drug treatment response. [0003] The current mainstream ctDNA mutation detection technology includes PCR amplification technology and DNA sequence detection technology. However, regardless of the sequencing technology, DNA needs to be enriched and extracted to reduce interf...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/28
Inventor 梁鑫淼李秀玲张小菲
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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