CN109929830B - Method for extracting circulating tumor DNA by SPE method - Google Patents
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- CN109929830B CN109929830B CN201711364691.3A CN201711364691A CN109929830B CN 109929830 B CN109929830 B CN 109929830B CN 201711364691 A CN201711364691 A CN 201711364691A CN 109929830 B CN109929830 B CN 109929830B
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Abstract
The invention provides a method for extracting circulating tumor DNA by an SPE method, belonging to the technical field of biology. The method comprises the following specific steps: 1) Loading the circulating tumor DNA enrichment material into a pipette tip or a gel sample loading suction head to form a micro solid phase extraction column, activating and balancing the micro solid phase extraction column by adopting eluent and sample loading liquid, dissolving a biological sample in the sample loading liquid, and loading the biological sample onto the micro solid phase extraction column, wherein the mass ratio of the circulating tumor DNA enrichment material to the biological sample is 100-1000; 2) Washing the extraction column with 5-200 times of eluate, wherein the pH of eluate is 0-7; 3) Eluting with 5-100 times column volume of eluent with pH of 10-12 to obtain circulating tumor DNA; the DNA enrichment material is a nanoparticle material with a metal core/carbon shell structure. The DNA enrichment material is a nanoparticle with a metal core/carbon shell structure. The method of the invention has the characteristics of high selectivity, high flux and the like in the aspect of extracting the circulating tumor DNA, and can realize the effective separation and enrichment of the circulating tumor DNA.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for extracting circulating tumor DNA by an SPE method.
Background
Circulating tumor DNA, ctDNA (circulating tumor DNA), refers to the release of DNA fragments produced by apoptosis or necrosis of tumor cells into the circulatory system. ctDNA is present in body fluids such as blood, synovial fluid, and cerebrospinal fluid. Since the half-life of ctDNA is about 1 hour, the current condition of the tumor can be accurately reflected, so ctDNA can be used as a non-invasive alternative for tissue biopsy. In addition, ctDNA plays a role in early diagnosis, prognosis, and determination of drug treatment response of cancer.
The ctDNA mutation detection technologies currently in the mainstream include two types, namely PCR-based amplification technologies and DNA sequence-based detection technologies. Regardless of the sequencing technique, however, it is desirable to enrich and extract DNA to reduce interference with amplification or subsequent sequencing.
The main extraction method of ctDNA is a solid phase extraction method based on silicon spheres and magnetic spheres and a liquid-liquid extraction method based on phenol-chloroform. The main problems are extraction efficiency and reproducibility.
Disclosure of Invention
Aiming at the problems, the invention provides a method for extracting circulating tumor DNA by an SPE method.
A method for extracting circulating tumor DNA by SPE method adopts a dispersed solid phase extraction mode, and comprises the following steps:
1) Loading the circulating tumor DNA enrichment material into a pipette tip or a gel sample loading suction head to form a micro solid phase extraction column, activating and balancing the micro solid phase extraction column by adopting eluent and sample loading liquid, dissolving a biological sample in the sample loading liquid, and loading the biological sample onto the micro solid phase extraction column, wherein the mass ratio of the circulating tumor DNA enrichment material to the biological sample is 100-1000;
2) Washing the extraction column with 5-200 times of eluate, wherein the pH of eluate is 0-7;
3) Eluting with 5-100 times column volume of eluent with pH of 10-12 to obtain circulating tumor DNA;
the whole process is carried out at 10-60 ℃;
the circulating tumor DNA enrichment material is a nanoparticle material with a metal core/carbon shell structure.
The core of the nano-particle material with the metal core/carbon shell structure is a metal nano-particle, and the structural formula of the shell of the nano-particle material is as follows:
the metal is Ag, ti, fe, ga, ni, au, pd or Pt. .
The biological sample is blood, synovial fluid, cerebrospinal fluid or urine.
The sample loading solution is a mixed solution of an organic solvent, organic acid and water, and the organic solvent is acetonitrile, methanol or ethanol; the organic acid is formic acid, acetic acid or trifluoroacetic acid, the volume of the organic solvent in the sample loading liquid is 10-50%, and the pH value is 0-7.
The eluent is a mixed solution of an organic solvent, organic acid and water, and the organic solvent is acetonitrile, methanol or ethanol; the organic acid is formic acid, acetic acid or trifluoroacetic acid, and the volume of the organic solvent in the eluent is 10-50% and the pH value is 10-12.
The leacheate is a mixed solution of an organic solvent, organic acid and water, and the organic solvent is acetonitrile, methanol or ethanol; the organic acid is formic acid, acetic acid or trifluoroacetic acid, the volume of the organic solvent in the leacheate is 0-50%, and the pH value is 0-7.
The invention has the advantages and beneficial effects that: the method of the invention has the characteristics of high selectivity, high flux and the like in the aspect of extracting the circulating tumor DNA, and can realize the effective separation and enrichment of the circulating tumor DNA.
Drawings
FIG. 1 is a schematic diagram of the preparation of the nanoparticle material with a metal core/carbon shell structure according to the present invention.
Detailed Description
The Noble Metal nano-particles with the Core/shell structure are prepared by adopting a preparation method in colloid Carbon Spheres and the Core/shell structures with Noble-Metal Nanoparticles, angew. Chem.2004,116,607-611, and the preparation process is shown in figure 1. Preparing a nano-particle material with a metal core/carbon shell structure, wherein the core of the nano-particle material is Ag, ti, fe, ga, ni, au, pd or Pt; the structural formula of the shell is as follows:
example 1
A method for extracting circulating tumor DNA by SPE method, adopt and disperse the solid phase extraction mode, the circulating tumor DNA enrichment material used is the nanoparticle material of the metal core/carbon shell layer structure, prepare by the above-mentioned method, wherein the shell is gold nanoparticle; the method comprises the following specific steps:
1) Loading the circulating tumor DNA enrichment material into a pipette tip or a gel sample loading suction head to form a micro solid phase extraction column, activating and balancing the micro solid phase extraction column by adopting eluent and sample loading liquid, dissolving a biological sample in the sample loading liquid, and loading the biological sample onto the micro solid phase extraction column, wherein the mass ratio of the circulating tumor DNA enrichment material to the biological sample is 100;
2) Washing the extraction column with 5-200 times of eluate, wherein the pH of eluate is 0-7;
3) Eluting with 5-100 times column volume of eluent with pH of 10-12 to obtain DNA;
the whole process is carried out at 60 ℃.
The sample loading liquid is a mixed liquid of an organic solvent, organic acid and water, and the organic solvent is ethanol; the organic acid is trifluoroacetic acid, the volume of the organic solvent in the sample loading liquid is 10-50%, and the pH value is 0-7.
The eluent is a mixed solution of an organic solvent, organic acid and water, and the organic solvent is ethanol; the organic acid is trifluoroacetic acid, the volume of the organic solvent in the eluent is 10-50%, and the pH value is 10-12.
The leacheate is a mixed solution of an organic solvent, organic acid and water, and the organic solvent is ethanol; the organic acid is trifluoroacetic acid, the volume of the organic solvent in the leacheate is 0-50%, and the pH value is 0-7.
After the extraction of the DNA-rich material, the recovery rate of the DNA extraction was 82% and the coverage was 84%, as evaluated by the standards.
Example 2
A method for extracting circulating tumor DNA by SPE method, adopt and disperse the solid phase extraction mode, the circulating tumor DNA enrichment material used is the nanoparticle material of the metal core/carbon shell layer structure, prepare by the above-mentioned method, wherein the shell is gold nanoparticle; the method comprises the following specific steps:
1) Loading the circulating tumor DNA enrichment material into a pipette tip or a gel sample loading suction head to form a micro solid phase extraction column, activating and balancing the micro solid phase extraction column by adopting eluent and sample loading liquid, dissolving a biological sample in the sample loading liquid, and loading the biological sample on the micro solid phase extraction column, wherein the mass ratio of the circulating tumor DNA enrichment material to the biological sample is 1000;
2) Washing the extraction column with 5-200 times of eluate, wherein the pH of eluate is 0-7;
3) Eluting with 5-100 times column volume of eluent with pH of 10-12 to obtain DNA;
the whole process is carried out at 10 ℃.
The sample loading liquid is a mixed liquid of an organic solvent, organic acid and water, and the organic solvent is acetonitrile; the organic acid is formic acid; the volume of the organic solvent in the sample liquid is 10-50%, and the pH value is 0-7.
The eluent is a mixed solution of an organic solvent, organic acid and water, and the organic solvent is acetonitrile; the organic acid is formic acid, the volume of the organic solvent in the eluent is 10-50%, and the pH value is 10-12.
The leacheate is a mixed solution of an organic solvent, an organic acid and water, the organic solvent is acetonitrile, the organic acid is formic acid, the volume of the organic solvent in the leacheate is 0-50%, and the pH value is 0-7.
After the extraction of the DNA-rich material, the extraction recovery rate of DNA was 81% and the coverage was 83%, as evaluated by standards.
Example 3
A method for extracting circulating tumor DNA by SPE method, adopt and disperse the solid phase extraction mode, the circulating tumor DNA enrichment material used is the nanoparticle material of the metal core/carbon shell layer structure, prepare by the above-mentioned method, wherein the shell is gold nanoparticle; the method comprises the following specific steps:
1) Loading the circulating tumor DNA enrichment material into a pipette tip or a gel sample loading suction head to form a micro solid phase extraction column, activating and balancing the micro solid phase extraction column by adopting eluent and sample loading liquid, dissolving a biological sample in the sample loading liquid, and loading the biological sample onto the micro solid phase extraction column, wherein the mass ratio of the circulating tumor DNA enrichment material to the biological sample is 500;
2) Washing the extraction column with 5-200 times of eluate, wherein the pH of eluate is 0-7;
3) Eluting with 5-100 times column volume of eluent with pH of 10-12 to obtain DNA;
the whole process is carried out at 30 ℃.
The sample loading liquid is a mixed liquid of an organic solvent, organic acid and water, and the organic solvent is methanol; the organic acid is acetic acid, the volume of the organic solvent in the sample liquid is 10-50%, and the pH value is 0-7.
The eluent is a mixed solution of an organic solvent, organic acid and water, and the organic solvent is methanol; the organic acid is acetic acid, the volume of the organic solvent in the eluent is 10-50%, and the pH value is 10-12.
The leacheate is a mixed solution of an organic solvent, an organic acid and water, wherein the organic solvent is methanol; the organic acid is acetic acid, the volume of the organic solvent in the leacheate is 0-50%, and the pH value is 0-7.
After the extraction of the DNA-rich material, the extraction recovery rate of DNA was 83% and the coverage was 85%, as evaluated by standards.
Claims (5)
1. A method for extracting circulating tumor DNA by SPE method is characterized by adopting a dispersed solid phase extraction mode, and comprises the following steps:
1) Loading the circulating tumor DNA enrichment material into a pipette tip or a gel sample loading suction head to form a micro solid phase extraction column, activating and balancing the micro solid phase extraction column by adopting eluent and sample loading liquid, dissolving a biological sample in the sample loading liquid, and loading the biological sample on the micro solid phase extraction column, wherein the mass ratio of the circulating tumor DNA enrichment material to the biological sample is 100-1000;
2) Washing the extraction column with 5-200 times of eluate, wherein the pH of eluate is 0-7;
3) Eluting with 5-100 times column volume of eluent with pH of 10-12 to obtain circulating tumor DNA;
the whole process is carried out at 10-60 ℃;
the circulating tumor DNA enrichment material is a nano-particle material with a metal core/carbon shell structure; the core of the nano-particle material with the metal core/carbon shell structure is a metal nano-particle, and the structural formula of the shell of the nano-particle material is as follows:
the metal is Ag, ti, fe, ga, ni, au, pd or Pt.
2. The SPE method for extracting circulating tumor DNA according to claim 1, wherein the biological sample is blood, synovial fluid, cerebrospinal fluid or urine.
3. The SPE method for extracting circulating tumor DNA according to claim 1, wherein the sample loading solution is a mixture of an organic solvent, an organic acid and water, the organic solvent is acetonitrile, methanol or ethanol; the organic acid is formic acid, acetic acid or trifluoroacetic acid, the volume of the organic solvent in the sample loading liquid is 10-50%, and the pH value is 0-7.
4. The method for extracting circulating tumor DNA by SPE method according to claim 1, wherein the eluent is a mixture of organic solvent, organic acid and water, the organic solvent is acetonitrile, methanol or ethanol; the organic acid is formic acid, acetic acid or trifluoroacetic acid, and the volume of the organic solvent in the eluent is 10-50% and the pH value is 10-12.
5. The SPE method of claim 1, wherein the circulating tumor DNA is extracted by the SPE method: the leacheate is a mixed solution of an organic solvent, organic acid and water, and the organic solvent is acetonitrile, methanol or ethanol; the organic acid is formic acid, acetic acid or trifluoroacetic acid, the volume of the organic solvent in the leacheate is 0-50%, and the pH value is 0-7.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104513819A (en) * | 2014-12-17 | 2015-04-15 | 吉林大学 | Method for extracting DNA selectively |
| CN106749884A (en) * | 2016-11-18 | 2017-05-31 | 武汉理工大学 | A kind of phosphorylation peptide gathering material and preparation method and application |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104513819A (en) * | 2014-12-17 | 2015-04-15 | 吉林大学 | Method for extracting DNA selectively |
| CN106749884A (en) * | 2016-11-18 | 2017-05-31 | 武汉理工大学 | A kind of phosphorylation peptide gathering material and preparation method and application |
Non-Patent Citations (1)
| Title |
|---|
| Xiaoming Sun等.Colloidal Carbon Spheres and Their Core/Shell Structures with Noble-Metal Nanoparticles.《Angew.Chem.Int.Ed.》.2001,597-601. * |
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