CN109929830A - A kind of method that SPE method extracts Circulating tumor DNA - Google Patents
A kind of method that SPE method extracts Circulating tumor DNA Download PDFInfo
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- CN109929830A CN109929830A CN201711364691.3A CN201711364691A CN109929830A CN 109929830 A CN109929830 A CN 109929830A CN 201711364691 A CN201711364691 A CN 201711364691A CN 109929830 A CN109929830 A CN 109929830A
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- circulating tumor
- tumor dna
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Abstract
The present invention provides a kind of method that SPE method extracts Circulating tumor DNA, belongs to field of biotechnology.Specific step is as follows by the present invention: 1) Circulating tumor DNA enrichment material being fitted into liquid-transfering gun pipette tips or gel loading suction nozzle, form micro- solid-phase extraction column, using eluent and sample solution activation and balance micro- solid-phase extraction column, biological sample is dissolved in sample solution, to on micro- solid-phase extraction column on and, the mass ratio of Circulating tumor DNA enrichment material and biological sample is 100~1000:1；2) extraction column, leacheate pH 0-7 are rinsed using the leacheate of 5-200 times of column volume；3) Circulating tumor DNA, eluent pH 10-12 are obtained using the elution of 5-100 times of column volume；The DNA enrichment material is metal inner core/carbon shell structurre nano-particle material.The DNA enrichment material is metal inner core/carbon shell structurre nano particle.Method of the invention shows the features such as highly selective and high-throughput in terms of extracting Circulating tumor DNA, and efficiently separating and being enriched with for Circulating tumor DNA may be implemented.
Description
Technical field
The present invention relates to a kind of methods that field of biotechnology more particularly to SPE method extract Circulating tumor DNA.
Background technique
Circulating tumor DNA, i.e. ctDNA (circulating tumor DNA), refer to tumour cell by Apoptosis or
The DNA fragmentation that person's necrosis generates discharges into the circulatory system.CtDNA is present in the body fluid such as blood, synovial fluid and cerebrospinal fluid.By
It is 1 hour or so in ctDNA half-life period, can accurately reflect tumour present case, so ctDNA can be used as the non-of tissue biopsy
Invasive alternative solution.In addition, ctDNA plays a role in early diagnosis of cancer, prognosis and measurement drug therapy reaction.
The ctDNA mutation detection techniques of mainstream include based on PCR amplification technique and based on DNA sequence dna detection technique two at present
Class.But whether which kind of sequencing technologies, requires all to be enriched with DNA and extracted, to reduce to amplification or subsequent sequencing
Interference.
The main extracting method of ctDNA is mainly based upon silicon ball, the solid phase extraction method of magnetic ball and based on phenol-chlorine
Imitative liquid-liquid extraction method.Main problem is extraction efficiency and property again.
Summary of the invention
In view of the above-mentioned problems, the present invention provides a kind of method that SPE method extracts Circulating tumor DNA.
A kind of method that SPE method extracts Circulating tumor DNA, using dispersive solid-phase extraction mode, the specific steps are as follows:
1) Circulating tumor DNA enrichment material is fitted into liquid-transfering gun pipette tips or gel loading suction nozzle, forms micro- solid phase extraction
Take column, activated using eluent and sample solution and balance micro- solid-phase extraction column, biological sample is dissolved in sample solution, and on arrive
On micro- solid-phase extraction column, the mass ratio of Circulating tumor DNA enrichment material and biological sample is 100~1000:1；
2) extraction column, leacheate pH 0-7 are rinsed using the leacheate of 5-200 times of column volume；
3) Circulating tumor DNA, eluent pH 10-12 are obtained using the elution of 5-100 times of column volume；
Above-mentioned whole process is carried out at 10-60 DEG C；
The Circulating tumor DNA enrichment material is metal inner core/carbon shell structurre nano-particle material.
The metal inner core/carbon shell structurre nano-particle material, kernel are metal nanoparticle, the knot of shell
Structure formula 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 solution is the mixed liquor of organic solvent, organic acid and water, and organic solvent is acetonitrile, methanol or ethyl alcohol；Have
Machine acid is formic acid, acetic acid or trifluoroacetic acid, and organic solvent volume is 10-50%, pH 0-7 in the sample solution.
The eluent is the mixed liquor of organic solvent, organic acid and water, and organic solvent is acetonitrile, methanol or ethyl alcohol；Have
Machine acid is formic acid, acetic acid or trifluoroacetic acid, and organic solvent volume is 10-50%, pH 10-12 in the eluent.
The leacheate is the mixed liquor of organic solvent, organic acid and water, and organic solvent is acetonitrile, methanol or ethyl alcohol；Have
Machine acid is formic acid, acetic acid or trifluoroacetic acid, and the volume of organic solvent is 0-50%, pH 0-7 in the leacheate.
The advantages and benefits of the present invention are: method of the invention shows height in terms of extracting Circulating tumor DNA
The features such as selectivity and high throughput, efficiently separating and being enriched with for Circulating tumor DNA may be implemented.
Detailed description of the invention
Fig. 1 is that metal inner core of the present invention/carbon shell structurre nano-particle material prepares schematic diagram.
Specific embodiment
The noble metal nano particles of core/shell structure of the present invention use " Colloidal Carbon Spheres
and Their Core/ShellStructures with Noble-Metal Nanoparticles》
Angew.Chem.2004, the preparation method in 116,607-611 are prepared, and the process of preparation is as shown in Figure 1.It prepares
Kernel is Ag, Ti, Fe, Ga, Ni, Au, metal inner core/carbon shell structurre nano-particle material of Pd or Pt nano particle；Its
The structural formula of shell is as follows:
Embodiment 1
A kind of method that SPE method extracts Circulating tumor DNA, using dispersive solid-phase extraction mode, Circulating tumor DNA used
Enrichment material is metal inner core/carbon shell structurre nano-particle material, is prepared using the above method, and shell therein is gold
Nano particle；, the specific steps are as follows:
1) Circulating tumor DNA enrichment material is fitted into liquid-transfering gun pipette tips or gel loading suction nozzle, forms micro- solid phase extraction
Take column, activated using eluent and sample solution and balance micro- solid-phase extraction column, biological sample is dissolved in sample solution, and on arrive
On micro- solid-phase extraction column, the mass ratio of Circulating tumor DNA enrichment material and biological sample is 100:1；
2) extraction column, leacheate pH 0-7 are rinsed using the leacheate of 5-200 times of column volume；
3) DNA, eluent pH 10-12 are obtained using the elution of 5-100 times of column volume；
Above-mentioned whole process is carried out at 60 DEG C.
The sample solution is the mixed liquor of organic solvent, organic acid and water, and organic solvent is ethyl alcohol；Organic acid is trifluoro second
Acid, organic solvent volume is 10-50%, pH 0-7 in the sample solution.
The eluent is the mixed liquor of organic solvent, organic acid and water, and organic solvent is ethyl alcohol；Organic acid is trifluoro second
Acid, organic solvent volume is 10-50%, pH 10-12 in the eluent.
The leacheate is the mixed liquor of organic solvent, organic acid and water, and organic solvent is ethyl alcohol；Organic acid is trifluoro second
Acid, the volume of organic solvent is 0-50%, pH 0-7 in the leacheate.
It is evaluated by standard items, after the extraction of ability DNA enrichment material, the extraction recovery of DNA is 82%, and coverage is
84%.
Embodiment 2
A kind of method that SPE method extracts Circulating tumor DNA, using dispersive solid-phase extraction mode, Circulating tumor DNA used
Enrichment material is metal inner core/carbon shell structurre nano-particle material, is prepared using the above method, and shell therein is gold
Nano particle；Specific step is as follows:
1) Circulating tumor DNA enrichment material is fitted into liquid-transfering gun pipette tips or gel loading suction nozzle, forms micro- solid phase extraction
Take column, activated using eluent and sample solution and balance micro- solid-phase extraction column, biological sample is dissolved in sample solution, and on arrive
On micro- solid-phase extraction column, the mass ratio of Circulating tumor DNA enrichment material and biological sample is 1000:1；
2) extraction column, leacheate pH 0-7 are rinsed using the leacheate of 5-200 times of column volume；
3) DNA, eluent pH 10-12 are obtained using the elution of 5-100 times of column volume；
Above-mentioned whole process is carried out at 10 DEG C.
The sample solution is the mixed liquor of organic solvent, organic acid and water, and organic solvent is acetonitrile；Organic acid is formic acid；
Organic solvent volume is 10-50%, pH 0-7 in the sample solution.
The eluent is the mixed liquor of organic solvent, organic acid and water, and organic solvent is acetonitrile；Organic acid is formic acid,
Organic solvent volume is 10-50%, pH 10-12 in the eluent.
The leacheate is the mixed liquor of organic solvent, organic acid and water, and organic solvent is that acetonitrile organic acid is formic acid, institute
The volume for stating organic solvent in leacheate is 0-50%, pH 0-7.
It is evaluated by standard items, after the extraction of ability DNA enrichment material, the extraction recovery of DNA is 81%, and coverage is
83%.
Embodiment 3
A kind of method that SPE method extracts Circulating tumor DNA, using dispersive solid-phase extraction mode, Circulating tumor DNA used
Enrichment material is metal inner core/carbon shell structurre nano-particle material, is prepared using the above method, and shell therein is gold
Nano particle；Specific step is as follows:
1) Circulating tumor DNA enrichment material is fitted into liquid-transfering gun pipette tips or gel loading suction nozzle, forms micro- solid phase extraction
Take column, activated using eluent and sample solution and balance micro- solid-phase extraction column, biological sample is dissolved in sample solution, and on arrive
On micro- solid-phase extraction column, the mass ratio of Circulating tumor DNA enrichment material and biological sample is 500:1；
2) extraction column, leacheate pH 0-7 are rinsed using the leacheate of 5-200 times of column volume；
3) DNA, eluent pH 10-12 are obtained using the elution of 5-100 times of column volume；
Above-mentioned whole process is carried out at 30 DEG C.
The sample solution is the mixed liquor of organic solvent, organic acid and water, and organic solvent is methanol；Organic acid is acetic acid,
Organic solvent volume is 10-50%, pH 0-7 in the sample solution.
The eluent is the mixed liquor of organic solvent, organic acid and water, and organic solvent is methanol；Organic acid is acetic acid,
Organic solvent volume is 10-50%, pH 10-12 in the eluent.
The leacheate is the mixed liquor of organic solvent, organic acid and water, and organic solvent is methanol；Organic acid is acetic acid,
The volume of organic solvent is 0-50%, pH 0-7 in the leacheate.
It is evaluated by standard items, after the extraction of ability DNA enrichment material, the extraction recovery of DNA is 83%, and coverage is
85%.
Claims (7)
1. a kind of method that SPE method extracts Circulating tumor DNA, it is characterised in that use dispersive solid-phase extraction mode, specific steps
It is as follows:
1) Circulating tumor DNA enrichment material is fitted into liquid-transfering gun pipette tips or gel loading suction nozzle, forms micro- solid-phase extraction column,
Using eluent and sample solution activation and micro- solid-phase extraction column is balanced, biological sample is dissolved in sample solution, and is upper to micro- solid
On phase extraction column, the mass ratio of Circulating tumor DNA enrichment material and biological sample is 100~1000:1；
2) extraction column, leacheate pH 0-7 are rinsed using the leacheate of 5-200 times of column volume；
3) Circulating tumor DNA, eluent pH 10-12 are obtained using the elution of 5-100 times of column volume；
Above-mentioned whole process is carried out at 10-60 DEG C；
The Circulating tumor DNA enrichment material is metal inner core/carbon shell structurre nano-particle material.
2. the method that a kind of SPE method according to claim 1 extracts Circulating tumor DNA, it is characterised in that: in the metal
Core/carbon shell structurre nano-particle material, kernel are metal nanoparticle, and the structural formula of shell is as follows:
3. the method that a kind of SPE method according to claim 2 extracts Circulating tumor DNA, it is characterised in that the metal is
Ag, Ti, Fe, Ga, Ni, Au, Pd or Pt.
4. the method that a kind of SPE method according to claim 1 extracts Circulating tumor DNA, it is characterised in that the biology sample
Product are blood, synovial fluid, cerebrospinal fluid or urine.
5. the method that a kind of SPE method according to claim 1 extracts Circulating tumor DNA, it is characterised in that the sample solution
For the mixed liquor of organic solvent, organic acid and water, organic solvent is acetonitrile, methanol or ethyl alcohol；Organic acid is formic acid, acetic acid or three
Fluoroacetic acid, organic solvent volume is 10-50%, pH 0-7 in the sample solution.
6. the method that a kind of SPE method according to claim 1 extracts Circulating tumor DNA, it is characterised in that the eluent
For the mixed liquor of organic solvent, organic acid and water, organic solvent is acetonitrile, methanol or ethyl alcohol；Organic acid is formic acid, acetic acid or three
Fluoroacetic acid, organic solvent volume is 10-50%, pH 10-12 in the eluent.
7. the method that a kind of SPE method according to claim 1 extracts Circulating tumor DNA, it is characterised in that: the leacheate
For the mixed liquor of organic solvent, organic acid and water, organic solvent is acetonitrile, methanol or ethyl alcohol；Organic acid is formic acid, acetic acid or three
Fluoroacetic acid, the volume of organic solvent is 0-50%, pH 0-7 in the leacheate.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201711364691.3A CN109929830B (en) | 2017-12-18 | 2017-12-18 | Method for extracting circulating tumor DNA by SPE method |
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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 |
-
2017
- 2017-12-18 CN CN201711364691.3A patent/CN109929830B/en active Active
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.》, 31 December 2001 (2001-12-31), pages 598 * |
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