CN108893525A - A kind of blood circulation Tumour DNA new method for extracting - Google Patents
A kind of blood circulation Tumour DNA new method for extracting Download PDFInfo
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- CN108893525A CN108893525A CN201810765822.7A CN201810765822A CN108893525A CN 108893525 A CN108893525 A CN 108893525A CN 201810765822 A CN201810765822 A CN 201810765822A CN 108893525 A CN108893525 A CN 108893525A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
Abstract
The present invention relates to bioscience technology fields, especially a kind of blood circulation Tumour DNA new method for extracting, including extracting blood sample, removal red blood cell, cracking leucocyte and digestible protein, removing protein, DNA absorption purification, DNA solution being gone to collect, erythrocyte cracked liquid is added into sample to be extracted, the red blood cell in sample to be extracted is cracked, is centrifugated, until red blood cell cracks completely, for extracting waste precipitating to remove red blood cell, erythrocyte cracked liquid includes following components：The trishydroxymethylaminomethane of the sodium chloride of 3-4mmol/L, 10-15g/L NH4Cl, 12-13mmol/L Tris-HCl and 0.02-0.03mol/L；The present invention Circulating tumor DNA, at low cost, ingenious in design, easy to operate, safety and environmental protection can be suitble to large-scale promotion application in rapidly extracting blood pressure.
Description
Technical field
The present invention relates to bioscience technology field more particularly to a kind of blood circulation Tumour DNA new method for extracting.
Background technique
There are episome group DNA in human plasma, these DNA moleculars are to be discharged into peripheral blood after Apoptosis or death
In, testing and analyzing free gDNA has critically important clinical meaning：Under many morbid states, such as apoplexy, myocardial infarction, glycosuria
Sick and many malignant tumor patient plasma free gDNA can be increased significantly.Therefore plasma free gDNA concentration can be used as disease into
One clinical indices of exhibition, it is more significant for the detection and analysis for the gDNA that dissociates in malignant tumor patient blood plasma, because
For these gDNA molecules directly from tumour cell, all information of malignancy can be obtained by carrying out genetic analysis to it,
Important foundation can be provided for the diagnosing and treating of tumour.Due to the free gDNA in maternal blood there are fetus, because
This, plasma free gDNA analyzes the important method for being also used as pre-natal diagnosis.
Since plasma free gDNA segment is small, content is low, is easily lost during the extraction process, and concentration is horizontal in ng/mL,
It can not be detected with ultraviolet spectrophotometry.Very high extraction efficiency is needed just to can apply to quantitative and related inspection to stable yield
It surveys.Traditional phenol/chloroform extraction process operator needs to contact these harmful substances of phenol, chloroform, influences human health.Also have
Patent uses silicon column absorption method, and principle can be extracted with silicon column reversible adsorption is collected using under the certain salinity of DNA molecular
Purify DNA.But this method extraction efficiency is lower, and it is cumbersome, dedicated DNA adsorption column is needed, can not be recycled, cost
It is high.
Therefore, it is necessary to a kind of circulating DNA extraction method, extraction efficiency is high, easy to operate, low in cost, safety collar
It protects.
Summary of the invention
The purpose of the present invention is to solve disadvantages existing in the prior art, and a kind of blood circulation tumour proposed
DNA new method for extracting.
To achieve the goals above, present invention employs following technical solutions：
Design a kind of blood circulation Tumour DNA new method for extracting, including to extract blood sample, removal red blood cell, cracking white
Cell and digestible protein go removing protein, DNA absorption purification, DNA solution to collect, and erythrocyte splitting is added into sample to be extracted
Liquid cracks the red blood cell in sample to be extracted, centrifuge separation, until red blood cell cracks completely, extracting waste is precipitated to remove red blood cell,
Erythrocyte cracked liquid includes following components：Sodium chloride, 10-15g/L NH4Cl, the 12-13mmol/L Tris- of 3-4mmol/L
The trishydroxymethylaminomethane of HCl and 0.02-0.03mol/L；
Cell pyrolysis liquid is added into white precipitate, cracks leucocyte and digestible protein, incubation reaction system, cell cracking
Liquid includes following component：Proclin300 20-30g/L,EDTA0.6-0.8g/L,SDS 20-30g/L,NaCl 5-10g/L,
Proteinase K 10g/L and urea 100-150g/L；
3mol/L sodium acetate solution is added to sodium acetate final concentration 0.3mol/L after removing removing protein, is added 2 times -20 DEG C of volume
The dehydrated alcohol of pre-cooling, 4 DEG C of 13000rpm are centrifuged 5min；Supernatant is abandoned, 70% ethanol washing of 2 times of -20 DEG C of volume pre-coolings is added
Precipitating, 4 DEG C of 13000rpm are centrifuged 5min, abandon supernatant, obtain DNA.
Preferably, it is as follows to extract blood sample process：1) it extracts 10ml venous blood and anti-coagulants is added；2) it turns upside down mixed
It is even, 5 DEG C of transports；3) venous blood is put into centrifuge, and 1500g, is centrifuged 12min by 4 DEG C；4) Aspirate supernatant is to new sterile 2.0ml
In centrifuge tube；5) 15000g, is centrifuged 12min by 4 DEG C；6) it sucts clearly into new sterile 2.0ml centrifuge tube.
Preferably, anti-coagulants is sodium citrate, Na2EDTA2H2O or heparin sodium；Sodium citrate final concentration of 0.38%,
Na2EDTA2H2O concentration is 1-1.3mg/mL.
Preferably, protein precipitant is added with precipitating proteins after cracking leucocyte and digestible protein, protein precipitant is：
1.5 parts of 5-sulphosalicylic acid, 80 parts of methanol and 22 parts of distilled water are made.
Preferably, the DNA drying at room temperature 15min obtained is placed on -20 DEG C or -80 DEG C long with elution buffer dissolving DNA
Phase saves.
A kind of blood circulation Tumour DNA new method for extracting proposed by the present invention, beneficial effect are：The present invention can be quick
Circulating tumor DNA in blood pressure is extracted, at low cost, ingenious in design, easy to operate, safety and environmental protection is suitble to large-scale promotion application.
Specific embodiment
The following is a clear and complete description of the technical scheme in the embodiments of the invention, it is clear that described embodiment
Only a part of the embodiment of the present invention, instead of all the embodiments.
Embodiment 1
A kind of blood circulation Tumour DNA new method for extracting, including extract blood sample, removal red blood cell, cracking leucocyte
And digestible protein, go removing protein, DNA absorption purification, DNA solution collect, erythrocyte cracked liquid is added into sample to be extracted, splits
The red blood cell in sample to be extracted is solved, is centrifugated, until red blood cell cracks completely, extracting waste precipitating is red thin to remove red blood cell
Cellular lysate liquid includes following components：Sodium chloride, 10g/L NH4Cl, 12mmol/LTris-HCl and the 0.02mol/L of 3mmol/L
Trishydroxymethylaminomethane；
Cell pyrolysis liquid is added into white precipitate, cracks leucocyte and digestible protein, incubation reaction system, cell cracking
Liquid includes following component：Proclin300 20g/L, EDTA 0.6g/L, SDS 20g/L, NaCl 5g/L, Proteinase K 10g/L
With urea 100g/L；
3mol/L sodium acetate solution is added to sodium acetate final concentration 0.3mol/L after removing removing protein, is added 2 times -20 DEG C of volume
The dehydrated alcohol of pre-cooling, 4 DEG C of 13000rpm are centrifuged 5min；Supernatant is abandoned, 70% ethanol washing of 2 times of -20 DEG C of volume pre-coolings is added
Precipitating, 4 DEG C of 13000rpm are centrifuged 5min, abandon supernatant, obtain DNA.
It is as follows to extract blood sample process：1) it extracts 10ml venous blood and anti-coagulants is added；2) it turns upside down mixing, 5 DEG C of fortune
It is defeated；3) venous blood is put into centrifuge, and 1500g, is centrifuged 12min by 4 DEG C；4) Aspirate supernatant is to new sterile 2.0ml centrifuge tube
In；5) 15000g, is centrifuged 12min by 4 DEG C；6) it sucts clearly into new sterile 2.0ml centrifuge tube.
Anti-coagulants is sodium citrate, Na2EDTA2H2O or heparin sodium；Sodium citrate final concentration of 0.38%,
Na2EDTA2H2O concentration is 1mg/mL.
Protein precipitant is added with precipitating proteins after cracking leucocyte and digestible protein, protein precipitant is：5- sulfo group
1.5 parts of salicylic acid, 80 parts of methanol and 22 parts of distilled water are made.
Obtained DNA drying at room temperature 15min is placed on -20 DEG C or -80 DEG C of long-term preservations with elution buffer dissolving DNA.
Embodiment 2
A kind of blood circulation Tumour DNA new method for extracting, including extract blood sample, removal red blood cell, cracking leucocyte
And digestible protein, go removing protein, DNA absorption purification, DNA solution collect, erythrocyte cracked liquid is added into sample to be extracted, splits
The red blood cell in sample to be extracted is solved, is centrifugated, until red blood cell cracks completely, extracting waste precipitating is red thin to remove red blood cell
Cellular lysate liquid includes following components：The sodium chloride of 3.2mmol/L, 12g/L NH4Cl, 12.5mmol/L Tris-HCl and
The trishydroxymethylaminomethane of 0.025mol/L；
Cell pyrolysis liquid is added into white precipitate, cracks leucocyte and digestible protein, incubation reaction system, cell cracking
Liquid includes following component：Proclin300 22g/L, EDTA 0.7g/L, SDS 22g/L, NaCl 6g/L, Proteinase K 10g/L
With urea 120g/L；
3mol/L sodium acetate solution is added to sodium acetate final concentration 0.3mol/L after removing removing protein, is added 2 times -20 DEG C of volume
The dehydrated alcohol of pre-cooling, 4 DEG C of 13000rpm are centrifuged 5min；Supernatant is abandoned, 70% ethanol washing of 2 times of -20 DEG C of volume pre-coolings is added
Precipitating, 4 DEG C of 13000rpm are centrifuged 5min, abandon supernatant, obtain DNA.
It is as follows to extract blood sample process：1) it extracts 10ml venous blood and anti-coagulants is added；2) it turns upside down mixing, 5 DEG C of fortune
It is defeated；3) venous blood is put into centrifuge, and 1500g, is centrifuged 12min by 4 DEG C；4) Aspirate supernatant is to new sterile 2.0ml centrifuge tube
In；5) 15000g, is centrifuged 12min by 4 DEG C；6) it sucts clearly into new sterile 2.0ml centrifuge tube.
Anti-coagulants is sodium citrate, Na2EDTA2H2O or heparin sodium；Sodium citrate final concentration of 0.38%,
Na2EDTA2H2O concentration is 1.2mg/mL.
Protein precipitant is added with precipitating proteins after cracking leucocyte and digestible protein, protein precipitant is：5- sulfo group
1.5 parts of salicylic acid, 80 parts of methanol and 22 parts of distilled water are made.
Obtained DNA drying at room temperature 15min is placed on -20 DEG C or -80 DEG C of long-term preservations with elution buffer dissolving DNA.
Embodiment 3
A kind of blood circulation Tumour DNA new method for extracting, including extract blood sample, removal red blood cell, cracking leucocyte
And digestible protein, go removing protein, DNA absorption purification, DNA solution collect, erythrocyte cracked liquid is added into sample to be extracted, splits
The red blood cell in sample to be extracted is solved, is centrifugated, until red blood cell cracks completely, extracting waste precipitating is red thin to remove red blood cell
Cellular lysate liquid includes following components：The sodium chloride of 3.5mmol/L, 13g/L NH4Cl, 12.5mmol/L Tris-HCl and
The trishydroxymethylaminomethane of 0.025mol/L；
Cell pyrolysis liquid is added into white precipitate, cracks leucocyte and digestible protein, incubation reaction system, cell cracking
Liquid includes following component：Proclin300 25g/L, EDTA 0.7g/L, SDS 25g/L, NaCl 8g/L, Proteinase K 10g/L
With urea 135g/L；
3mol/L sodium acetate solution is added to sodium acetate final concentration 0.3mol/L after removing removing protein, is added 2 times -20 DEG C of volume
The dehydrated alcohol of pre-cooling, 4 DEG C of 13000rpm are centrifuged 5min；Supernatant is abandoned, 70% ethanol washing of 2 times of -20 DEG C of volume pre-coolings is added
Precipitating, 4 DEG C of 13000rpm are centrifuged 5min, abandon supernatant, obtain DNA.
It is as follows to extract blood sample process：1) it extracts 10ml venous blood and anti-coagulants is added；2) it turns upside down mixing, 5 DEG C of fortune
It is defeated；3) venous blood is put into centrifuge, and 1500g, is centrifuged 12min by 4 DEG C；4) Aspirate supernatant is to new sterile 2.0ml centrifuge tube
In；5) 15000g, is centrifuged 12min by 4 DEG C；6) it sucts clearly into new sterile 2.0ml centrifuge tube.
Anti-coagulants is sodium citrate, Na2EDTA2H2O or heparin sodium；Sodium citrate final concentration of 0.38%,
Na2EDTA2H2O concentration is 1.2mg/mL.
Protein precipitant is added with precipitating proteins after cracking leucocyte and digestible protein, protein precipitant is：5- sulfo group
1.5 parts of salicylic acid, 80 parts of methanol and 22 parts of distilled water are made.
Obtained DNA drying at room temperature 15min is placed on -20 DEG C or -80 DEG C of long-term preservations with elution buffer dissolving DNA.
Embodiment 4
A kind of blood circulation Tumour DNA new method for extracting, including extract blood sample, removal red blood cell, cracking leucocyte
And digestible protein, go removing protein, DNA absorption purification, DNA solution collect, erythrocyte cracked liquid is added into sample to be extracted, splits
The red blood cell in sample to be extracted is solved, is centrifugated, until red blood cell cracks completely, extracting waste precipitating is red thin to remove red blood cell
Cellular lysate liquid includes following components：Sodium chloride, 15g/L NH4Cl, 13mmol/LTris-HCl and the 0.03mol/L of 4mmol/L
Trishydroxymethylaminomethane；
Cell pyrolysis liquid is added into white precipitate, cracks leucocyte and digestible protein, incubation reaction system, cell cracking
Liquid includes following component：Proclin300 30g/L, EDTA 0.8g/L, SDS 30g/L, NaCl 10g/L, Proteinase K 10g/
L and urea 150g/L；
3mol/L sodium acetate solution is added to sodium acetate final concentration 0.3mol/L after removing removing protein, is added 2 times -20 DEG C of volume
The dehydrated alcohol of pre-cooling, 4 DEG C of 13000rpm are centrifuged 5min；Supernatant is abandoned, 70% ethanol washing of 2 times of -20 DEG C of volume pre-coolings is added
Precipitating, 4 DEG C of 13000rpm are centrifuged 5min, abandon supernatant, obtain DNA.
It is as follows to extract blood sample process：1) it extracts 10ml venous blood and anti-coagulants is added；2) it turns upside down mixing, 5 DEG C of fortune
It is defeated；3) venous blood is put into centrifuge, and 1500g, is centrifuged 12min by 4 DEG C；4) Aspirate supernatant is to new sterile 2.0ml centrifuge tube
In；5) 15000g, is centrifuged 12min by 4 DEG C；6) it sucts clearly into new sterile 2.0ml centrifuge tube.
Anti-coagulants is sodium citrate, Na2EDTA2H2O or heparin sodium；Sodium citrate final concentration of 0.38%,
Na2EDTA2H2O concentration is 1.3mg/mL.
Protein precipitant is added with precipitating proteins after cracking leucocyte and digestible protein, protein precipitant is：5- sulfo group
1.5 parts of salicylic acid, 80 parts of methanol and 22 parts of distilled water are made.
Obtained DNA drying at room temperature 15min is placed on -20 DEG C or -80 DEG C of long-term preservations with elution buffer dissolving DNA.
The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto,
Anyone skilled in the art in the technical scope disclosed by the present invention, according to the technique and scheme of the present invention and its
Inventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.
Claims (5)
1. a kind of blood circulation Tumour DNA new method for extracting, including extract blood sample, removal red blood cell, cracking leucocyte and
Digestible protein goes removing protein, DNA absorption purification, DNA solution to collect, it is characterised in that：Red blood cell is added into sample to be extracted
Lysate cracks the red blood cell in sample to be extracted, centrifuge separation, until red blood cell cracks completely, extracting waste precipitating is red to remove
Cell, erythrocyte cracked liquid include following components：Sodium chloride, 10-15g/L NH4Cl, the 12-13mmol/L of 3-4mmol/L
The trishydroxymethylaminomethane of Tris-HCl and 0.02-0.03mol/L；
Cell pyrolysis liquid is added into white precipitate, cracks leucocyte and digestible protein, incubation reaction system, cell pyrolysis liquid packet
Containing following component：Proclin300 20-30g/L, EDTA 0.6-0.8g/L, SDS 20-30g/L, NaCl 5-10g/L, albumen
Enzyme K 10g/L and urea 100-150g/L；
3mol/L sodium acetate solution is added to sodium acetate final concentration 0.3mol/L after removing removing protein, 2 times of -20 DEG C of volume pre-coolings are added
Dehydrated alcohol, 4 DEG C of 13000rpm are centrifuged 5min；Supernatant is abandoned, the 70% ethanol washing precipitating of 2 times of -20 DEG C of volume pre-coolings is added,
4 DEG C of 13000rpm are centrifuged 5min, abandon supernatant, obtain DNA.
2. a kind of blood circulation Tumour DNA new method for extracting according to claim 1, which is characterized in that extract blood sample
This process is as follows：1) it extracts 10ml venous blood and anti-coagulants is added；2) it turns upside down mixing, 5 DEG C of transports；3) venous blood is put into centrifugation
Machine, 1500g, are centrifuged 12min by 4 DEG C；4) Aspirate supernatant is into new sterile 2.0ml centrifuge tube；5) 15000g, 4 DEG C, centrifugation
12min；6) it sucts clearly into new sterile 2.0ml centrifuge tube.
3. a kind of blood circulation Tumour DNA new method for extracting according to claim 2, it is characterised in that：Anti-coagulants is Chinese holly
Rafter acid sodium, Na2EDTA2H2O or heparin sodium；Final concentration of 0.38%, the Na2EDTA2H2O concentration of sodium citrate is 1-
1.3mg/mL。
4. a kind of blood circulation Tumour DNA new method for extracting according to claim 1, it is characterised in that：Crack leucocyte
And protein precipitant is added after digestible protein with precipitating proteins, protein precipitant is：1.5 parts of 5-sulphosalicylic acid, methanol 80
Part is made with 22 parts of distilled water.
5. a kind of blood circulation Tumour DNA new method for extracting according to claim 1, it is characterised in that：The obtained room DNA
The dry 15min of temperature is placed on -20 DEG C or -80 DEG C of long-term preservations with elution buffer dissolving DNA.
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Cited By (1)
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| CN113234717A (en) * | 2021-04-12 | 2021-08-10 | 南京医科大学附属南京医院 | Separation and enrichment method of circulating tumor DNA |
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