CN103571826A - Efficient extraction method for whole blood genome DNA (deoxyribonucleic acid) - Google Patents
Efficient extraction method for whole blood genome DNA (deoxyribonucleic acid) Download PDFInfo
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- CN103571826A CN103571826A CN201310593112.8A CN201310593112A CN103571826A CN 103571826 A CN103571826 A CN 103571826A CN 201310593112 A CN201310593112 A CN 201310593112A CN 103571826 A CN103571826 A CN 103571826A
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Abstract
The invention relates to a method for quickly and efficiently extracting whole blood genome DNA (deoxyribonucleic acid), and belongs to the field of nucleic acid purification. The method comprises the following steps: adding a red cell lysis solution into whole blood, eddying until red cells are completely split, removing supernatant, and sucking all residual liquid; adding a white cell lysis solution and protease K, and splitting white cells in a water bath of 37 DEG C until the white cells are deposited and dissolved; adding sodium chloride and chloroform, carrying out centrifugal separation on DNA, and transferring the supernatant to another clean EP (epoxy) pipe; adding ammonium acetate and absolute ethanol for depositing DNA, washing by 75% of ethanol, naturally drying, and adding TE (Tris-EDTA(ethylene diamine tetraacetic acid)) for dissolving to obtain DNA. The method has the characteristics of high speed, high efficiency and non-toxicity. The extracted DNA can be used for various subsequent molecular biology studies such as polymerase chain reaction (PCR), methylation detection and gene cloning.
Description
Technical field
The present invention relates to a kind of quick, efficient Whole Blood Genomic DNA extracting method, belong to field of nucleic acid purification.
Background technology
At present, Molecular Biology and technical development are very rapid,, with the close combination of a plurality of subjects such as biology, medical science, genetics and zoology, obtaining high purity and complete genomic dna is the basis of carrying out subsequent molecular, is the prerequisite of other research work.Peripheral blood can be used as molecular marker in disease of hematopoietic system research, and for the exploitation of extracorporeal diagnostic system, also can be used for the molecular monitoring of the outer numerous systemic diseases of blood system.From peripheral blood, extract DNA, the research of carrying out nucleic acid level is the important research means of clinical molecule biology.
In the researchs such as heredopathia and tumour and cardiovascular and cerebrovascular disease, can utilize peripheral blood as biomaterial, detect the DNA difference between diseased individuals and normal health individuality, for example DNA methylation analysis and detection in Gene Mutation.Finding quick, economic DNA extraction method is the matter of utmost importance that colony's molecular biology experiment faces.Yet extracting one of difficult problem that DNA faces from blood is the pollution that DNA is vulnerable to many kinds of substance, and DNA purity is declined, and causes experimental result accuracy to reduce.Some traditional peripheral blood DNA extracting method is used the objectionable impuritiess such as saturated phenol, and complicated operation, and process is loaded down with trivial details, and the used time is long; Open automatization DNA extraction device, although do not re-use the objectionable impuritiess such as saturated phenol, operating process is also relatively simple, instrument and reagent are very expensive, are not suitable for the extraction of the extensive DNA of common lab.
Summary of the invention
The object of the present invention is to provide a kind of quick, high efficiency extraction methods for genomic DNA, guarantee that the genomic dna extracting is complete, and make leaching process efficient, quick, nontoxic.
For achieving the above object, the technical solution used in the present invention is:
A Whole Blood Genomic DNA extracting method, comprises the following steps:
(1) press whole blood: the volume ratio of erythrocyte cracked liquid=1:1, in the whole blood in EP pipe, add erythrocyte cracked liquid, vortex is standing more than 5 minutes after 2 minutes, until solution clear, then solution is carried out to centrifugation, rotating speed is 4000 revs/min, and centrifugation time is 2 minutes, abandoning supernatant;
(2) in the EP pipe of step (1), add the erythrocyte cracked liquid that accounts for volume of whole blood 1/3, vortex 1 minute, until precipitation is scattered completely, then carries out centrifugation to solution again, and rotating speed is 4000 revs/min, and disengaging time is 1 minute, abandoning supernatant; Repeat this step once, and blot residual liquid with thieving paper;
(3) in the EP pipe of step (2), add the white corpuscle diluent that accounts for volume of whole blood 1/3, separately add the Proteinase K that accounts for volume of whole blood 1/600, mix 37
oc water-bath 60-90 minute, until precipitation is dissolved completely;
(4) in the EP pipe of step (3), add respectively the 5 mol/L sodium chloride solutions that account for volume of whole blood 1/12 and the chloroform that accounts for volume of whole blood 2/3, fully shake up, then solution is carried out to centrifugation, centrifugal rotational speed is 12000 revs/min, centrifugation time is 5 minutes, and supernatant liquid is transferred in another clean EP pipe;
(5) to adding in the clean EP pipe of step (4), account for the Spirit of Mindererus of solution 1/10 volume and the ice ethanol of 2 times of volumes in pipe, fully shake up, precipitation DNA, then solution is carried out to centrifugation, the rotating speed of centrifugation is 4000 revs/min, and disengaging time is 1 minute, abandoning supernatant;
(6) in the clean EP pipe of step (5), add 75% ethanol that accounts for volume of whole blood 1/3, to DNA washing of precipitate, then carry out centrifugation, rotating speed is 12000 revs/min, and centrifugation time is 1 minute, abandoning supernatant; Repeat this step twice;
(7) the clean EP pipe of step (6) is stood upside down and is placed on the horizontal experiment table that is placed with thieving paper, seasoning 5 minutes;
(8) to the TE damping fluid adding in the clean EP pipe of step (7) with whole blood equal volume, dissolving DNA precipitation.
Adopt the present invention of technique scheme, compared with prior art, its advantage is:
1. the centrifugation time, the centrifuge speed that in the present invention's step, relate to, make the leaching process of genomic dna in whole blood have efficient, quick, easy feature;
2. the whole process of the present invention does not need to use the reagent of poisonous phenol, and all reagent costs are lower, makes whole process not only safe and reliable, and both economical;
2. use the genomic dna integrity of the inventive method purifying good, purity is high, can be used for polymerase chain reaction (PCR), real-time polymerase chain reaction (Real Time PCR), chip analysis, molecular cloning equimolecular biological experiment.
As preferably, the present invention further technical scheme is:
In described erythrocyte cracked liquid, the formula of each component is:
Tris-HCl (PH7.6 or 8.0) 0.01M
Sucrose 320mM
MgCl
2 5mM
Triton×100 1%。
In described write cell lysis buffer, the formula of each component is:
Tris-HCl (PH7.6 or 8.0) 0.01M
EDTA (PH8.0) 4mM
SDS 0.2%
NaCl 50mM。
In described TE damping fluid, the formula of each component is:
Tris-HCl (PH7.6 or 8.0) 1M
EDTA (PH8.0) 0.5M。
Embodiment
Below in conjunction with embodiment, the invention will be further described, and object is only to understand better content of the present invention.Therefore, the cited case does not limit the scope of the invention.
Embodiment 1: from people's whole blood, extract DNA.
(1) in 600 microlitre people whole bloods, add erythrocyte cracked liquid 600 microlitres, the volume ratio adding is whole blood: erythrocyte cracked liquid=1:1, and vortex 2 minutes is standing more than 5 minutes, until solution clear.Solution is carried out to centrifugation, and rotating speed is 4000 revs/min, and centrifugation time is 2 minutes, abandoning supernatant.Wherein erythrocyte cracked liquid is prepared as follows: get 10ml1MTris-HCl (PH7.6), 109.54g Sucrose, 1.01g MgCl
2, add 10ml Triton * 100, add water to 800ml, dissolve and be settled to 1000ml.
(2), to the erythrocyte cracked liquid that adds 200 microlitres in the EP pipe of step (1), vortex 1 minute, until precipitation is scattered completely.Solution is carried out to centrifugation, and rotating speed is 4000 revs/min, and disengaging time is 1 minute, abandons supernatant.Repeat once, and blot residual liquid with thieving paper.
(3) in the EP pipe of step (2), add 200 microlitre white corpuscle diluents, 1 microlitre Proteinase K, mixes 37
oc water-bath 60-90 minute, until precipitation is dissolved completely.Wherein write cell lysis buffer is prepared as follows: add 10ml 1MTris-HCl (PH7.6), and 8ml 0.5 M EDTA, 20ml 10% SDS, 10ml 5M NaCl, is settled to 1000ml.
(4) in the EP pipe of step (3), add 50 microlitre 5 mol/L sodium chloride solutions and 400 microlitre chloroforms, fully shake up.Solution is carried out to centrifugation, and centrifugal rotational speed is 12000 revs/min, and centrifugation time is 5 minutes.Supernatant liquid is transferred in another clean EP pipe.
(5) in the EP pipe of step (4), add 1/10 volume Spirit of Mindererus and 2 times of volume ice ethanol, fully shake up, precipitation DNA.Solution is carried out to centrifugation, and the rotating speed of centrifugation is 4000 revs/min, and disengaging time is 1 minute, abandons supernatant.
(6) in the EP pipe of step (5), add 200 milliliter of 75% ethanol, to DNA washing of precipitate, in 12000 revs/min centrifugal 1 minute, abandon supernatant.Repeat twice.
(7) the EP pipe of step (6) is stood upside down and places and be placed with on the horizontal experiment table of thieving paper seasoning 5 minutes.
(8) add 600 microlitre TE dissolving DNA precipitations.Wherein TE damping fluid is prepared as follows: get 10ml 1M Tris-HCl (PH8.6), 2ml 0.5 M EDTA, adds water to 1000ml.
The DNA extracting from 600 microlitre people whole bloods according to method described in embodiment 1, is detected and is found that DNA has a single bright wisp band at 23130bp place by its 1% agarose gel electrophoresis.
Embodiment 2: from the whole blood of mouse, extract DNA.
(1) in 600 microliters of mouse whole bloods, add erythrocyte cracked liquid 600 microlitres, the volume ratio adding is whole blood: erythrocyte cracked liquid=1:1, and vortex 2 minutes is standing more than 5 minutes, until solution clear.Solution is carried out to centrifugation, and rotating speed is 4000 revs/min, and centrifugation time is 2 minutes, abandoning supernatant.Erythrocyte cracked liquid wherein, its preparation method is: get 10ml1MTris-HCl (PH7.6), 109.54g Sucrose, 1.01g MgCl
2, add 10ml Triton * 100, add water to 800ml, dissolve and be settled to 1000ml.
(2), to the erythrocyte cracked liquid that adds 200 microlitres in the EP pipe of step (1), vortex 1 minute, until precipitation is scattered completely.Solution is carried out to centrifugation, and rotating speed is 4000 revs/min, and disengaging time is 1 minute, abandons supernatant.Repeat once, and blot residual liquid with thieving paper.
(3) in the EP pipe of step (2), add 200 microlitre white corpuscle diluents, 1 microlitre Proteinase K, mixes 37
oc water-bath 60-90 minute, until precipitation is dissolved completely.Write cell lysis buffer wherein, its preparation method is: add 10ml 1MTris-HCl (PH7.6), 8ml 0.5 M EDTA, 20ml 10% SDS, 10ml 5M NaCl, is settled to 1000ml.
(4) in the EP pipe of step (3), add 50 microlitre 5 mol/L sodium chloride solutions and 400 microlitre chloroforms, fully shake up.Solution is carried out to centrifugation, and centrifugal rotational speed is 12000 revs/min, and centrifugation time is 5 minutes.Supernatant liquid is transferred in another clean EP pipe.
(5) in the EP pipe of step (4), add 1/10 volume Spirit of Mindererus and 2 times of volume ice ethanol, fully shake up, precipitation DNA.Solution is carried out to centrifugation, and the rotating speed of centrifugation is 4000 revs/min, and disengaging time is 1 minute, abandons supernatant.
(6) in the EP pipe of step (5), add 200 milliliter of 75% ethanol, to DNA washing of precipitate, in 12000 revs/min centrifugal 1 minute, abandon supernatant.Repeat twice.
(7) the EP pipe of step (6) is stood upside down and places and be placed with on the horizontal experiment table of thieving paper seasoning 5 minutes.
(8) add 600 microlitre TE dissolving DNA precipitations.TE damping fluid wherein, its preparation method is: get 10ml 1M Tris-HCl (PH8.6), 2ml 0.5 M EDTA, adds water to 1000ml.
DNA according to method described in embodiment 2 from extracting from 600 microliters of mouse whole bloods, by its 1% agarose gel electrophoresis detect DNA at 23130bp place, have one single compared with bright band.
Claims (4)
1. an efficient Whole Blood Genomic DNA extracting method, is characterized in that, comprises the following steps:
(1) press whole blood: the volume ratio of erythrocyte cracked liquid=1:1, in the whole blood in EP pipe, add erythrocyte cracked liquid, vortex is standing more than 5 minutes after 2 minutes, until solution clear, then solution is carried out to centrifugation, rotating speed is 4000 revs/min, and centrifugation time is 2 minutes, abandoning supernatant;
(2) in the EP pipe of step (1), add the erythrocyte cracked liquid that accounts for volume of whole blood 1/3, vortex 1 minute, until precipitation is scattered completely, then carries out centrifugation to solution again, and rotating speed is 4000 revs/min, and disengaging time is 1 minute, abandoning supernatant; Repeat this step once, and blot residual liquid with thieving paper;
(3) in the EP pipe of step (2), add the white corpuscle diluent that accounts for volume of whole blood 1/3, separately add the Proteinase K that accounts for volume of whole blood 1/600, mix 37
oc water-bath 60-90 minute, until precipitation is dissolved completely;
(4) in the EP pipe of step (3), add respectively the 5 mol/L sodium chloride solutions that account for volume of whole blood 1/12 and the chloroform that accounts for volume of whole blood 2/3, fully shake up, then solution is carried out to centrifugation, centrifugal rotational speed is 12000 revs/min, centrifugation time is 5 minutes, and supernatant liquid is transferred in another clean EP pipe;
(5) to adding in the clean EP pipe of step (4), account for the Spirit of Mindererus of solution 1/10 volume and the ice ethanol of 2 times of volumes in pipe, fully shake up, precipitation DNA, then solution is carried out to centrifugation, the rotating speed of centrifugation is 4000 revs/min, and disengaging time is 1 minute, abandoning supernatant;
(6) in the clean EP pipe of step (5), add 75% ethanol that accounts for volume of whole blood 1/3, to DNA washing of precipitate, then carry out centrifugation, rotating speed is 12000 revs/min, and centrifugation time is 1 minute, abandoning supernatant; Repeat this step twice;
(7) the clean EP pipe of step (6) is stood upside down and is placed on the horizontal experiment table that is placed with thieving paper, seasoning 5 minutes;
(8) to the TE damping fluid adding in the clean EP pipe of step (7) with whole blood equal volume, dissolving DNA precipitation.
2. efficient Whole Blood Genomic DNA extracting method as claimed in claim 1, is characterized in that, in described erythrocyte cracked liquid, the formula of each component is:
Tris-HCl (PH7.6 or 8.0) 0.01M
Sucrose 320mM
MgCl
2 5mM
Triton×100 1%。
3. efficient Whole Blood Genomic DNA extracting method as claimed in claim 1, is characterized in that, in described write cell lysis buffer, the formula of each component is:
Tris-HCl (PH7.6 or 8.0) 0.01M
EDTA (PH8.0) 4mM
SDS 0.2%
NaCl 50mM。
4. efficient Whole Blood Genomic DNA extracting method as claimed in claim 1, is characterized in that, in described TE damping fluid, the formula of each component is:
Tris-HCl (PH7.6 or 8.0) 1M
EDTA (PH8.0) 0.5M。
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Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103898096A (en) * | 2014-03-27 | 2014-07-02 | 江苏佰龄全基因生物医学技术有限公司 | Mammal blood genome DNA extraction kit and method for extracting mammal blood genome DNA |
| CN104017800A (en) * | 2014-06-20 | 2014-09-03 | 益百尚(北京)生物技术有限责任公司 | Whole genome DNA (Deoxyribonucleic Acid) extraction kit for blood and method thereof |
| CN104109663A (en) * | 2014-05-13 | 2014-10-22 | 中国农业科学院特产研究所 | Simple and efficient animal blood DNA extraction method |
| CN105548568A (en) * | 2016-01-27 | 2016-05-04 | 中国人民解放军第三军医大学 | Method for detecting deoxyribonucleic acid (DNA)-protein cross link based on enzyme-linked immuno sorbent assay (ELISA) |
| CN105961375A (en) * | 2016-07-13 | 2016-09-28 | 北京中科唯新生物医学研究所有限公司 | Saliva preserving fluid, preparation method and usage thereof |
| CN107677823A (en) * | 2016-08-02 | 2018-02-09 | 北京金沐医疗科技有限公司 | A kind of protein chip kit and its detection method for being used to detect tumour antigen |
| CN107677820A (en) * | 2016-08-02 | 2018-02-09 | 北京金沐医疗科技有限公司 | A kind of kit and its detection method for detecting tumour antigen |
| CN109355286A (en) * | 2018-12-14 | 2019-02-19 | 长春市志昂生物科技有限公司 | A method of exempting from pre-treatment automation and extracts large volume whole blood DNA |
| CN109691432A (en) * | 2017-10-24 | 2019-04-30 | 深圳乐土生物科技有限公司 | A kind of buccal swab saves liquid and its preparation method and application |
| CN109706143A (en) * | 2019-02-28 | 2019-05-03 | 武汉华大医学检验所有限公司 | A kind of extracting method of peripheral blood high molecular weight genomic DNA |
| CN109852607A (en) * | 2018-12-30 | 2019-06-07 | 上海星耀医学科技发展有限公司 | It the reagent of erythroplastid and is applied in DNA extraction in a kind of removal biological sample |
| CN111304289A (en) * | 2020-02-21 | 2020-06-19 | 金陵科技学院 | DNA template preparation solution and DNA template preparation method |
| CN114317522A (en) * | 2021-12-16 | 2022-04-12 | 力因精准医疗产品（上海）有限公司 | Whole blood DNA extraction kit and nucleic acid extraction method |
| CN114350650A (en) * | 2021-12-16 | 2022-04-15 | 力因精准医疗产品（上海）有限公司 | Kit for extracting RNA from blood without DNA residue and method for extracting nucleic acid |
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2013
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Cited By (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103898096A (en) * | 2014-03-27 | 2014-07-02 | 江苏佰龄全基因生物医学技术有限公司 | Mammal blood genome DNA extraction kit and method for extracting mammal blood genome DNA |
| CN104109663A (en) * | 2014-05-13 | 2014-10-22 | 中国农业科学院特产研究所 | Simple and efficient animal blood DNA extraction method |
| CN104017800A (en) * | 2014-06-20 | 2014-09-03 | 益百尚(北京)生物技术有限责任公司 | Whole genome DNA (Deoxyribonucleic Acid) extraction kit for blood and method thereof |
| CN104017800B (en) * | 2014-06-20 | 2017-01-11 | 益百尚(北京)生物技术有限责任公司 | Whole genome DNA (Deoxyribonucleic Acid) extraction kit for blood and method thereof |
| CN105548568A (en) * | 2016-01-27 | 2016-05-04 | 中国人民解放军第三军医大学 | Method for detecting deoxyribonucleic acid (DNA)-protein cross link based on enzyme-linked immuno sorbent assay (ELISA) |
| CN105961375A (en) * | 2016-07-13 | 2016-09-28 | 北京中科唯新生物医学研究所有限公司 | Saliva preserving fluid, preparation method and usage thereof |
| CN107677823A (en) * | 2016-08-02 | 2018-02-09 | 北京金沐医疗科技有限公司 | A kind of protein chip kit and its detection method for being used to detect tumour antigen |
| CN107677820A (en) * | 2016-08-02 | 2018-02-09 | 北京金沐医疗科技有限公司 | A kind of kit and its detection method for detecting tumour antigen |
| CN109691432A (en) * | 2017-10-24 | 2019-04-30 | 深圳乐土生物科技有限公司 | A kind of buccal swab saves liquid and its preparation method and application |
| CN109355286A (en) * | 2018-12-14 | 2019-02-19 | 长春市志昂生物科技有限公司 | A method of exempting from pre-treatment automation and extracts large volume whole blood DNA |
| CN109852607A (en) * | 2018-12-30 | 2019-06-07 | 上海星耀医学科技发展有限公司 | It the reagent of erythroplastid and is applied in DNA extraction in a kind of removal biological sample |
| CN109706143A (en) * | 2019-02-28 | 2019-05-03 | 武汉华大医学检验所有限公司 | A kind of extracting method of peripheral blood high molecular weight genomic DNA |
| CN111304289A (en) * | 2020-02-21 | 2020-06-19 | 金陵科技学院 | DNA template preparation solution and DNA template preparation method |
| CN111304289B (en) * | 2020-02-21 | 2023-06-20 | 金陵科技学院 | DNA template preparation liquid and DNA template preparation method |
| CN114317522A (en) * | 2021-12-16 | 2022-04-12 | 力因精准医疗产品（上海）有限公司 | Whole blood DNA extraction kit and nucleic acid extraction method |
| CN114350650A (en) * | 2021-12-16 | 2022-04-15 | 力因精准医疗产品（上海）有限公司 | Kit for extracting RNA from blood without DNA residue and method for extracting nucleic acid |
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Application publication date: 20140212 |