CN102321613B - Extraction method of clotting genomic DNA (deoxyribonucleic acid) - Google Patents
Extraction method of clotting genomic DNA (deoxyribonucleic acid) Download PDFInfo
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- CN102321613B CN102321613B CN 201110259405 CN201110259405A CN102321613B CN 102321613 B CN102321613 B CN 102321613B CN 201110259405 CN201110259405 CN 201110259405 CN 201110259405 A CN201110259405 A CN 201110259405A CN 102321613 B CN102321613 B CN 102321613B
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Abstract
The invention provides an extraction method of clotting genomic DNA (deoxyribonucleic acid), which comprises the following steps that: nattokinase is added to a sample before extraction, and fibrin can be quickly decomposed by utilizing the thrombolytic force of the nattokinase several times more than that of a thrombolytic agent UK (urokinase), and further blood clots are separated, so that the blood clots become a homogeneous solution; and the obstacle of blood DNA extraction caused by sludged blood is removed, thereby the extraction speed of blood DNA is obviously improved, and simultaneously the purity and the yield of DNA product are greatly increased. The method provided by the invention is applicable to the extraction of DNA from sludged blood from a crime scene or dead bodies, the extraction of high-throughput blood gDNA and the extraction of blood gDNA in an antibody analysis experiment.
Description
Technical field
The present invention relates to the extracting method of genomic dna, be specifically related to a kind of method of from people's sludged blood, extracting genomic dna.
Background technology
Blood extracts DNA can be widely used in medical diagnosis on disease, gene analysis, identification etc.Check diagnosis assisting therapy develops rapidly medical science from traditional therapeutic treatment toward molecular biosciences.In the blood DNA leaching process, especially carry out in the high-throughput blood DNA leaching process, for various reasons, individual samples blood often can occur produce sludged blood.In addition, the residual blood sample of crime scene also can produce a large amount of sludged bloods for a long time owing to preserving.The sludged blood that produces will hinder carrying out smoothly of high-throughout blood extraction.Now general blood DNA extracts needs to adopt mechanical grinding method to grind sludged blood, then extracts blood DNA, consuming time like this, operation inconvenience and can't remove the residual sludged blood that produces because of fibrinogen fully, and experimental result is also had a greatly reduced quality.
Traditional sludged blood processing mode mainly contains following two kinds: 1) polishing centrifugal filtration process: sludged blood is ground to form the as far as possible solution of homogeneous, but because the scleroproein in the sludged blood is unusually obstinate, very difficult grinding, and grinding can cause a large amount of degradeds of blood DNA, causes dna fragmentation length greatly to reduce.2) washing agent method: adopt washing agent Triton x-100, remove red corpuscle and some albumen impurity as far as possible, again with the dissolving sludged blood that spends the night of chemical cracking liquid by force, but because the fibrinogen that thrombin action forms in Parenogen is to dissolve with general chemical process, therefore, this method also causes DNA yield and purity all undesirable.
Summary of the invention
The object of the present invention is to provide a kind of method of extracting genomic dna from sludged blood, contain the whole blood of sludged blood with solution when extracting genomic dna, DNA yield and purity is a undesirable difficult problem all.
Technical problem solved by the invention is achieved through the following technical solutions:
A kind of method of extracting genomic dna from sludged blood may further comprise the steps:
1) adding Nattokinase to final concentration in the blood clot is 40-80mg/ml, room temperature is placed 5min and is treated that sludged blood disappears, the Proteinase K that adds 1/10 times of sample volume, then lysis buffer (the lysate prescription: 20%SDS that adds 1 times of sample volume, 1%Triton-x100,100mM sodium-acetate, 50mMEDTA, PH5.2), vibration mixing;
2) 65 ℃ of lower temperature are bathed 10min, during the vibration mixing once, then add the dehydrated alcohol of 1.04 times of sample volumes, the vibration mixing;
3) the DNA adsorption column is inserted in the collection tube, sample liquid is added in the adsorption column, centrifugal 30 seconds of 12,000rpm outwells waste liquid;
4) add the lavation buffer solution of 2 times of sample volumes in the adsorption column, centrifugal 30 seconds of 12,000rpm, washing buffer liquid formula: 8M Guanidinium hydrochloride: ethanol=13: 17;
5) the DNA adsorption column is reinserted in the collection tube, add 70% ethanol of 2 times of sample volumes in the DNA adsorption column, centrifugal 30 seconds of 12,000rpm repeats above step 1 time;
6) 12, the centrifugal 1min of 000rpm inserts the DNA adsorption column in the centrifuge tube, adds the elutriant of 0.4-0.6 times of sample volume preheating in the adsorption column, and room temperature leaves standstill 2min;
7) 13, the centrifugal 1min eluted dna of 000rpm, wash-out obtains 60-70%DNA.
Preferably, described 1) add the RNase A of 0.02 times of sample volume in the step.
Preferably, described 1) adding Nattokinase to final concentration in the step is 60mg/ml.
Preferably, described 5) centrifugal selection is uncapped centrifugal in the step.
Preferably, described 6) Eluant temperature of preheating is 65 ℃ in the step.
Preferably, described 6) after step finished, the elutriant of again using 0.4 times of sample volume preheating is eluted dna again, and wash-out is gathered in the crops other 20% DNA.
The invention has the beneficial effects as follows:
The present invention adopts the method that adds Nattokinase in sample, because Nattokinase has the above thrombolysis power of thrombolytic agent UK several times, utilize its enzymic activity can promptly decompose fibrinogen, and then separation sludged blood, make sludged blood become the solution of homogeneous, removal causes the obstacle of blood DNA extraction because of sludged blood, thereby significantly improves the extraction rate of blood DNA, and the colleague increases purity and the output of DNA product greatly.
The inventive method is applicable to following situation:
1) crime scene gathers sludged blood: when the crime scene gathers sludged blood, because blood flow volume is little and all condensed into fritter, adopt the inventive method can utilize to greatest extent sludged blood to extract DNA, take full advantage of valuable material;
2) high-throughput blood gDNA extracts: occur owing to generally can be attended by a small amount of sludged blood in the high-throughput blood gDNA leaching process, may cause the DNA experiment to interrupt, or cause final purified product purity to cross low or yield descends greatly, adopt the inventive method to process and to remove sludged blood residual in the blood, guarantee to test and carry out smoothly;
3) adopt solidification blood from dead corpse: because the blood of dead corpse solidifies, gather its solidification blood with it after, adopt the inventive method to process and can propose smoothly blood gDNA;
4) blood gDNA extracts in the antibody analysis experiment: a lot of antibody extract in the blood of testing and do not add antithrombotics, then get blood supernatant Dispersal risk, cause blood coagulation, be difficult to carry out the extraction of genomic dna, can finely address this problem with the inventive method.
Description of drawings
Fig. 1 is for adopting the inventive method to extract the race glue figure of blood gDNA;
1, sample one is fresh sludged blood (serum is arranged) rabbit blood; 2, sample two is fresh sludged blood (serum-free) rabbit blood; 3, sample three is-20 ℃ of people's sludged bloods of preserving one month; 4, sample four is-20 ℃ of people's sludged bloods of preserving a week, and application of sample is 5ul.
Fig. 2 is for adopting mechanical milling method to extract the race glue figure of blood gDNA.
Embodiment
For technique means, creation characteristic that the present invention is realized, reach purpose and effect is easy to bright understanding, below in conjunction with concrete diagram, further set forth the present invention.
Embodiment 1:
Material source: Nattokinase, 40000Fu/g, river bend and reach company's purchase in Shanghai; Proteinase K adopts the sigma Proteinase K.
Sample source: sample one: fresh sludged blood (serum is arranged) rabbit blood; Sample two: fresh sludged blood (serum-free) rabbit blood; Sample three :-20 ℃ of people's sludged bloods of preserving one month; Sample four :-20 ℃ of people's sludged bloods of preserving a week.The sample collecting amount is 250 μ L, is handled as follows:
1, sample is transferred to the 1.5mL centrifuge tube, if the not enough 250ul of sample size adds PBS or TE Buffer and mends to 250 μ L, adds Nattokinase 20mg, room temperature is placed 5min and is treated that grumeleuse disappears; Add 25 μ L Proteinase Ks and 250 μ L lysates in centrifuge tube, the 10 seconds mixings that vibrate add 5 μ L RNase A; The lysate prescription is: 20%SDS, 1%Triton-x100,100mM sodium-acetate, 50mM EDTA, PH5.2;
2,65 ℃ of temperature were bathed 10 minutes, during the vibration mixing once; In lysate, add 260 μ L dehydrated alcohols, vibration mixing, the liquid that instantaneous centrifugal collection tube covers;
3, a DNA adsorption column is inserted the 2mL collection tube, sample liquid is added in the adsorption column, centrifugal 30 seconds of 12,000rpm outwells waste liquid;
4, in adsorption column, add 500 μ L lavation buffer solutions, centrifugal 30 seconds of 12,000rpm, washing buffer liquid formula: 8M Guanidinium hydrochloride: ethanol=13: 17;
5, add 500 μ L, 70% ethanol in the adsorption column, centrifugal 30 seconds of 12,000rpm abandons collection tube and waste liquid, repeats this step 1 time;
6,12,000rpm opened lid centrifugal 1 minute, and the DNA adsorption column is inserted a new 1.5ml centrifuge tube, added 100-150 μ L preheating (65 ℃) elutriant in adsorption column, room temperature leaves standstill 2-5 minute, or 65 ℃ of temperature baths can greatly improve yield in 3 minutes and remove residual ethanol;
7, 〉=13, centrifugal 1 minute eluted dna of 000rpm, wash-out can obtain 60-70%DNA usually for the first time, uses again eluted dna of 100ul preheating (65 ℃) elutriant, and the 2nd time wash-out can be gathered in the crops other 20% DNA.
The said extracted time is about half hour, adds the half hour of detection, all finishes in one hour altogether.
Extract DNA race glue and the results are shown in accompanying drawing 1; The race glue that adopts the conventional mechanical polishing to extract blood gDNA the results are shown in accompanying drawing 2.As a result, the DNA band is clear without conditions of streaking among Fig. 1, and the DNA band is not concentrated and obvious conditions of streaking is arranged among Fig. 2, illustrate that to adopt the DNA purity of the inventive method extraction higher.
The DNA experimental data that above-mentioned four kinds of samples adopt the inventive method to extract is as follows:
According to above-mentioned data analysis, all more than 9.3ug, the yield of sample one, sample two is slightly low for the yield of sample three, sample four, but also far above adopting the conventional mechanical polishing to extract the yield of blood gDNA.
Above demonstration and described ultimate principle of the present invention and principal character and advantage of the present invention.The technician of the industry should understand; the present invention is not restricted to the described embodiments; that describes in above-described embodiment and the specification sheets just illustrates principle of the present invention; without departing from the spirit and scope of the present invention; the present invention also has various changes and modifications, and these changes and improvements all fall in the claimed scope of the invention.The claimed scope of the present invention is defined by appending claims and equivalent.
Claims (5)
1. a method of extracting genomic dna from sludged blood is characterized in that, may further comprise the steps:
1) adding Nattokinase to final concentration in the blood clot is 40-80mg/ml, room temperature is placed 5min and is treated that sludged blood disappears, the Proteinase K that adds 1/10 times of sample volume, then the lysate damping fluid that adds 1 times of sample volume, described lysis buffer prescription is: 20%SDS, 1% Triton X-100, the 100mM sodium-acetate, 50mM EDTA, pH5.2, the RNase A of 0.02 times of sample volume of adding behind the vibration mixing;
2) 65 ℃ of lower temperature are bathed 10min, during the vibration mixing once, then add the dehydrated alcohol of 1.04 times of sample volumes, vibration mixing, the liquid that instantaneous centrifugal collection tube covers;
3) the DNA adsorption column is inserted in the collection tube, sample liquid is added in the adsorption column, centrifugal 30 seconds of 12,000rpm outwells waste liquid;
4) add the lavation buffer solution of 2 times of sample volumes in the adsorption column, centrifugal 30 seconds of 12,000rpm, washing buffer liquid formula: 8M Guanidinium hydrochloride: ethanol=13: 17;
5) the DNA adsorption column is reinserted in the collection tube, add 70% ethanol of 2 times of sample volumes in the DNA adsorption column, centrifugal 30 seconds of 12,000rpm repeats this step 1 time;
6) 12, the centrifugal 1min of 000rpm inserts the DNA adsorption column in the centrifuge tube, adds the elutriant of 0.4-0.6 times of sample volume preheating in the adsorption column, and room temperature leaves standstill 2min;
7) 13, the centrifugal 1min eluted dna of 000rpm, wash-out obtains 60-70%DNA.
2. a kind of method of extracting genomic dna from sludged blood according to claim 1 is characterized in that described 1) to add Nattokinase to final concentration in the step be 60mg/ml.
3. a kind of method of extracting genomic dna from sludged blood according to claim 1 is characterized in that described 6) in the step centrifugal selection uncap centrifugal.
4. a kind of method of extracting genomic dna from sludged blood according to claim 1 is characterized in that described 6) Eluant temperature of preheating is 65 ℃ in the step.
5. a kind of method of extracting genomic dna from sludged blood according to claim 1 is characterized in that described 7) after step finished, the elutriant of again using 0.4 times of sample volume preheating is eluted dna again, and wash-out is gathered in the crops other 20% DNA.
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| CN 201110259405 CN102321613B (en) | 2011-09-05 | 2011-09-05 | Extraction method of clotting genomic DNA (deoxyribonucleic acid) |
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| CN 201110259405 CN102321613B (en) | 2011-09-05 | 2011-09-05 | Extraction method of clotting genomic DNA (deoxyribonucleic acid) |
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Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104673914A (en) * | 2015-02-13 | 2015-06-03 | 重庆京因生物科技有限责任公司 | Cell lysis solution for rapid gene detection |
| CN105002159A (en) * | 2015-07-03 | 2015-10-28 | 陈立波 | DNA extraction kit used for extracting human body/animal blood and tissue DNA |
| CN106754871A (en) * | 2016-12-01 | 2017-05-31 | 厦门大学附属中山医院 | A kind of method of the DNA rapid extraction from clot |
| CN107245486A (en) * | 2017-06-21 | 2017-10-13 | 长沙金域医学检验所有限公司 | Poba gene group DNA extraction method |
| CN107904229A (en) * | 2017-07-25 | 2018-04-13 | 武汉菲思特生物科技有限公司 | Genome DNA extracting method and extracts kit |
| CN107988204A (en) * | 2017-11-24 | 2018-05-04 | 广州基迪奥生物科技有限公司 | A kind of whole blood DNA rapid extracting method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020068280A1 (en) * | 2000-12-06 | 2002-06-06 | Jeff Fairman | Compositions and methods for DNA purification from whole blood |
| CN101173274A (en) * | 2006-10-30 | 2008-05-07 | 张伟 | Bacterium and blood genome DNA extracting reagent kit |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020068280A1 (en) * | 2000-12-06 | 2002-06-06 | Jeff Fairman | Compositions and methods for DNA purification from whole blood |
| CN101173274A (en) * | 2006-10-30 | 2008-05-07 | 张伟 | Bacterium and blood genome DNA extracting reagent kit |
Non-Patent Citations (2)
| Title |
|---|
| Eszter Pais et al..Effects of nattokinase,a pro-fibrinolytic enzyme,on red blood cell aggregation and whole blood viscosity.《Clinical Hemorheology and Microcirculation》.2006,第35卷139-142. * |
| 曹果清等.酚/氯仿抽提法提取绵羊凝血块中基因组DNA.《安徽农业科学》.2009,第37卷(第34期),16771-16772. * |
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