CN106754869A - A kind of extracting method of poba gene group DNA - Google Patents
A kind of extracting method of poba gene group DNA Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
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- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
Abstract
The invention provides a kind of extracting method of genomic DNA, the method is improved to traditional phenol chloroform method, significantly improves the purity and content of put forward DNA, and operation is relatively easy, convenient and with low cost, environment-friendly.
Description
Technical field
The invention belongs to field of medical molecular biology, a kind of quick, high efficiency extraction human peripheral's blood is specifically provided
The method of genomic DNA.
Background technology
Protocols in Molecular Biology has penetrated into biology, preclinical medicine and clinical medicine many aspects.Human genome
The extraction of DNA is a basic technology of molecular biology research, is also that molecular biology beginner and clinician are intended to carry out
Various molecular biology correlative studys enter gate technique.
Genomic DNA as inhereditary material carrier, quick, economical, safe efficiently from clinical human's blood preparation
The genomic DNA of high yield, high-purity is extracted, for the gene diagnosis of genetic disease, especially by maternal peripheral blood
The early stage of liquid diagnosing fetal genetic disease and sex identification, tumour and communicable disease etc. makes a definite diagnosis, and other molecules
Biological experiment all it is critical that.Used as the committed step of gene studies, the extraction and purifying of genomic DNA always are
One takes time and effort and automaticity process not high, thus how quick, economic acquisition high-quality genomic DNA
Just tool is of great significance.
The basis that genomic DNA is most of molecular biology experiment is extracted from the whole blood of mammal.Genomic DNA
Extraction be generally used for building genomic library, Southern hybridization and PCR isolated genes etc..For the genetic analysis of crowd
And the HLA Genotypings work of hematopoiesis stem cell donors person's data bank is required for extracting gene from large batch of whole blood sample
Group DNA.And DNA extract purity and yield will directly influence PCR amplification, the endonuclease reaction of restriction endonuclease and
The success or failure of the experiments such as molecular cloning.Such as SNP (SNP) analysis, genomic library construction and determined dna sequence
Deng.
The operating method of molecular biology experiment is also more and more；For carrying out the beginner of molecular biology work and facing
It is sometimes difficult to select any operating method to be more applicable conveniently for bed doctor.At present, common DNA extraction method has many
Kind, but step is all more, and need the long period to complete.Representational method such as phenol-chloroform extraction process, deposits in practice
In more problem and shortage, such as：The protease K digesting time typically at least needs several hours, and time-consuming；Isopropanol is repeatedly taken out
Carry, one is the degree increase for polluting DNA, and two is the loss for easily causing DNA；Because of repeatedly transfer, sample contamination is easily caused, this
It is that PCR reactions the major reason of false positive occur；DNA after separation will also be purified by ethanol precipitation, trivial operations, workload
Greatly；Purity is relatively low；The process of cracking is inadequate fully and completely, so as to cause the DNA in extracted sample with substantial amounts of egg
White matter；Extract DNA have to varying degrees degraded etc. defect.
The DNA extraction kit that this year occurs can effectively extract DNA, but expense is of a relatively high,
To realize saving experiment manpower and cost, while DNA is fast and efficiently extracted, there is a need in the field to provide a kind of new
Can accelerate whole experimental work progress, improve extraction efficiency extraction genomic DNA method.The present invention provides big
The content of the invention
The purpose of the present invention includes：
A kind of method that DNA is taken from blood is provided；
A kind of method that genomic DNA is extracted during whole blood from large sample is provided；
A kind of human peripheral's blood-based of rapidly and efficiently extracting is provided because of the method for group DNA, etc..
Specifically, the invention provides a kind of method for extracting genomic DNA, comprising the following steps：
1. draw blood is in centrifuge tube；
2. buffer solution is added, is mixed；The buffer solution contains 2.5% dodecyl sodium sulfonate in terms of mass-volume concentration
Sodium；
3. sodium chloride solution is added, is mixed；
4. the mixed solution of Tris balance phenols and chloroform is added, is mixed；
5. it is centrifuged, Aspirate supernatant is in another centrifuge tube；
6. add and the isometric isopropanol of supernatant, mixing；
7. it is centrifuged, discards supernatant；
8. 70% ethanol solution is added, is mixed, centrifugation discards supernatant；Repeat step is 8.
9. centrifuge tube is upside down on clean blotting paper and stops；
10. the TE buffer solutions plus containing RNase, obtain DNA extracts.
One embodiment of the present invention is that the extracting method is comprised the following steps：
1. absorption 100ul blood is in centrifuge tube；
2. 600ul buffer solutions are added, mixing 10 times of turning upside down；The buffer solution is contained in terms of mass-volume concentration
2.5% dodecyl sodium sulfate；The buffer solution also contains composition described in table 2 below simultaneously；
Table 2：TKM-2 buffers
| Amount of substance concentration | Reagent name |
| 10mmol/ml | The Tris-HCl of pH value 7.6 |
| 10mmol/ml | KCl |
| 10mmol/ml | MgCl2 |
| 2mmol/ml | EDTA |
| 0.4mol/ml | NaCl |
| Supplement system | ddH2O |
3. the sodium chloride solution of 21ul 6mmol/mL is added, mixing 10 times of turning upside down；
4. the Tris balance phenols of 300ul and the mixed solution of chloroform are added, in vibration 10 seconds on desk-top vortex oscillator；
5. it is centrifuged 2 minutes under the conditions of 12000rpm, slowly draws the supernatant of transparent color in another centrifuge tube；
6. add and the isometric isopropanol of supernatant, vibration 10 seconds on desk-top vortex oscillator；
7. 12000rpm/2min is centrifuged, supernatant is discarded；
8. the ethanol solution of volumetric concentration 70% is added, is mixed, centrifugation discards supernatant；
9. centrifuge tube is upside down in and 1min is stopped on blotting paper, it is ensured that be deposited in pipe；
10. 1 × TE the buffer solutions for adding 100ul to contain RNase；The formula of 1 × TE buffer solutions is as shown in table 3 below；Institute
It is the RNase of 1 × TE buffer solutions containing 3ul of 1ml that 1 × TE buffer solutions are stated with the proportioning of RNase.
Table 3：1 × TE Buffer 1L are prepared
| Title | Volume (mL) |
| 1M Tris-HCl Buffer(pH7.6) | 10 |
| 500mM EDTA(pH8.0) | 20 |
| ddH2O | 970 |
| Total | 1000 |
Preferably, the centrifuge tube is 1.5mlEP pipes.
Preferably, the step 1. blood behaviour venous blood.
Preferably, the 5. described slow absorption of step is to be drawn using pipettor.
Preferably, the 7. described supernatant that discards of step is slowly to outwell supernatant.
Preferably, the 8. described ethanol solution of step is the ethanol solution of volumetric concentration 70%；Step 8. after the completion of weight
Carry out once again.
It is preferred that the 4. described Tris balance phenols of step and chloroform mixed solution, with volume basis, Tris balance phenols：Chlorine
Imitative=1:1.
One embodiment of the present invention is, including following extraction step：
1. absorption 100ul people's venous blood is in 1.5mlEP；
2. 600ul buffer solutions are added, mixing 10 times of turning upside down；The buffer solution is contained in terms of mass-volume concentration
2.5% dodecyl sodium sulfate；The buffer solution also contains composition described in table 2 below simultaneously；
Table 2：TKM-2 buffers
| Amount of substance concentration | Reagent name |
| 10mmol/ml | The Tris-HCl of pH value 7.6 |
| 10mmol/ml | KCl |
| 10mmol/ml | MgCl2 |
| 2mmol/ml | EDTA |
| 0.4mol/ml | NaCl |
| Supplement system | ddH2O |
3. the sodium chloride solution of 21ul 6mmol/mL is added, mixing 10 times of turning upside down；
4. the Tris balance phenols of 300ul and the mixed solution of chloroform are added, in vibration 10 seconds on desk-top vortex oscillator；Institute
The mixed solution of Tris balance phenols and chloroform is stated, with volume basis, Tris balance phenols：Chloroform=1:1；
5. it is centrifuged 2 minutes under the conditions of 12000rpm, the supernatant of transparent color is slowly drawn using pipettor in another
In branch centrifuge tube；
6. add and the isometric isopropanol of supernatant, vibration 10 seconds on desk-top vortex oscillator；
7. 12000rpm/2min is centrifuged, supernatant is slowly outwelled；
8. the ethanol solution of volumetric concentration 70% is added, is mixed, centrifugation discards supernatant；Then, volumetric concentration is added
70% ethanol solution, mixes, and centrifugation discards supernatant；
9. centrifuge tube is upside down in and 1min is stopped on blotting paper, it is ensured that be deposited in pipe；
10. 1 × TE the buffer solutions for adding 100ul to contain RNase,；The formula of 1 × TE buffer solutions is as shown in table 3 below；
1 × TE buffer solutions are with the proportioning of RNase, 1ml1 × TE buffer solution RNases containing 3ul.
Table 3：1 × TE Buffer 1L are prepared
| Title | Volume (mL) |
| 1M Tris-HCl Buffer(pH7.6) | 10 |
| 500mM EDTA(pH8.0) | 20 |
| ddH2O | 970 |
| Total | 1000 |
The poba gene group DNA extraction method that the present invention is provided, can be described as improveing " phenol chloroform " extraction method.
Extract the deficiency of reference method for conventional method/kit, the genome DNA extracting method that the present invention is provided,
It is more practical, economical, efficient；More existing DNA extraction method, the improvement of extracting method of the present invention includes：
1. the use of Proteinase K, reduces cost are removed；
2. chloroform is used in extractive process, to make protein sufficiently be separated from DNP；
3. Direct Pyrolysis leucocyte film, nuclear membrane in a short time, DNA is discharged, through conventional precipitation, washing,
Less than the extraction that genomic DNA can be completed in 30min；
4. losses of the DNA in extraction process is reduced, the concentration of DNA is improved；
5. it is as few as possible to make the DNA degradation for extracting；
6. eliminate and extract the muddy phenomenons of the DNA for obtaining, while avoiding what the band in agarose gel electrophoresis trailed
Phenomenon.
The poba gene group DNA extraction method that the present invention is provided, can be described as improvement " phenol chloroform " and extracts genomic DNA
Method, its financial cost is cheap, and the DNA mass of acquisition preferably, can meet PCR amplifications and restriction endonuclease digestion experiment
Need, thus can consider that the method can completely replace RNA isolation kit, be that one kind is applied to clinical high-volume sample genomic dna
The good method extracted.
Furthermore, the TKM-2 in the lysate can change the surface tension of cell membrane, and lysed erythrocyte can also be removed blood red
Porphyrin compound in albumen, so as to eliminate the suppression that the compound is reacted PCR.After erythrocyte splitting, the whole of DNA is extracted
Individual process is carried out in two Ep pipes, and avoid the continuous extracting of the time-consuming phenol/chloroform of repetition causes DNA to lose and pollute,
Ensure that the quality of PCR reactions.The method is simple and effective, can mass disposal sample, avoid what isoamyl alcohol was caused to human body
Harm.
In addition, it is ensured that the integrality of DNA primary structures, other large biological molecules (protein, polysaccharide, lipid etc.) are excluded
Pollution and nucleic acid molecules between pollution when DNA molecular (extract, RNA molecule should be removed), and should not exist highly concentrated
The organic solvent and metal ion of degree, also avoid chemical factor to the destruction of phosphodiester bond in nucleic acid chains and anti-to follow-up enzyme
Inhibitory action should be produced.Also simplify operating procedure simultaneously, not only can be time-consuming, more importantly, in can reducing extraction process
Destruction of the various adverse factors to nucleic acid.
Beneficial effect
Genomic DNA method is extracted in the improvement " phenol chloroform " that the present invention is provided, and its financial cost is cheap, the DNA matter of acquisition
Amount is preferable, the need for meeting PCR amplifications and restriction endonuclease digestion experiment, thus can consider that the method can be complete
Substitution RNA isolation kit, is a kind of good method extracted suitable for clinical high-volume sample genomic dna.
Furthermore, the TKM-2 in the lysate can change the surface tension of cell membrane, and lysed erythrocyte can also be removed blood red
Porphyrin compound in albumen, so as to eliminate the suppression that the compound is reacted PCR.After erythrocyte splitting, the whole of DNA is extracted
Individual process is carried out in two Ep pipes, and avoid the continuous extracting of the time-consuming phenol/chloroform of repetition causes DNA to lose and pollute,
Ensure that the quality of PCR reactions.The method is simple and effective, can mass disposal sample, avoid what isoamyl alcohol was caused to human body
Harm.
In addition, it is ensured that the integrality of DNA primary structures, other large biological molecules (protein, polysaccharide, lipid etc.) are excluded
Pollution and nucleic acid molecules between pollution when DNA molecular (extract, RNA molecule should be removed), and should not exist highly concentrated
The organic solvent and metal ion of degree, also avoid chemical factor to the destruction of phosphodiester bond in nucleic acid chains and anti-to follow-up enzyme
Inhibitory action should be produced.Also simplify operating procedure simultaneously, not only can be time-consuming, more importantly, in can reducing extraction process
Destruction of the various adverse factors to nucleic acid.
Brief description of the drawings
The electrophoresis result that Fig. 1 kits are extracted to human peripheral's blood-based by group DNA
The contrast test electrophoresis result that Fig. 2 is extracted to human peripheral's blood-based by group DNA
Wherein, 1 is sample a# isolation kit methods, and 2 is sample b# isolation kit methods,
3 is that sample a# improves " phenol chloroform " method, and 4 is that sample b# improves " phenol chloroform " method
Specific embodiment
One Rela of embodiment × GeneBlood DNA System poba genes group DNA extraction systems (0.1-20ml) examinations
Agent box (non-centrifugal column, Tiangeng company) method extracts experiment
Experimental technique
1. is overturned and mixed to 750ul cell pyrolysis liquid CLA are added in blood (blood sample A) of the 300ul containing anti-coagulants
20 times.
2. .12000rpm is centrifuged 1min.Supernatant is abandoned, centrifuge tube is upside down on clean blotting paper and is stopped 2min, really
Guarantor is deposited in pipe.
3. mixed liquor (the 200ul buffer solutions FGA+1.5ul of now with the current buffer solution FGA and Proteinse K
Proteinse K)
4. adds the mixed liquor of 200ul buffer solutions FGA and Proteinase K, and acutely concussion or vortex are mixed up and down immediately
It is even to solution without agglomerate.
5. .65 DEG C of water-bath 10min, overturns mix for several times therebetween.
6. adds 200ul isopropanols, and the mixing 50 times of turning upside down is to there is thread or tufted genomic DNA.
7. .12000rmp centrifugations 5min, abandons supernatant.Centrifuge tube is upside down on clean blotting paper, it is ensured that be deposited in
Guan Zhong.
8. adds 300ul70% ethanol, and vortex oscillation 5s, 12000rmp centrifugation 2min abandons supernatant.
9. be upside down in for centrifuge tube at least 5min stopped on clean blotting paper by, it is ensured that is deposited in pipe.
10. is air-dried DNA precipitations until all of evaporate clean (at least 5min)
200ul buffer solution TB, low speed vortex 5s, 65 DEG C of heating 20min dissolving DNAs is added to flick help for several times therebetween
It is molten.
Separately, blood sample B is taken, DNA is extracted by the extracting method of blood sample sample A.
The detection of DNA purity and yield
Determine the OD of DNA sample260/OD280Absorbance, calculates this purity that gets sample.DNA total amounts are with OD260/OD280
Value is reference, and the computing formula of DNA total amounts is：DNA
(μ g/ml)=OD260× 50 × extension rate.
DNA integrity detections
Agarose gel electrophoresis is detected：The Ago-Gel of configuration 1%, takes 3ul Marker, DNA, 120V that 2ul is extracted
30min, gel imaging system analytical electrophoresis result is simultaneously imaged, and integrity detection is carried out to extracting DNA.
Experimental result
Result is as shown in Figure of description Fig. 1 and table 1-1, table 1-2：
Purity and concentration results that the method for table 1-1 embodiments one is extracted to human peripheral's blood-based by group DNA
Purity and concentration results that the method for table 1-2 embodiments one is extracted to human peripheral's blood-based by group DNA
Above-mentioned conventional method/kit extract the extraction of reference method test result indicate that, existing extracting method is existed
Following problems：
1. the protease K digesting time typically at least need several hours, time-consuming；
2. the extracting repeatedly of isopropanol, one is the degree increase for polluting DNA, and two is the loss for easily causing DNA；
3. because of repeatedly transfer, sample contamination is easily caused, this is also that PCR reactions the major reason of false positive occur；
4. the DNA after separating will also be purified by ethanol precipitation, and trivial operations, workload is big；
5. purity does not meet OD260/OD280=1.8 ± 0.1 requirement, purity is relatively low；
6. the process for cracking is inadequate fully and completely, so as to cause the DNA in extracted sample with substantial amounts of protein；
7. extract DNA has degraded to varying degrees.
Present invention improvement " phenol chloroform " method of embodiment two extracts DNA experiments
Experimental technique
1. absorption 100ul is grown up venous blood in 1.5ml EP pipes；
2. add 600ul TKM-2 (with2.5%SDS), mixing 10 times of turning upside down (TKM2 is prepared and seen below table 2)
3. 21ul 6M NaCl solutions are added, mixing 10 times of turning upside down
4. (volume ratio of Tris balance phenols and chloroform is 1 to be eventually adding the Tris balances phenol-chloroform of 300ul:1), in platform
10s is vibrated on formula vortex oscillator
5. 2min is centrifuged under the conditions of 12000rpm, slow Aspirate supernatant (transparent color) is in (this in another Ep pipe
Step answers careful operation, to avoid being drawn onto phenol, it is recommended to use 100ml pipettors are drawn repeatedly to realize)
6. isopropanol (the V isometric with supernatant is addedSupernatant∶VIsopropanol=1: 1), vibrated on desk-top vortex oscillator
10s (adds isopropanol that thread or cotton-shaped genomic DNA occurs, it should to examine)
7. 12000rpm/2min is centrifuged, discards supernatant and (notes：Being precipitated in the case of number of poles to relax, so
Will slowly fall supernatant)
8. the ethanol of 1ml 70% is added, turning upside down makes precipitation suspend.12000rpm/1min is then centrifuged for, is discarded
Supernatant
9. above-mentioned step is repeated 8. once
9. Ep pipes are upside down on clean blotting paper and stop 1min, it is ensured that be deposited in pipe
10. plus 1 × TE of 100ul Buffer (containing RNase, referring to rear table 3),
Table 2：TKM-2Buffer is prepared
| Amount of substance concentration | Reagent name |
| 10mmol/ml | Tris-HCl(pH 7.6) |
| 10mmol/ml | KCl |
| 10mmol/ml | MgCl2 |
| 2mmol/ml | EDTA |
| 0.4mol/ml | NaCl |
| Supplement system | ddH2O |
Table 3：1 × TE Buffer 1L are prepared
| Title | Volume (mL) |
| 1M Tris-HCl Buffer(pH7.6) | 10 |
| 500mM EDTA(pH8.0) | 20 |
| ddH2O | 970 |
| Total | 1000 |
Note：That is the RNase of the 1 × TEBuffer+3ul of 1ml of 1 × TE Buffer and RNase
The detection of DNA purity and yield
Determine the OD of DNA sample260/OD280Absorbance, calculates this purity that gets sample.DNA total amounts are with OD260/OD280
Value is reference, and the computing formula of DNA total amounts is：DNA (μ g/ml)=OD260× 50 × extension rate.
DNA integrity detections
Agarose gel electrophoresis is detected：The Ago-Gel of configuration 1%, takes 3ul Marker, DNA, 120V that 2ul is extracted
30min, gel imaging system analytical electrophoresis result is simultaneously imaged, and integrity detection is carried out to extracting DNA.
Result such as table 4-1, the table 4-2 institutes extracted by group DNA to human peripheral's blood-based using improvement " phenol chloroform " method
Show：
Purity of table 4-1 present invention improvement " phenol chloroform " method to human blood sample a# peripheral blood extracting genome DNAs
And concentration results
Table 4-2 present invention improvement " phenol chloroform " methods are to the pure of human blood sample b# peripheral blood extracting genome DNAs
Degree and concentration results
Because DNA extraction kit operation is relatively complicated and expensive, while be not suitable for substantial amounts of Gene Experiments grinding
Study carefully.It is with low cost and extracting method of the present invention is without using DNA extraction kit；Meanwhile, more conventional phenol-chloroform extraction process is carried
DNA extraction method is taken, easy to operate, extraction effect is more preferable.Improvement phenol chloroform " the DNA purity that method is obtained that the present invention is provided
Height, DNA content is dramatically increased.
The present invention of embodiment three improvement " phenol chloroform " method is tested with kit extraction process Contrast on effect
With reference to embodiment one and the extracting method of embodiment two, DNA is extracted respectively, enter row agarose gel electrophoresis detection；
Testing result is as shown in Figure of description 2.
We carry out improvement addition SDS and NaCl to traditional phenol-chloroform extraction process to carry out nucleus in our current research
The rupture of film discharges nuclear dna, promotes protein denaturation, prevents DNA to be degraded while suppressing DNA enzymatic activity；Using DNA and
RNA differences of solubility in the NaCl solution of high concentration make it be effectively separated；Phenol chloroform-isopropanol is taken out repeatedly
Carrying can be clean by the removal of the impurity components such as cell fragment, it is ensured that the purity of DNA.
Find in an experiment, precipitated through absolute ethyl alcohol and through the DNA for the obtaining precipitations after the washing of 70% ethanol in colourless
Bright shape, it is soluble.Preferably, electrophoresis detection is hardly visible conditions of streaking, and loading wells to the genomic DNA quality that the method is obtained
More totally without shinny phenomenon, albumen obtains thoroughly removing very much in showing sample, and not only DNA output is of a relatively high at place, while
The need for downstream experiment can be met.
Claims (9)
1. a kind of method for extracting genomic DNA, comprises the following steps：
1. draw blood is in centrifuge tube；
2. buffer solution is added, is mixed；The buffer solution contains 2.5% dodecyl sodium sulfate in terms of mass-volume concentration；
3. sodium chloride solution is added, is mixed；
4. the mixed solution of Tris balance phenols and chloroform is added, is mixed；
5. it is centrifuged, Aspirate supernatant is in another centrifuge tube；
6. add and the isometric isopropanol of supernatant, mixing；
7. it is centrifuged, discards supernatant；
8. ethanol solution is added, is mixed, centrifugation discards supernatant；Then, ethanol solution is added, is mixed, centrifugation is discarded
Clear liquid；
9. centrifuge tube is upside down on clean blotting paper and stops；
10. the TE buffer solutions plus containing RNase, obtain DNA extracts.
2. method according to claim 1, it is characterised in that the described method comprises the following steps：
1. absorption 100ul blood is in centrifuge tube；
2. 600ul buffer solutions are added, mixing 10 times of turning upside down；The buffer solution contains 2.5% in terms of mass-volume concentration
Dodecyl sodium sulfate；The buffer solution also contains composition described in table 2 below simultaneously；
Table 2：TKM-2 buffers
3. the sodium chloride solution of 21ul 6mmol/mL is added, mixing 10 times of turning upside down；
4. the mixed solution of 300ul Tris balance phenols and chloroform is added, in vibration 10 seconds on table-type low-speed vortex oscillator；
5. it is centrifuged 2 minutes under the conditions of 12000rpm, slowly draws the supernatant of transparent color in another centrifuge tube；
6. add and the isometric isopropanol of supernatant, vibration 10 seconds on desk-top vortex oscillator；
7. 12000rpm/2min is centrifuged, supernatant is discarded；
8. the ethanol solution of volumetric concentration 70% is added, is mixed, 12000rpm/min centrifugation 1min discard supernatant；Then, then
Ethanol solution is added, is mixed, centrifugation discards supernatant；
9. centrifuge tube is upside down in and 1min is stopped on blotting paper, it is ensured that be deposited in centrifuge tube；
10. 1 × TE the buffer solutions for adding 100ul to contain RNase；The formula of 1 × TE buffer solutions is as shown in table 3 below；Described 1
× TE buffer solutions are the RNase of 1 × TE buffer solutions containing 3ul of 1ml with the proportioning of RNase；
Table 3：1 × TE Buffer 1L are prepared
3. method according to claim 2, it is characterised in that the centrifuge tube is 1.5mlEP pipes.
4. method according to claim 2, it is characterised in that the step 1. blood behaviour venous blood.
5. method according to claim 2, it is characterised in that the 5. described slow absorption of step is to be drawn using pipettor.
6. method according to claim 2, it is characterised in that step is 7. described to discard supernatant slowly to outwell supernatant
Liquid.
7. method according to claim 2, it is characterised in that the 8. described ethanol solution of step is volumetric concentration 70%
Ethanol solution.
8. method according to claim 2, it is characterised in that the mixing of the 4. described Tris balance phenols of step and chloroform is molten
Liquid, with volume basis, Tris balance phenols：Chloroform=1:1.
9. method according to claim 1 and 2, it is characterised in that the described method comprises the following steps：
1. absorption 100ul people's venous blood is in 1.5mlEP；
2. 600ul buffer solutions are added, mixing 10 times of turning upside down；The buffer solution contains 2.5% in terms of mass-volume concentration
Dodecyl sodium sulfate；The buffer solution also contains composition described in table 2 below simultaneously；
Table 2：TKM-2 buffers
3. the sodium chloride solution of 21ul 6mmol/mL is added, mixing 10 times of turning upside down；
4. the Tris balance phenols of 300ul and the mixed solution of chloroform are added, in vibration 10 seconds on desk-top vortex oscillator；It is described
The mixed solution of Tris balance phenols and chloroform, with volume basis, Tris balance phenols：Chloroform=1:1；
5. it is centrifuged 2 minutes under the conditions of 12000rpm, the supernatant of transparent color is slowly drawn using pipettor in another fragmented
In heart pipe；
6. add and the isometric isopropanol of supernatant, vibration 10 seconds on desk-top vortex oscillator；
7. 12000rpm/2min is centrifuged, supernatant is slowly outwelled；
8. the ethanol solution of volumetric concentration 70% is added, is mixed, centrifugation discards supernatant；Then, volumetric concentration 70% is added
Ethanol solution, mix, centrifugation, discard supernatant；
9. centrifuge tube is upside down in and 1min is stopped on blotting paper, it is ensured that be deposited in pipe；
10. 1 × TE the buffer solutions for adding 100ul to contain RNase；The formula of 1 × TE buffer solutions is as shown in table 3 below；Described 1
× TE buffer solutions are the RNase of 1 × TE buffer solutions containing 3ul of 1ml with the proportioning of RNase；
Table 3：1 × TE Buffer 1L are prepared
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