CN109971753A - A kind of trace amount DNA extracting method based on dried blood spot - Google Patents
A kind of trace amount DNA extracting method based on dried blood spot Download PDFInfo
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- CN109971753A CN109971753A CN201910368794.XA CN201910368794A CN109971753A CN 109971753 A CN109971753 A CN 109971753A CN 201910368794 A CN201910368794 A CN 201910368794A CN 109971753 A CN109971753 A CN 109971753A
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- dna
- trace amount
- dried blood
- blood spot
- method based
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- 238000000034 method Methods 0.000 title claims abstract description 38
- 239000008280 blood Substances 0.000 title claims abstract description 16
- 210000004369 blood Anatomy 0.000 title claims abstract description 16
- 239000006228 supernatant Substances 0.000 claims abstract description 20
- 239000007791 liquid phase Substances 0.000 claims abstract description 14
- YTRQFSDWAXHJCC-UHFFFAOYSA-N chloroform;phenol Chemical compound ClC(Cl)Cl.OC1=CC=CC=C1 YTRQFSDWAXHJCC-UHFFFAOYSA-N 0.000 claims abstract description 8
- 238000000703 high-speed centrifugation Methods 0.000 claims abstract description 8
- 239000007788 liquid Substances 0.000 claims abstract description 8
- 238000002156 mixing Methods 0.000 claims abstract description 5
- 238000007400 DNA extraction Methods 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 22
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 9
- 238000005119 centrifugation Methods 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 229910052681 coesite Inorganic materials 0.000 claims description 5
- 229910052906 cristobalite Inorganic materials 0.000 claims description 5
- 239000000377 silicon dioxide Substances 0.000 claims description 5
- 235000012239 silicon dioxide Nutrition 0.000 claims description 5
- 229910052682 stishovite Inorganic materials 0.000 claims description 5
- 229910052905 tridymite Inorganic materials 0.000 claims description 5
- 125000000118 dimethyl group Chemical group [H]C([H])([H])* 0.000 claims description 4
- 229920005573 silicon-containing polymer Polymers 0.000 claims description 4
- 108091005804 Peptidases Proteins 0.000 claims description 3
- 239000004365 Protease Substances 0.000 claims description 3
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 3
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 3
- 239000011324 bead Substances 0.000 claims description 3
- 230000009514 concussion Effects 0.000 claims description 3
- 229960000935 dehydrated alcohol Drugs 0.000 claims description 3
- 238000004090 dissolution Methods 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 3
- 229960004756 ethanol Drugs 0.000 claims description 3
- 239000004519 grease Substances 0.000 claims description 3
- 239000006166 lysate Substances 0.000 claims description 3
- 229910052751 metal Inorganic materials 0.000 claims description 3
- 239000002184 metal Substances 0.000 claims description 3
- 238000001821 nucleic acid purification Methods 0.000 claims description 3
- 238000005191 phase separation Methods 0.000 claims description 3
- 238000000926 separation method Methods 0.000 claims description 3
- 235000017281 sodium acetate Nutrition 0.000 claims description 3
- 239000001632 sodium acetate Substances 0.000 claims description 3
- 239000012224 working solution Substances 0.000 claims description 3
- 238000001556 precipitation Methods 0.000 claims description 2
- -1 dimethyl siloxane Chemical class 0.000 claims 1
- 229920001296 polysiloxane Polymers 0.000 claims 1
- 108020004414 DNA Proteins 0.000 description 26
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 6
- 238000007796 conventional method Methods 0.000 description 5
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 3
- XYHKNCXZYYTLRG-UHFFFAOYSA-N 1h-imidazole-2-carbaldehyde Chemical compound O=CC1=NC=CN1 XYHKNCXZYYTLRG-UHFFFAOYSA-N 0.000 description 2
- GWYFCOCPABKNJV-UHFFFAOYSA-M 3-Methylbutanoic acid Natural products CC(C)CC([O-])=O GWYFCOCPABKNJV-UHFFFAOYSA-M 0.000 description 2
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 2
- 238000002105 Southern blotting Methods 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- GWYFCOCPABKNJV-UHFFFAOYSA-N beta-methyl-butyric acid Natural products CC(C)CC(O)=O GWYFCOCPABKNJV-UHFFFAOYSA-N 0.000 description 2
- 238000001917 fluorescence detection Methods 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 238000004064 recycling Methods 0.000 description 2
- 229910052710 silicon Inorganic materials 0.000 description 2
- 239000010703 silicon Substances 0.000 description 2
- 108091029865 Exogenous DNA Proteins 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000012869 ethanol precipitation Methods 0.000 description 1
- 238000002795 fluorescence method Methods 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
Classifications
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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 invention patent relates to arrive a kind of trace amount DNA extracting method based on dried blood spot, it is characterized mainly in that, efficient liquid phase separating pipe is prepared in trace amount DNA extraction process, and add 100ng carrier RNA mixing after DNA solution collection, isometric phenol chloroform is added, and it is transferred to efficient liquid phase separating pipe, high speed centrifugation carries out stratified liquid, recycles 98% supernatant.There is the invention patent supernatant can all shift and feature easy to operate.
Description
Technical field
The invention belongs to Trace DNA analysis fields, are related specifically to a kind of trace amount DNA extracting method based on dried blood spot.
Background technique
Trace amount DNA refers to content down to the other DNA of pieck stage, on forensic, criminal case and micro biology, spirit
Quick, accurate DNA quantitative approach plays an important role in biological study and application field, according to 2005 editions " China
Pharmacopeia 2005 editions (three) " using Southern blotting hybrid method, this method can detect detection exogenous DNA content
The DNA of pg level, but the method for sxemiquantitative.In addition, also there is related researcher to establish trace amount DNA fluorescence detection method,
This method extracting DNA plays inspissation, and the DNA in sample can achieve the level of fluorescence detection after concentration.Fluorescence
Method and Southern blotting hybrid method compare, more rapidly, precision it is higher.
However, there is supernatants to extract the deficiencies of incomplete and recovery rate is not high and DNA purity is not high for the above method.
Summary of the invention
In consideration of it, it is necessary to provide a kind of trace amount DNA extracting method based on dried blood spot regarding to the issue above.Skill of the present invention
Art scheme is as follows:
A kind of trace amount DNA extracting method based on dried blood spot, mainly prepares efficient liquid phase in trace amount DNA extraction process
Separating pipe, and add 100ng carrier RNA mixing after DNA solution collection, isometric phenol chloroform is added, and be transferred to efficiently
Liquid phase separation pipe, high speed centrifugation carry out stratified liquid, recycle 98% supernatant.
Further, the extracting method step is predominantly as described below:
(1) take the dried blood spot sample of 3 × 3mm of three pieces into the centrifuge tube of 1.5ml.
(2) the protease k working solution (20mg/ml) of 20 μ l is added, adds the lysate of 240ul-300ul.
(3) it after the concussion 10sec-15sec that is vortexed is mixed, is put into the constant temperature for be preheated to 50-60 DEG C overnight.
(4) rear liquid overnight is transferred to double layer separation pipe together with filter paper, reduces remaining DNA solution on the scraps of paper, can returns
Receive 98% or more DNA solution amount.
(5) efficient liquid phase separating pipe is prepared, dimethyl silicone polymer+SiO2 solution 200- is added into 2ml centrifuge tube
300ul vacuum silicon grease is uniformly mixed, and quickly high speed centrifugation -60 seconds 30 seconds.
(6) after DNA solution is collected plus 100ng carrier RNA is mixed, and isometric phenol chloroform is added, and be transferred to efficiently
Liquid phase separation pipe, high speed centrifugation carry out stratified liquid, recycle 98% supernatant；
(7) dehydrated alcohol of 1.3ml-1.5ml volume, 100ul 3M sodium acetate PH5.2,4 ° of maximum (top) speed centrifugations are added
60sec abandons supernatant；
(8) add 4 ° of the maximum (top) speed of 75%-80% ethanol wash one time centrifugation 60sec again, abandon supernatant.Repeat the step 1
It is secondary；
(9) water dissolution is added after drying, obtains initial DNA；
(10) the initial DNA obtained is purified by Beckman XP Beads.
Further, the final concentration of 10-50% of the dimethyl silicone polymer), the final concentration of 1- of SiO2 solution
10%.
Further, the thermostatic mode is constant temperature water bath, sand-bath or metal bath；
Further, the carrier RNA is as yeast tRNA solution, as the load in nucleic acid purification and precipitation process
Body.
Further, the extracting method obtains the DNA of high-purity, purity can reach 260/280=1.8-2.1 it
Between.
Be compared with the prior art, present approach provides in supernatant transfer process, conventional method be commonly from
Supernatant is drawn in heart pipe plus after the centrifugation layering of phenol chloroform, recycling supernatant is not thorough, and supernatant can be shifted all, and separated
During increase 100ng carrier RNA mixing, realize recovery rate and be significantly increased, precision is higher, meanwhile, we
Equipment of the method when mentioning high-precision, there is no original method is increased.
Detailed description of the invention
A kind of Fig. 1 trace amount DNA extracting method key step based on dried blood spot provided by the invention；
Increase the efficient liquid phase separating pipe and preparation flow of carried RNA in Fig. 2 present invention；
Fig. 3 the invention patent example structure comparison diagram；
Specific embodiment
Further detailed, complete explanation is made to the present invention below with reference to embodiment.
Embodiment:
According to the shown process of Fig. 1 and Fig. 2, the next main invention using conventional method and the invention patent is carried out pair
Than operation, the operating procedure of new method is mainly introduced, as follows:
Steps are as follows for conventional method:
L. isometric and phenol or phenol/chloroform (1:1) mixed liquor is added in sample
2. turn upside down very even
3. room temperature, 12000 revs/min, when being centrifuged that 5 minutes such as protein is more or boundary is unclear, can be centrifuged 10 minutes.
4. drawing in another new centrifuge tube of upper layer (water layer) dislocation
5. isometric phenol/chloroform/isovaleric acid (25:24:1) solution, which is added, repeats step 2,3,4.
6. isometric chloroform/isovaleric acid (24:1) solution, which is added, repeats step 2,3,4.
Ethanol precipitation nucleic acid 7. (see the deposition and purification of nucleic acid)
The invention patent method and step:
1. taking the dried blood spot sample of 3 × 3mm of three pieces into the centrifuge tube (providing for oneself) of 1.5ml (101).
2. the protease k working solution (20mg/ml) of 20 μ l is added, the lysate (102) of 240ul-300ul is added.
3. being put into after the concussion 10sec-15sec that is vortexed is mixed and being preheated to 50-60 DEG C of constant temperature (metal bath, water-bath, sand-bath)
In overnight (103).
4. rear liquid overnight is transferred to double layer separation pipe together with filter paper, remaining DNA solution on the scraps of paper is reduced, can be recycled
98% or more DNA solution amount (104).
5. preparing efficient liquid phase separating pipe (105): dimethyl silicone polymer (final concentration 10- being added into 2ml centrifuge tube
50%)+SiO2 solution (final concentration 1-10%)+200-300ul vacuum silicon grease (201) is uniformly mixed (202), and quickly high speed
It is centrifuged -60 seconds 30 seconds (203).
6.DNA solution adds 100ng carrier RNA (106) after collecting (yeast tRNA solution is used as nucleic acid purification and sinks
Carrier during shallow lake) mixing, isometric phenol chloroform is added, and be transferred to efficient liquid phase separating pipe, high speed centrifugation carries out liquid phase
Layering, (conventional method is to draw supernatant in common centrifuge tube plus after the centrifugation layering of phenol chloroform to 98% supernatant of recycling, recycles supernatant
It is not thorough)；
7. the dehydrated alcohol of 1.3ml-1.5ml volume is added, 100ul 3M sodium acetate PH5.2,4 ° of maximum (top) speed centrifugations
60sec is abandoned supernatant (107).
8. adding 4 ° of the maximum (top) speed of 75%-80% ethanol wash one time centrifugation 60sec again, supernatant is abandoned.It is primary to repeat the step
(108)。
9. water dissolution is added after drying, initial DNA (109) are obtained.
The initial DNA obtained is purified by Beckman XP Beads, obtains the DNA of high-purity, purity can reach
Between 260/280=1.8-2.1.
By the comparison of both the above method, obtain as shown in Figure 3.
Above-mentioned experimental result absolutely proves the novelty of this method, has surmounted conventional method significantly.
Finally, it is necessary to illustratively, above-described embodiment is made further specifically just for technical solution of the present invention
It is bright, can not be interpreted as protection scope of the present invention, those skilled in the art's above content according to the present invention make one
A little non-intrinsically safes change and adjustment all belongs to the scope of protection of the present invention.
Claims (6)
1. a kind of trace amount DNA extracting method based on dried blood spot, is characterized mainly in that, prepared in trace amount DNA extraction process high
Liquid phase separation pipe is imitated, and adds 100ng carrier RNA mixing after DNA solution collection, isometric phenol chloroform is added, and shift
To efficient liquid phase separating pipe, high speed centrifugation carries out stratified liquid, recycles 98% supernatant.
2. a kind of trace amount DNA extracting method based on dried blood spot according to claim 1, is characterized mainly in that, described to mention
Take method and step predominantly as described below:
(1) take the dried blood spot sample of 3 × 3mm of three pieces into the centrifuge tube of 1.5ml.
(2) the protease k working solution (20mg/ml) of 20 μ l is added, adds the lysate of 240ul-300ul.
(3) it after the concussion 10sec-15sec that is vortexed is mixed, is put into the constant temperature for be preheated to 50-60 DEG C overnight.
(4) rear liquid overnight is transferred to double layer separation pipe together with filter paper, reduces remaining DNA solution on the scraps of paper, can be recycled
98% or more DNA solution amount.
(5) efficient liquid phase separating pipe is prepared, it is true that dimethyl silicone polymer+SiO2 solution 200-300ul is added into 2ml centrifuge tube
Empty silicone grease is uniformly mixed, and quickly high speed centrifugation -60 seconds 30 seconds.
(6) after DNA solution is collected plus 100ng carrier RNA is mixed, and isometric phenol chloroform is added, and be transferred to efficient liquid phase
Separating pipe, high speed centrifugation carry out stratified liquid, recycle 98% supernatant；
(7) dehydrated alcohol of addition 1.3ml-1.5ml volume, 100ul3M sodium acetate PH5.2,4 ° of centrifugation 60sec of maximum (top) speed,
Abandon supernatant；
(8) add 4 ° of the maximum (top) speed of 75%-80% ethanol wash one time centrifugation 60sec again, abandon supernatant.It is primary to repeat the step；
(9) water dissolution is added after drying, obtains initial DNA；
(10) the initial DNA obtained is purified by Beckman XP Beads.
3. a kind of trace amount DNA extracting method based on dried blood spot according to claim 1, is characterized mainly in that, described poly-
The final concentration of 10-50% of dimethyl siloxane), the final concentration of 1-10% of SiO2 solution.
4. a kind of trace amount DNA extracting method based on dried blood spot according to claim 1, is characterized mainly in that, the perseverance
Warm mode is constant temperature water bath, sand-bath or metal bath.
5. a kind of trace amount DNA extracting method based on dried blood spot according to claim 1, is characterized mainly in that, described
Carrier RNA is as yeast tRNA solution, as the carrier in nucleic acid purification and precipitation process.
6. a kind of trace amount DNA extracting method based on dried blood spot according to claim 1, is characterized mainly in that, described to mention
Method is taken to obtain the DNA of high-purity, purity can reach between 260/280=1.8-2.1.
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Cited By (2)
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| CN111206030A (en) * | 2020-02-18 | 2020-05-29 | 南京农业大学 | Virus nucleic acid extraction method based on glass fiber filter paper |
| WO2022006857A1 (en) * | 2020-07-10 | 2022-01-13 | 南京亿科人群健康研究院有限公司 | Dried blood spot-based trace dna extraction method |
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| CN111206030A (en) * | 2020-02-18 | 2020-05-29 | 南京农业大学 | Virus nucleic acid extraction method based on glass fiber filter paper |
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Inventor after: He Yuanlin Inventor after: Jiang Yue Inventor after: Dai Juncheng Inventor before: He Yuanlin Inventor before: Yue Jiang Inventor before: Dai Juncheng |
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Effective date of registration: 20210622 Address after: Unit e466, 5th floor, Lecheng Plaza, phase II, biomedical industrial park, No. 218, Sangtian street, Suzhou Industrial Park, Suzhou area, China (Jiangsu) pilot Free Trade Zone, Suzhou, Jiangsu 215024 Applicant after: Ecobet (Suzhou) Biotechnology Co.,Ltd. Address before: 210000 A, 10 level, phase 1, yangchuang Chuang Chuang, 211 Pu Pu Road, Jiangbei new district, Nanjing, Jiangsu. Applicant before: Hundred million Co.,Ltd. of population health research institute of section of Nanjing |
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