CN114525275B - Eutectic solvent applied to DNA extraction, DNA extraction method and kit - Google Patents
Eutectic solvent applied to DNA extraction, DNA extraction method and kit Download PDFInfo
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- 230000005496 eutectics Effects 0.000 title claims abstract description 78
- 239000002904 solvent Substances 0.000 title claims abstract description 75
- 238000000034 method Methods 0.000 title claims abstract description 32
- 238000007400 DNA extraction Methods 0.000 title abstract description 21
- 239000001763 2-hydroxyethyl(trimethyl)azanium Substances 0.000 claims abstract description 44
- 235000019743 Choline chloride Nutrition 0.000 claims abstract description 44
- SGMZJAMFUVOLNK-UHFFFAOYSA-M choline chloride Chemical group [Cl-].C[N+](C)(C)CCO SGMZJAMFUVOLNK-UHFFFAOYSA-M 0.000 claims abstract description 44
- 229960003178 choline chloride Drugs 0.000 claims abstract description 44
- 229910052739 hydrogen Inorganic materials 0.000 claims abstract description 18
- 239000001257 hydrogen Substances 0.000 claims abstract description 18
- BYEAHWXPCBROCE-UHFFFAOYSA-N 1,1,1,3,3,3-hexafluoropropan-2-ol Chemical compound FC(F)(F)C(O)C(F)(F)F BYEAHWXPCBROCE-UHFFFAOYSA-N 0.000 claims abstract description 16
- 238000000605 extraction Methods 0.000 claims description 41
- 239000006228 supernatant Substances 0.000 claims description 30
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 20
- 239000007984 Tris EDTA buffer Substances 0.000 claims description 17
- 238000002156 mixing Methods 0.000 claims description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 claims description 12
- 108090000623 proteins and genes Proteins 0.000 claims description 12
- 238000010438 heat treatment Methods 0.000 claims description 11
- 239000000203 mixture Substances 0.000 claims description 11
- 102000004169 proteins and genes Human genes 0.000 claims description 11
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 10
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 10
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 10
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 10
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 10
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 10
- 239000012071 phase Substances 0.000 claims description 10
- 229910052938 sodium sulfate Inorganic materials 0.000 claims description 10
- 235000011152 sodium sulphate Nutrition 0.000 claims description 10
- WBHQBSYUUJJSRZ-UHFFFAOYSA-M sodium bisulfate Chemical compound [Na+].OS([O-])(=O)=O WBHQBSYUUJJSRZ-UHFFFAOYSA-M 0.000 claims description 9
- 229910000342 sodium bisulfate Inorganic materials 0.000 claims description 9
- 238000003756 stirring Methods 0.000 claims description 8
- 239000000243 solution Substances 0.000 claims description 7
- 239000003795 chemical substances by application Substances 0.000 claims description 6
- 239000008346 aqueous phase Substances 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 3
- 229910052751 metal Inorganic materials 0.000 claims description 3
- 239000002184 metal Substances 0.000 claims description 3
- 239000013592 cell lysate Substances 0.000 claims description 2
- 230000001376 precipitating effect Effects 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 210000004369 blood Anatomy 0.000 abstract description 28
- 239000008280 blood Substances 0.000 abstract description 28
- 238000002474 experimental method Methods 0.000 abstract description 23
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 6
- 238000011084 recovery Methods 0.000 abstract description 3
- 102000053602 DNA Human genes 0.000 description 92
- 108020004414 DNA Proteins 0.000 description 92
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 15
- 229940098773 bovine serum albumin Drugs 0.000 description 15
- 239000000523 sample Substances 0.000 description 14
- 238000005119 centrifugation Methods 0.000 description 12
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 11
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 11
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 238000012408 PCR amplification Methods 0.000 description 8
- 238000002835 absorbance Methods 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 239000012535 impurity Substances 0.000 description 6
- 230000006037 cell lysis Effects 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 108010067770 Endopeptidase K Proteins 0.000 description 4
- 230000003321 amplification Effects 0.000 description 4
- 230000001909 effect on DNA Effects 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 239000003960 organic solvent Substances 0.000 description 3
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 239000012472 biological sample Substances 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 102100025064 Cellular tumor antigen p53 Human genes 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 231100000086 high toxicity Toxicity 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 238000005192 partition Methods 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 235000019419 proteases Nutrition 0.000 description 1
- 238000000751 protein extraction Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000008227 sterile water for injection Substances 0.000 description 1
- 150000003467 sulfuric acid derivatives Chemical class 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
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- 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
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- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
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Abstract
The invention discloses an application of a eutectic solvent in DNA extraction, wherein the eutectic solvent is a choline chloride system eutectic solvent, and particularly the molar ratio of choline chloride to hydrogen bond donors is 1:1-5, and the hydrogen bond donors comprise hexafluoroisopropanol; the invention also discloses a method for extracting DNA by using the eutectic reagent, which adopts the choline chloride system eutectic solvent to extract DNA in whole blood, the recovery rate of the method for extracting DNA can reach more than 95%, the concentration of the extracted DNA is more than 50 mug/mL, the method can extract and obtain DNA sample at one time, and the method is efficient, safe and environment-friendly, can meet the requirement of mass experiments, and in addition, the invention also discloses a DNA extraction kit which comprises the choline chloride system eutectic solvent.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to application of a eutectic solvent in DNA extraction, a DNA extraction method and a kit.
Background
Deoxyribonucleic acid (DNA) is an important biological molecule, contains almost all genetic material required by the life of organisms, is a carrier of genetic information, and has important application value in the field of life science. Extraction of DNA from complex biological samples is a fundamental process in biology and is also the fundamental step in initiating other downstream activities such as sequencing, amplification, hybridization, ligation, cloning and biological detection. The extraction of DNA from whole blood is a relatively simple method for obtaining genome genetic information of organisms, and is also an important means and technology for carrying out gene correlation research. However, the extraction of high quality DNA from complex biological samples containing proteins, polysaccharides and various metabolites is very challenging, and the quality and amount of DNA sample extracted directly affects the accuracy of subsequent experiments.
At present, the conventional method for extracting DNA by using an organic solvent causes a series of problems, which are mainly characterized in that the sample size of the DNA obtained by one extraction is small and the toxicity of an extraction reagent is high. Therefore, it is important to find a safer and more efficient DNA extraction and purification method.
Disclosure of Invention
Aiming at the defects or improvement demands of the prior art, the invention provides a method and a kit for extracting DNA by using a eutectic agent, and aims to find out the specific enrichment effect of the eutectic solvent on DNA in whole blood and apply the eutectic agent to extract DNA in blood, thereby solving the technical problems of small amount of DNA extracted at one time and high toxicity of the extraction agent in the existing extraction method.
In order to achieve the above object, according to one aspect of the present invention, there is provided a use of a eutectic solvent in extracting DNA, wherein the eutectic solvent is a choline chloride system eutectic solvent, which is used as a DNA extractant.
Preferably, the eutectic solvent is used for extracting DNA, and the choline chloride system eutectic solvent is the eutectic solvent with the molar ratio of choline chloride to hydrogen bond donor reagent being 1:1-5.
Preferably, the use of the eutectic solvent in DNA extraction, wherein the hydrogen bond donor agent is hexafluoroisopropanol.
According to another aspect of the present invention, there is provided a method of extracting DNA by a eutectic solvent, comprising the steps of:
taking the obtained cell lysate supernatant containing the DNA to be extracted, adding a protein denaturing agent, uniformly mixing, fully denaturing and precipitating the protein, and centrifuging and layering; taking supernatant, adding TE buffer solution, sulfate and choline chloride system eutectic solvent to form a double-aqueous phase extraction system; mixing uniformly and standing at room temperature; after layering, taking the lower eutectic solvent phase, adding water for back extraction, centrifuging and layering to obtain supernatant; the supernatant contains the extracted DNA.
Preferably, the method for extracting DNA by the eutectic solvent comprises the two-aqueous phase extraction system, wherein the sulfate concentration is 0.08-0.5 g/mL; the volume ratio of the choline chloride system eutectic solvent is 60-400 mu L/mL.
Preferably, the sulfate is one or more of ammonium sulfate, sodium sulfate, magnesium sulfate or sodium bisulfate, wherein the concentration of the ammonium sulfate or the sodium bisulfate is 0.12-0.5 g/mL; the concentration of the sodium sulfate or the magnesium sulfate is 0.08-0.5 g/mL.
Preferably, in the method for extracting DNA by using the eutectic solvent, the hydrogen bond donor is hexafluoroisopropanol, and the molar ratio of the two is 1:1-5.
Preferably, in the method for extracting DNA by using the eutectic solvent, water is added and uniformly mixed according to the volume ratio of 1:1-4.
Preferably, the method for extracting DNA by using the eutectic solvent comprises the following steps of:
mixing the choline chloride and the hydrogen bond donor according to a molar ratio of 1:1-10, then placing the mixture in a closed pressure-resistant pipe, placing the pressure-resistant pipe in a magnetic heating stirrer, and heating and stirring until transparent liquid is formed, namely the eutectic solvent; the heating and stirring are carried out under the conditions of a metal bath or an oil bath at 60-100 ℃, the stirring speed is 150-1000 rpm, and the heating time is 1-12 h.
According to another aspect of the present invention, there is also provided a DNA extraction kit comprising the choline chloride system eutectic solvent according to the present invention.
In general, compared with the prior art, the technical proposal which is conceived by the invention can obtain the following beneficial effects due to the adoption of the choline chloride system eutectic solvent for extracting DNA:
the eutectic solvent is a green chemical reagent, is commonly used for separating organic matters, has a specific enrichment effect on DNA, has an extraction rate of 80% or more on DNA, can be applied to DNA extraction, particularly has a molar ratio of choline chloride to hydrogen bond donors of 1:1-5, particularly has hexafluoroisopropanol as the hydrogen bond donor, has a recovery rate of 95% or more on DNA extraction in blood, and can be used for preparing a DNA extraction kit.
In addition, the method for extracting DNA by using the eutectic solvent provided by the invention adopts a nontoxic choline chloride system eutectic solvent as an extractant, the concentration of the extracted DNA is more than 50 mug/mL, and the sample size of the extracted DNA at one time can meet the requirements of multiple experiments or large-scale amplification, so that the method is safer and more efficient.
Drawings
FIG. 1 shows the results of PCR amplification in example 1;
FIG. 2 shows the PCR amplification result in example 2;
FIG. 3 shows the result of PCR amplification in example 3;
FIG. 4 shows the result of PCR amplification in example 4;
fig. 5 shows the extraction yield of the eutectic solvent to DNA, BSA, RNA.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.
In the existing whole blood DNA extraction method, the volume of a sample after 200 mu L of whole blood sample is extracted and eluted is 50-80 mu L, and the traditional DNA extraction method generally adopts an organic solvent such as chloroform, so that potential safety hazards exist in the existing extraction method due to the toxicity of the organic solvent, and the importance of finding a safer and more effective DNA extraction reagent is significant along with the importance of people on green chemistry.
Experiments show that the DNA has proper distribution ratio in the choline chloride eutectic solvents, and the distribution of other components in blood in the eutectic solvents is relatively low, and the experimental results show that the RNA extraction rate is lower than 20%, the protein extraction rate is lower than 50%, and the DAN extraction rate can reach 80% or more; it can be seen that the partition ratio of DNA in this kind of eutectic solvent is significantly different from that of other components in blood, and it has a specific enrichment effect on DNA, and can be used for extracting DNA in blood.
Particularly, a eutectic solvent with the molar ratio of choline chloride to hydrogen bond donor reagent of 1:1-5 is adopted, the hydrogen bond donor content is too high, the eutectic solvent has certain volatility, the separation and the extraction are not facilitated, the proportion of the choline chloride to the hydrogen bond donor is proper, the separation and the extraction effects are good, and the cost is lower; wherein the hydrogen bond donor, preferably hexafluoroisopropanol, has better enrichment effect on DNA by the choline chloride system eutectic solvent.
The pure eutectic solvent forms a stable and uniform phase, and forms a double-water-phase system, namely a two-phase system under the proper ion type and concentration conditions. However, experiments using the choline chloride system eutectic solvent to extract DNA show that sulfate has a greater effect on the DNA extraction effect of the eutectic solvent than phosphate and carbonate, wherein sulfate comprises one or more of ammonium sulfate, sodium sulfate, magnesium sulfate, or sodium bisulfate; further experiments show that the volume ratio of the eutectic solvent of the choline chloride system is preferably 60-400 mu L/mL, the mass concentration of sulfate is 0.08-0.5 g/mL, and the eutectic solvent is difficult to form two phases under the condition of too low ion concentration; in particular, it should be noted that, under the condition of too high an example concentration, the lower phase volume is larger and the water content is high, so that the extraction rate of DNA is reduced, which is unfavorable for the extraction of DNA.
The proper concentration of different sulfates is different, wherein the preferable concentration of ammonium sulfate or sodium bisulfate is 0.12-0.5 g/mL; sodium sulfate or magnesium sulfate, preferably at a concentration of 0.08 to 0.5g/mL; the recovery rate of the extraction method for extracting the DNA can reach more than 95%, and the extraction of the DNA with high purity, high efficiency, strong enrichment, safety and environmental protection can be realized.
The concentration of the DNA extracted by the method is more than 50 mug/mL; experiments also show that the concentration of 350 to 2000 mu L obtained by final elution of 200 mu L of whole blood sample can be directly used for the subsequent PCR reaction; and the whole blood DNA can be recovered by adopting an elution method for downstream analysis, so that good effects can be obtained. Therefore, the method for extracting DNA by the eutectic solvent can meet the requirements of multiple experiments or large-scale amplification by extracting the sample amount of the DNA at one time, and the extracted DNA has high purity.
The invention provides an application of a eutectic solvent in DNA extraction, wherein the eutectic solvent is a choline chloride system eutectic solvent, preferably the molar ratio of choline chloride to hydrogen bond donors is 1:1-5, and the hydrogen bond donors comprise hexafluoroisopropanol.
In addition, the invention also provides a method for extracting DNA by using the eutectic solvent, which comprises the following steps:
(1) Cell lysis: taking a whole blood sample, adopting surfactant treatment to fully crack, and adopting protease treatment to fully release DNA to obtain a whole blood crack sample;
(2) Removing impurities: under the protection of a pH buffer solution, adding sufficient alcohol into the whole blood cracked sample to denature and precipitate proteins, and taking supernatant containing DNA; the nucleic acid molecule has better stability in a solution with pH value less than 5 and less than 9, preferably TE buffer with pH value between 7 and 9, has the protection function on the base of DNA by removing impurities in weak alkaline environment, and is beneficial to protecting the integrity of DNA molecules.
(3) DNA extraction: taking the supernatant containing DNA in the step (2), adding TE buffer solution, sulfate and choline chloride system eutectic solvent to form a double-aqueous phase extraction system, wherein the volume ratio of the choline chloride system eutectic solvent in the double-aqueous phase extraction system is 60-400 mu L/mL, and the mass concentration of the sulfate is 0.08-0.5 g/mL; standing and layering; taking the lower eutectic solvent phase, adding water, mixing uniformly, standing at constant temperature, back extracting, centrifuging and layering to obtain supernatant, wherein the supernatant contains the extracted DNA.
The sulfate comprises one or more of ammonium sulfate, sodium sulfate, magnesium sulfate, or sodium bisulfate, wherein the ammonium sulfate or sodium bisulfate has a preferred concentration of 0.12-0.5 g/mL; the concentration of the sodium sulfate or the magnesium sulfate is preferably 0.08-0.5 g/mL;
the molar ratio of the choline chloride to hexafluoroisopropanol is preferably 1:1-5;
the TE buffer solution is added, preferably, the TE buffer solution is added according to the volume ratio of 1:1-6;
and adding water for uniform mixing, preferably adding water for uniform mixing according to the volume ratio of the lower eutectic solvent phase to water of 1:1-4.
The choline chloride system eutectic solvent is prepared according to the following preparation method:
choline chloride and a hydrogen bond donor are mixed according to a molar ratio of 1:1-10 and then placed in a closed pressure-resistant pipe, the pressure-resistant pipe is placed in a magnetic heating stirrer, and the mixture is heated and stirred until transparent liquid is formed, namely the eutectic solvent; the heating and stirring are carried out under the conditions of a metal bath or an oil bath at 60-100 ℃, the stirring speed is 150-1000 rpm, and the heating time is 1-12 h.
In addition, the invention also provides a DNA extraction kit, which comprises the choline chloride system eutectic solvent.
The following are examples:
example 1 extraction of DNA from whole blood samples the following steps are performed:
(1) Cell lysis: 200 mu L of whole blood is added with 20 mu L of TritonX-100 and 20 mu L of 100mg/mL proteinase K, and the mixture is placed at 70 ℃ for 10min;
(2) Removing impurities such as protein: adding 0.76mL TE buffer (pH 8.0, 10mM Tris-HCl, 1mM EDTA) to the treated whole blood sample, centrifuging at 12000rpm for 2min, and separating by centrifugation; taking 900 mu L of supernatant after centrifugation, adding 900 mu L of methanol, uniformly mixing, centrifuging at 12000rpm for 2min, and layering by centrifugation;
(3) Extracting DNA: taking 1mL of supernatant after centrifugation, adding 4mL of TE buffer (pH 8.0, 10mM Tris-HCl, 1mM EDTA), adding 1.4g of ammonium sulfate, adding 0.7mL of eutectic solvent (choline chloride: hexafluoroisopropanol=1:2.5, mol/mol), mixing uniformly, and standing for 2min; 200. Mu.L of the lower layer was taken and subjected to back extraction with 400. Mu.L of water, left at 90℃for 5min, and centrifuged at 12000rpm for 1min, and the supernatant containing the extracted DNA was collected, and PCR was performed using the supernatant as a DNA template, as described below.
Example 2 extraction of DNA from whole blood samples the following steps are performed:
(1) Cell lysis: 200 mu L of whole blood is added with 20 mu L of TritonX-100 and 20 mu L of 100mg/mL proteinase K, and the mixture is placed at 70 ℃ for 10min;
(2) Removing impurities such as protein: adding 0.76mL TE buffer (pH 8.0, 10mM Tris-HCl, 1mM EDTA) to the treated whole blood sample, centrifuging at 12000rpm for 2min, and separating by centrifugation; taking 900 mu L of supernatant after centrifugation, adding 900 mu L of methanol, uniformly mixing, centrifuging at 12000rpm for 2min, and layering by centrifugation;
(3) Extracting DNA: taking 1mL of supernatant after centrifugation, adding 4mL of TE buffer (pH 8.0, 10mM Tris-HCl, 1mM EDTA), adding 0.6g of sodium sulfate, adding 0.7mL of eutectic solvent (choline chloride: hexafluoroisopropanol=1:2.5, mol/mol), mixing uniformly, and standing for 2min; 200. Mu.L of the lower layer was back-extracted with 400. Mu.L of water, left at 90℃for 5min, and centrifuged at 12000rpm for 1min, and the supernatant was collected, and the supernatant contained the extracted DNA.
Example 3 extraction of DNA from whole blood samples the following steps are performed:
(1) Cell lysis: 200 mu L of whole blood is added with 20 mu L of TritonX-100 and 20 mu L of 100mg/mL proteinase K, and the mixture is placed at 56 ℃ for 10min;
(2) Removing impurities such as protein: 0.76mL TE buffer (pH 8.0, 10mM Tris-HCl, 1mM EDTA) was added to the treated whole blood sample, and the mixture was centrifuged at 12000rpm for 5min; taking 900 mu L of supernatant after centrifugation, adding 900 mu L of methanol, uniformly mixing, and centrifuging at 12000rpm for 5min;
(3) Extracting DNA: taking 1mL of supernatant after centrifugation, adding 4mL of TE buffer (pH 8.0, 10mM Tris-HCl, 1mM EDTA), adding 1.2g of magnesium sulfate, adding 1.0mL of eutectic solvent (choline chloride: hexafluoroisopropanol=1:3, mol/mol), mixing uniformly, and standing for 5min; 200. Mu.L of the lower layer was back-extracted with 400. Mu.L of water, left at 90℃for 5min, and centrifuged at 12000rpm for 5min to obtain a supernatant containing the extracted DNA.
Example 4 extraction of DNA from whole blood samples the following steps are performed:
(1) Cell lysis: 200 mu L of whole blood is added with 20 mu L of TritonX-100 and 20 mu L of 100mg/mL proteinase K, and the mixture is placed at 56 ℃ for 10min;
(2) Removing impurities such as protein: 0.76mL TE buffer (pH 8.0, 10mM Tris-HCl, 1mM EDTA) was added to the treated whole blood sample, and the mixture was centrifuged at 12000rpm for 5min; taking 900 mu L of supernatant after centrifugation, adding 900 mu L of ethanol, uniformly mixing, and centrifuging at 12000rpm for 5min;
(3) Extracting DNA: taking 1mL of supernatant after centrifugation, adding 4mL of TE buffer (pH 8.0, 10mM Tris-HCl, 1mM EDTA), adding 1.0g of sodium bisulfate, adding 1.0mL of eutectic solvent (choline chloride: hexafluoroisopropanol=1:4, mol/mol), mixing uniformly, and standing for 5min; 200. Mu.L of the lower layer was back-extracted with 400. Mu.L of water, left at 90℃for 5min, and centrifuged at 12000rpm for 5min to obtain a supernatant containing the extracted DNA.
Experiment 1 detection of the DNA extracted in example 1
PCR was performed using the DNA-containing supernatant obtained in example 1 as a DNA template, and specifically as follows:
(1) PCR primer P53 sequence
(2) PCR reaction system
| Component (A) | Volume of |
| 2×PCRMix | 12.5μL |
| F/R | 0.5 mu L each |
| DNA | 1μL |
| Sterilized water for injection | 10.5μL |
| Total volume of | 25μL |
(3) PCR reaction procedure
The results of the PCR amplification are shown in FIG. 1.
Experiment 2 detection example 2 extraction of DNA
PCR was performed using the DNA-containing supernatant obtained in example 2 as a DNA template, and the PCR primer sequences, the PCR reaction system and the PCR reaction procedure were the same as those of experiment 1, and the PCR amplification results are shown in FIG. 2.
Experiment 3 detection example 3 extraction of DNA
PCR was performed using the DNA-containing supernatant obtained in example 3 as a DNA template, and specifically as follows:
(1) PCR primer sequences
(2) The PCR reaction system is the same as in experiment 1;
(3) The PCR reaction procedure was the same as in experiment 1, and the PCR amplification results are shown in FIG. 3.
Experiment 4 detection example 4 extraction of DNA
PCR was performed using the DNA-containing supernatant obtained in example 4 as a DNA template, and specifically as follows:
(1) PCR primer sequences
(2) The PCR reaction system is the same as in experiment 1;
(3) The PCR reaction procedure was the same as in experiment 1, and the PCR amplification results are shown in FIG. 4.
In the above examples, the DNA concentration obtained by detecting the extraction of the DNA is 50 mug/mL or more, which can be used for multiple experiments or large-scale amplification.
Experiment 5 extraction ratio of the eutectic solvent of the choline chloride System to DNA, RNA and BSA
Experiment 5-1
5mL of TE buffer (pH is DNA, RNA or bovine serum albumin at a conventional proper pH value, 10mM Tris-HCl, 1mM EDTA), 0.6g of sodium sulfate is added, 0.7mL of eutectic solvent (choline chloride: hexafluoroisopropanol=1:3, mol/mol) is added, 10 mug/mL of DNA or 10 mug/mL of RNA or 0.2mg/mL of Bovine Serum Albumin (BSA) is added, uniformly mixed, and the mixture is left stand for 2min; 200 μl of the lower layer was removed, and the absorbance of DNA and RNA was measured at a wavelength of 260nm by an ultraviolet detector, and the absorbance of bovine serum albumin was measured at a wavelength of 280nm, and the extraction rate of DNA, RNA, BSA was calculated according to the following formula, and the results are shown in FIG. 5 (top).
Experiment 5-2
5mL TE buffer (pH is DNA, RNA or bovine serum albumin with a conventional proper pH value, 10mM Tris-HCl and 1mM EDTA), 1.4g of ammonium sulfate is added, 0.7mL of eutectic solvent (choline chloride: hexafluoroisopropanol=1:3, mol/mol) is added, and the mixture is stirred evenly and kept stand for 2min; 200. Mu.L of the lower layer was removed, 200. Mu.L of the lower layer was placed in an ultraviolet detector to measure the absorbance of DNA and RNA at a wavelength of 260nm and the absorbance of bovine serum albumin at a wavelength of 280nm, and the extraction rate of DNA, RNA, BSA was calculated according to the following formula, respectively, and the result was shown in FIG. 5 (middle).
Experiment 5-3
5mL TE buffer (pH is DNA, RNA or bovine serum albumin with a conventional proper pH value, 10mM Tris-HCl, 1mM EDTA), 1.2g magnesium sulfate is added, 1mL eutectic solvent (choline chloride: hexafluoroisopropanol=1:3, mol/mol) is added, mixed evenly, and left stand for 2min; 200 μl of the lower layer was removed, and the absorbance of DNA and RNA was measured at a wavelength of 260nm by an ultraviolet detector, and the absorbance of bovine serum albumin was measured at a wavelength of 280nm, and the extraction rate of DNA, RNA, BSA was calculated according to the following formula, and the results are shown in FIG. 5 (bottom).
The extraction rates of DNA, RNA and BSA by the eutectic solvents in the above 3 experiments are shown in FIG. 5.
As can be seen from FIG. 5, the extraction rate of the choline chloride system eutectic solvent of the invention to DNA, RNA, BSA is significantly different, the choline chloride system eutectic solvent has an enrichment effect on DNA, and the extraction rate of DNA can be up to more than 95%, and can be used as a DNA extractant for extracting DNA in blood.
It will be readily appreciated by those skilled in the art that the foregoing description is merely a preferred embodiment of the invention and is not intended to limit the invention, but any modifications, equivalents, improvements or alternatives falling within the spirit and principles of the invention are intended to be included within the scope of the invention.
Claims (4)
1. A method for extracting DNA by a eutectic solvent, comprising the steps of:
taking the obtained cell lysate supernatant containing the DNA to be extracted, adding a protein denaturing agent, uniformly mixing, fully denaturing and precipitating the protein, and centrifuging and layering; taking supernatant, adding TE buffer solution, sulfate and choline chloride system eutectic solvent to form a double-aqueous phase extraction system; mixing uniformly and standing at room temperature; after layering, taking the lower eutectic solvent phase, adding water for back extraction, and separating the supernatant; the supernatant contains the extracted DNA;
the hydrogen bond donor of the choline chloride system eutectic solvent is hexafluoroisopropanol, and the molar ratio of the hydrogen bond donor to the hexafluoroisopropanol is 1:1-5; the aqueous two-phase extraction system comprises a sulfate concentration of 0.08-0.5 g/mL; the volume ratio of the choline chloride system eutectic solvent is 60-400 mu L/mL.
2. The method for extracting DNA by using the eutectic solvent according to claim 1, wherein the sulfate is one or more of ammonium sulfate, sodium sulfate, magnesium sulfate or sodium bisulfate, and the concentration of the ammonium sulfate or the sodium bisulfate is 0.12-0.5 g/mL; the concentration of the sodium sulfate or the magnesium sulfate is 0.08-0.5 g/mL.
3. The method for extracting DNA by using the eutectic solvent according to claim 2, wherein the water is added and mixed uniformly according to a volume ratio of 1:1-4.
4. The method for extracting DNA by using the eutectic solvent according to claim 2, wherein the choline chloride system eutectic solvent is obtained by the following preparation method:
mixing choline chloride and a hydrogen bond donor according to a molar ratio of 1:1-10, then placing the mixture in a closed pressure-resistant pipe, placing the pressure-resistant pipe in a magnetic heating stirrer, and heating and stirring until a transparent liquid is formed, namely the choline chloride system eutectic solvent; the heating and stirring are carried out under the conditions of a metal bath or an oil bath at 60-100 ℃, the stirring speed is 150-1000 rpm, and the heating time is 1-12 h.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102604935A (en) * | 2012-04-09 | 2012-07-25 | 邢秀梅 | Method for safely and quickly extracting genomic deoxyribose nucleic acid (DNA) from blood |
| CN110003323A (en) * | 2019-04-02 | 2019-07-12 | 武汉大学 | A kind of method that double-aqueous phase system isolates and purifies protein |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102604935A (en) * | 2012-04-09 | 2012-07-25 | 邢秀梅 | Method for safely and quickly extracting genomic deoxyribose nucleic acid (DNA) from blood |
| CN110003323A (en) * | 2019-04-02 | 2019-07-12 | 武汉大学 | A kind of method that double-aqueous phase system isolates and purifies protein |
Non-Patent Citations (7)
| Title |
|---|
| Facile and environmentally friendly halogenation of BODIPYs in deep eutectic solvent;Liang Wang等;Dyes and Pigments;第112卷;第274-279页 * |
| Hexafluoroisopropanol-Based Deep Eutectic Solvent/Salt Aqueous Two-Phase Systems for Extraction of Anthraquinones from Rhei Radix et Rhizoma Samples;Wen-Wen Deng等;ACS Sustainable Chem. Eng.;第5卷(第5期);第4267-4275页 * |
| Hexafluoroisopropanol-based deep eutectic solvents for high-performance DNA extraction;Jia Xu等;RSC Adv.;第13卷;文章号9595 * |
| High-performance of deep eutectic solvent based aqueous bi-phasic systems for the extraction of DNA;Na Li等;RSC Adv.;第6卷;文章号84406 * |
| Na Li等.High-performance of deep eutectic solvent based aqueous bi-phasic systems for the extraction of DNA.RSC Adv..2016,第6卷文章号84406. * |
| 低共熔溶剂双水相体系的构建及其用于萃取分离生物大分子和染料的研究;许攀丽;中国优秀硕士学位论文全文数据库工程科技I辑(第6期);文章号B014-1038 * |
| 基于六氟异丙醇的新型低共熔溶剂在蛋白质纯化与分析中的应用研究;王璇璇;中国优秀硕士学位论文全文数据库基础科学辑(第3期);文章号A006-93 * |
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