CN113755487A - A method for extracting genomic deoxyribonucleic acid - Google Patents
A method for extracting genomic deoxyribonucleic acid Download PDFInfo
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Abstract
本发明涉及基因组技术领域，具体涉及一种提取基因组脱氧核糖核酸的方法包括将生物组织进行研磨、涡旋混匀，得到第一浑浊液，采用缓冲液清洗第一浑浊液，离心分离，倒弃上清液，得到细胞沉淀；向细胞沉淀中加入裂解液，进行混匀、裂解、孵育，直至溶液无团块，得到悬浮液；向悬浮液中加入前处理后的磁性微球，涡旋混匀，振摇吸附10min，在磁场上分离磁性微球并用吸管取出上清液，采用乙醇洗涤磁性微球，得到吸附微球；向吸附微球中加入TE缓冲液进行脱附，磁场下分离磁性微球获得含有脱氧核糖核酸的洗脱液，本发明方法的操作步骤简单，无需使用对人体有害的有机物质，能够节省时间和资源，并获得高纯度的DNA片段，从而具有比较好的效果和产率。The invention relates to the technical field of genomes, in particular to a method for extracting genomic deoxyribonucleic acid, comprising grinding biological tissues, vortexing and mixing to obtain a first turbid solution, washing the first turbid solution with a buffer, centrifuging, and discarding Supernatant to obtain cell pellet; add lysate to the cell pellet, mix, lyse, and incubate until the solution is free of clumps to obtain a suspension; add pre-treated magnetic microspheres to the suspension, and vortex to mix The magnetic microspheres were separated on the magnetic field and the supernatant was taken out with a pipette. The magnetic microspheres were washed with ethanol to obtain the adsorption microspheres; TE buffer was added to the adsorption microspheres for desorption, and the magnetic microspheres were separated under the magnetic field. The microspheres obtain the eluate containing deoxyribonucleic acid, the method of the present invention has simple operation steps, does not need to use organic substances harmful to the human body, can save time and resources, and obtain high-purity DNA fragments, thereby having relatively good effects and efficiency. Yield.
Description
Technical Field
The invention relates to the technical field of genome, in particular to a method for extracting genome deoxyribonucleic acid.
Background
In the traditional gene typing, transgenic detection and strain detection tests, people conventionally adopt manual extraction or purchase of a general genome deoxyribonucleic acid (DNA) extraction kit, but no matter the manual extraction or purchase of the kit is adopted for extraction, a certain amount of materials are taken in the whole extraction process, and the genome deoxyribonucleic acid is finally obtained through the processes of liquid nitrogen grinding, lysate cracking, incubation, centrifugation, organic extraction, rinsing, airing and dissolving.
However, even if the risk of frostbite of operators caused by grinding with liquid nitrogen is not considered, the whole extraction process is complicated in steps, uses numerous instruments and has long waiting time.
For example, patent publication No. CN101792756B discloses a method for extracting genomic dna, which comprises adding lysis solution to biological tissue, adding proteinase k for incubation, and extracting dna by centrifugation, but dna extracted by this method is prone to have certain small-molecule impurities, and is difficult to be directly used for detection, and the whole extraction process is complicated in steps, and many instruments are used, and the waiting time is long.
Disclosure of Invention
The invention aims to provide a method for extracting genome deoxyribonucleic acid with good effect, weak irritation and good yield.
In order to solve the technical problems, the invention adopts the technical scheme that: a method for extracting genome deoxyribonucleic acid, comprising the following steps:
the method comprises the following steps: grinding and uniformly mixing biological tissues to obtain a first turbid solution, cleaning the first turbid solution by adopting a buffer solution, carrying out centrifugal separation, and pouring and discarding a supernatant to obtain a cell precipitate;
step two: adding lysis solution into the cell sediment in the step one, uniformly mixing, lysing and incubating until the solution has no lumps to obtain suspension;
step three: adding the pretreated magnetic microspheres into the suspension in the second step, uniformly mixing, shaking for adsorption, separating the magnetic microspheres on a magnetic field, taking out supernatant by using a straw, and washing the magnetic microspheres by using ethanol to obtain adsorption microspheres;
step four: and (3) adding TE buffer solution into the adsorption microspheres in the third step for desorption, and separating the magnetic microspheres under a magnetic field to obtain the eluent containing the deoxyribonucleic acid.
Further, the lysis solution comprises the following components: CHAPS with the volume fraction of 1-4%, EDTA with the volume fraction of 2-4mol/L, guanidine hydrochloride or guanidine thiocyanate with the volume fraction of 2-4mol/L and urea with the volume fraction of 6-10mol/L, wherein the CHAPS can destroy the spatial structure of protein molecules and carry out enzymolysis on the protein into small-segment polypeptides, the urea can compete with the polypeptides for hydrogen bonds in solution and simultaneously destroy the secondary structure of the protein, the solubility of the nonpolar side chain of the protein molecules in water is increased, the hydrophobic interaction of the three-dimensional structure is reduced, the conformation of the protein molecules is changed, the guanidine hydrochloride or guanidine thiocyanate substances can induce the protein to be unfolded and the solubility of the protein is increased, the EDTA serving as a complexing agent and a detergent can destroy cell membranes, a lysate can dissolve the protein on the cell walls to form cell wall cavities, and the cell membranes can also be broken by a lysate entering from the cell wall pore structures, and can decompose the protein combined with the DNA to expose the DNA molecules and dissolve the DNA molecules in the lysate.
Further, the incubation condition is that the incubation is carried out for 5-30min at 55-65 ℃, and the heating is carried out for 3-10min at 80-95 ℃.
Further, the biological tissue includes one of plant tissue, animal tissue, or bacteria.
Further, the magnetic microspheres are made of silicon dioxide, chitosan or agarose coated ferroferric oxide.
Furthermore, the surface of the magnetic microsphere is subjected to amination modification and carboxylation modification, the magnetic microsphere with the modified surface can adsorb DNA, and has the characteristic of strong magnetic responsiveness and large specific surface area.
Further, the pretreatment of the magnetic microspheres comprises the following steps: the magnetic microspheres are placed in a centrifuge tube, washed with buffer solution, shaken up, the centrifuge tube is placed on a magnetic separation rack for separation, and the suspension is removed by a pipette.
Compared with the prior art, the invention has the advantages and positive effects that:
the method for extracting the genome deoxyribonucleic acid can safely and quickly obtain DNA fragments, the lysate can dissolve protein on cell walls to form cell wall cavities, cell membranes can also be broken from the lysate entering from cell wall pore structures, and the protein combined with the DNA can be decomposed to expose DNA molecules to be dissolved in the lysate, the DNA can be adsorbed to the surface of the DNA through a magnetic separation technology, and the DNA can be separated from a sample through an external magnetic field.
Detailed Description
For a better understanding of the present invention, the present invention is further described below in conjunction with specific embodiments.
Example 1:
the method comprises the following steps: grinding plant tissues, uniformly mixing in a vortex mode to obtain a first turbid solution, cleaning the first turbid solution by adopting a buffer solution, carrying out centrifugal separation, and pouring and discarding supernatant to obtain cell sediment;
step two: adding lysis solution containing 1% by volume of CHAPS, 2mol/L of EDTA, 2mol/L of guanidine hydrochloride and 6mol/L of urea into the cell sediment in the first step, uniformly mixing, incubating at 55 ℃ for 5min, and heating at 80 ℃ for 3min until the solution has no lumps to obtain suspension;
step three: performing amination modification and carboxylation modification on the surface of a magnetic microsphere prepared by silicon dioxide coated ferroferric oxide, then placing the magnetic microsphere in a centrifugal tube, washing the magnetic microsphere with a buffer solution, shaking the magnetic microsphere uniformly, placing the centrifugal tube on a magnetic separation frame for separation, removing the suspension with a suction tube, adding the pretreated magnetic microsphere into the suspension obtained in the step two, uniformly mixing the mixture by vortex, performing shaking adsorption for 10min, separating the magnetic microsphere on a magnetic field, taking out supernatant with the suction tube, and washing the magnetic microsphere with ethanol to obtain an adsorption microsphere;
step four: adding TE buffer solution with the same volume as the cracking liquid into the adsorption microspheres in the third step for desorption, and separating the magnetic microspheres under a magnetic field to obtain the eluent containing the deoxyribonucleic acid.
Example 2:
the method comprises the following steps: grinding animal tissues, uniformly mixing in a vortex mode to obtain a first turbid liquid, cleaning the first turbid liquid by adopting a buffer solution, carrying out centrifugal separation, and pouring and discarding supernatant liquid to obtain cell sediment;
step two: adding lysis solution containing 2% of CHAPS by volume fraction, 3mol/L of EDTA, 3mol/L of guanidine thiocyanate and 8mol/L of urea into the cell sediment in the first step, uniformly mixing, incubating at 60 ℃ for 22min, and heating at 90 ℃ for 6min until the solution has no lumps to obtain suspension;
the third step, the fourth step, is the same as the example 1, except that the magnetic microspheres are made of chitosan-coated ferroferric oxide.
Example 3:
the method comprises the following steps: grinding the bacteria, uniformly mixing in a vortex mode to obtain a first turbid liquid, cleaning the first turbid liquid by adopting a buffer solution, carrying out centrifugal separation, and pouring and discarding supernatant to obtain cell sediment;
step two: adding lysis solution comprising CHAPS with volume fraction of 4%, EDTA with volume fraction of 4mol/L, guanidine hydrochloride with volume fraction of 4mol/L and urea with volume fraction of 10mol/L into the cell sediment obtained in the first step, uniformly mixing, incubating at 65 ℃ for 30min, and heating at 95 ℃ for 10min until the solution has no lumps to obtain suspension;
step three-step four is the same as in example 1, except that the magnetic microspheres are made of agarose coated ferroferric oxide.
Experimental example 1:
this example examined the effect of the concentration of each component of the lysate on the yield of DNA.
Blank control group a: PBS buffer solution is adopted;
experiment groups A, B, C, D and E, genome deoxyribonucleic acid was extracted according to the method of example 2, except that the volume fractions of CHAPS were 0%, 1%, 3%, 4% and 5%, respectively;
experiment group F, experiment group G, experiment group H, experiment group I: genomic deoxyribonucleic acid was extracted according to the method of example 2, except that the concentrations of EDTA were 0, 2, 4, 5mol/L, respectively;
experiment group J, experiment group K, experiment group L, experiment group M: genomic deoxyribonucleic acid was extracted according to the method of example 2, except that the concentrations of guanidine hydrochloride were 0, 2, 4, and 5mol/L, respectively;
experiment group N, experiment group O, experiment group P, experiment group Q: genomic deoxyribonucleic acid was extracted according to the method of example 2, except that the concentrations of urea were 0, 6, 10 and 12mol/L, respectively.
The DNA concentration in the eluate was quantitatively determined by means of an ultraviolet spectrophotometer, and the yield thereof was calculated, the results are shown in Table 1.
TABLE 1 determination of DNA yield (unit: mg/g)
| Group of | CHAPS(％) | EDTA(mol/L) | Guanidine hydrochloride (mol/L) | Urea (mol/L) | Yield (mg/g) |
| Blank control group | -- | -- | -- | -- | 0.0014 |
| Experimental group A | 0 | 3 | 3 | 8 | 0.1257 |
| Experimental group B | 1 | 3 | 3 | 8 | 0.1452 |
| Experimental group C | 3 | 3 | 3 | 8 | 0.1656 |
| Experimental group D | 4 | 3 | 3 | 8 | 0.1831 |
| Experimental group E | 5 | 3 | 3 | 8 | 0.1826 |
| Experimental group F | 3 | 0 | 3 | 8 | 0.1211 |
| Experimental group G | 3 | 2 | 3 | 8 | 0.1723 |
| Experimental group H | 3 | 4 | 3 | 8 | 0.1845 |
| Experimental group I | 3 | 5 | 3 | 8 | 0.1840 |
| Experimental group J | 3 | 3 | 0 | 8 | 0.1171 |
| Experimental group K | 3 | 3 | 2 | 8 | 0.1693 |
| Experimental group L | 3 | 3 | 4 | 8 | 0.1822 |
| Experimental group M | 3 | 3 | 5 | 8 | 0.1823 |
| Experimental group N | 3 | 3 | 3 | 0 | 0.1261 |
| Experimental group O | 3 | 3 | 3 | 6 | 0.1623 |
| Experimental group P | 3 | 3 | 3 | 10 | 0.1831 |
| Experimental group Q | 3 | 3 | 3 | 12 | 0.1833 |
From the measurement of DNA yields, it can be seen that the yields of DNA in experimental groups A-E increased with the increase in volume fraction of CHAPS, reaching a peak when the volume fraction of CHAPS increased to 4%, and the yields of DNA were lower when the lysate did not contain CHAPS;
the yield of DNA in the experimental group F-I increases with the increase of the concentration of EDTA, reaches a peak value when the concentration of EDTA increases to 4mol/L, is not different from 4mol/L when the concentration of EDTA increases to 5mol/L, and is lower when the lysate does not contain EDTA;
experimental groups J-M: the yield of DNA is increased along with the increase of the concentration of guanidine hydrochloride, the peak value is reached when the concentration of guanidine hydrochloride is increased to 4mol/L, the difference between the peak value and the peak value is not large when the concentration of guanidine hydrochloride is increased to 5mol/L, and the yield of DNA is low when the lysate does not contain guanidine hydrochloride;
experimental groups N-Q: the yield of DNA increases with the increase of the concentration of urea, reaches a peak when the concentration of urea increases to 10mol/L, does not differ much from 10mol/L when the concentration of urea increases to 12mol/L, and is lower when the lysis solution does not contain urea.
The CHAPS can destroy the space structure of protein molecules, proteolysis is carried out to obtain small-fragment polypeptide, urea can compete with polypeptide for hydrogen bonds in solution, the secondary structure of protein can be destroyed, the solubility of the nonpolar side chain of the protein molecules in water is increased, the hydrophobic interaction of the three-dimensional structure is reduced, the conformation of the protein molecules is changed, guanidine hydrochloride or guanidine thiocyanate substances can induce the protein to be unfolded, the solubility of the protein is increased, EDTA (ethylene diamine tetraacetic acid) serving as a complexing agent and a detergent can destroy cell membranes, the protein on the cell walls can be dissolved by lysate to form cell wall cavities, the cell membranes can also be broken by the lysate entering from the cell wall pore structures, and the protein combined with DNA can be decomposed to expose the DNA molecules and be dissolved in the lysate.
Experimental example 2:
this example examined the comparison of magnetic microspheres with conventional extraction for DNA yield.
Control groups A-C: the DNA extracted by the traditional extraction method is adopted, and the traditional extraction method is the prior art, so the description is not repeated, and the difference is that the biological tissues respectively adopt poplar leaves, calf thymus and saccharomyces cerevisiae;
experimental group R, experimental group S, experimental group T: extracting genome deoxyribonucleic acid according to the method of the embodiment 2, except that the biological tissues adopt poplar leaves, calf thymus and saccharomyces cerevisiae respectively;
experiment group U, experiment group V, experiment group W: the genomic deoxyribonucleic acid was extracted according to the method of example 2, except that the magnetic microspheres were silica microspheres, chitosan microspheres, and agarose microspheres, respectively.
The DNA concentration in the eluate was quantitatively determined by means of an ultraviolet spectrophotometer, and the yield thereof was calculated, the results are shown in Table 2.
TABLE 2 determination of DNA yield (unit: mg/g)
| Group of | Biological tissue | Magnetic microspheres | Yield (mg/g) |
| Control group A | Poplar leaf | -- | 0.1591 |
| Control group B | Calf thymus | -- | 0.1610 |
| Control group C | Saccharomyces cerevisiae | -- | 0.1588 |
| Experimental group R | Poplar leaf | Chitosan microspheres | 0.1626 |
| Experimental group S | Calf thymus | Chitosan microspheres | 0.1674 |
| Experimental group T | Saccharomyces cerevisiae | Shell polymerSugar microspheres | 0.1659 |
| Experimental group U | Calf thymus | Silica microspheres | 0.1677 |
| Experimental group V | Calf thymus | Agarose microspheres | 0.1667 |
Through the determination of the DNA yield, only the difference of extraction modes between the experimental group R-T and the control group A-C can be seen, the yield of the DNA is not greatly different, the magnetic microspheres with the modified surfaces can adsorb the DNA, the effect is slightly better than that of the traditional extraction method, the effect of animal materials of calf thymus is slightly better than that of plants and bacteria, because animal tissues do not have cell walls, and the experimental group T-U can see that the types of the magnetic microspheres have almost no difference on the yield of the DNA;
the method for extracting the genome deoxyribonucleic acid can safely and quickly obtain DNA fragments, the lysate can dissolve protein on cell walls to form cell wall cavities, cell membranes can also be broken from the lysate entering from cell wall pore structures, and the protein combined with the DNA can be decomposed to expose DNA molecules to be dissolved in the lysate, the DNA can be adsorbed to the surface of the DNA through a magnetic separation technology, and the DNA can be separated from a sample through an external magnetic field.
The embodiments of the present invention have been described in detail, but the description is only for the preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made within the scope of the present invention should be covered by the present patent.
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