WO2018223657A1 - Chaotropic agent and method for extracting genomic dna by using same - Google Patents

Chaotropic agent and method for extracting genomic dna by using same Download PDF

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WO2018223657A1
WO2018223657A1 PCT/CN2017/116735 CN2017116735W WO2018223657A1 WO 2018223657 A1 WO2018223657 A1 WO 2018223657A1 CN 2017116735 W CN2017116735 W CN 2017116735W WO 2018223657 A1 WO2018223657 A1 WO 2018223657A1
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genomic dna
chaotropic agent
dna
mol
extracting
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PCT/CN2017/116735
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French (fr)
Chinese (zh)
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陈礼平
王海英
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陈礼平
王海英
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Priority to JP2019515850A priority Critical patent/JP6704563B2/en
Publication of WO2018223657A1 publication Critical patent/WO2018223657A1/en

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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

Definitions

  • the present invention relates to the field of biotechnology, and in particular to a chaotropic agent for rapidly separating histones from genomic DNA, and a method for extracting DNA.
  • genomic DNA of an organism refers to all genetic information contained in the DNA of the organism. Genomic DNA and histones are bound together by non-covalent bonds. Therefore, how to separate DNA and histones is the key and difficult point in the process of genomic DNA extraction. In fact, different genomic DNA extraction methods, pre-sample pretreatment (cell lysis) and post-DNA precipitation, washing and other steps are similar, the core difference between the different methods is how to effectively separate DNA and histones.
  • the Protease K method is the most classical method that uses proteinase K to degrade histones, thereby releasing genomic DNA.
  • the disadvantage of this method is that: (1) The incubation temperature of proteinase K is 50 to 65 ° C, requiring additional heating equipment. (2) Proteinase K requires time to digest histones, often 0.5h-overnight, and the duration of the experiment. (3) With the prolongation of the standing time and the increase of the number of repeated freeze-thaw cycles, the enzyme activity of proteinase K will decrease, and the experimental repeatability and stability are poor.
  • a chaotropic agent is often used in practical applications to separate histones and genomic DNA.
  • the chaotropic agent can break the non-covalent interaction between histone and genomic DNA (hydrogen bond, dipole-dipole interaction force, hydrophobic interaction force), reversibly denature the histone, and release genomic DNA.
  • the purpose of genomic DNA extraction include guanidine hydrochloride, guanidinium thiocyanate, sodium perchlorate, and lithium perchlorate.
  • the concentration of the reagent used in the guanidine hydrochloride and guanidinium thiocyanate method is high, and the concentration of the reagent used in the sodium perchlorate and the lithium perchlorate method is low, but the valence of the chlorine element in the perchlorate is high, which is +7, the reagent Strong oxidizing.
  • the object of the present invention is to overcome the deficiencies of the proteinase K method and the perchlorate method, and to provide a chaotropic agent for rapidly separating histones from genomic DNA, and a method for extracting DNA.
  • Chaotropic agents are a class of compounds that break the non-covalent interaction between histones and genomic DNA, which can reversibly denature histones, thereby releasing genomic DNA and achieving genomic DNA extraction.
  • the present invention provides a chaotropic agent, characterized in that the chaotropic agent contains a bromate.
  • the bromate is sodium bromate.
  • the final concentration of the sodium bromate is 0.1 to 3 mol/L.
  • the final concentration of the sodium bromate is 0.2-3 mol/L.
  • the final concentration of the sodium bromate is 1-3 mol/L.
  • the present invention also provides a method of extracting genomic DNA using the chaotropic agent, the DNA being bound to histone before extraction; wherein the method comprises the following steps:
  • the supernatant obtained by extracting the protein is mixed with a DNA precipitating agent, and centrifuged to obtain a DNA precipitate.
  • the method also includes washing and redissolving the DNA pellet.
  • the present invention replaces the histone and genomic DNA with the chemical substance sodium bromate (NaBrO 3 ) instead of the protease K.
  • This method has the following advantages: (1) no need to use the proteinase K, avoid heating and need to be mixed from time to time. Uniform, easy to operate, and no heating equipment required. (2) Avoid prolonged digestion of proteinase K, saving operating time. (3) Treatment of samples with chemical reagents to avoid insufficient disadvantages of proteinase K digestion, rapid and sufficient separation of histones and DNA, high DNA yield and high purity. (4) It avoids the defect that the activity of proteinase K is easy to change, has good repeatability and high stability. Compared with the conventional proteinase K digestion method, the present invention can extract eukaryotic genomic DNA including humans efficiently, quickly and simply.
  • the purity and concentration of the genomic DNA extracted by the method of the present invention are comparable to those of the sodium perchlorate or lithium perchlorate method, but the chaotropic agent used has a lower halogenation valence (+5 valence) and the reagent oxidizing property is weaker.
  • the purity and concentration of the extracted DNA can meet the requirements of molecular biology related experiments such as PCR gene amplification, chip analysis, molecular cloning, gene (group) sequencing/detection.
  • Fig. 1 shows the results of electrophoretic detection of sodium bromate method (NaBrO 3 method) and proteinase K method (digestion for 40 min) for extracting salivary method and buccal swab method for collecting oral mucosal epithelial cells.
  • Fig. 2 shows the results of electrophoretic detection of the extraction of DNA from oral mucosal epithelial cells by saliva extraction by sodium bromate method (NaBrO 3 method) and proteinase K method (digestion only for 5 min).
  • Figure 3 shows the results of electrophoretic detection of human leukocyte DNA extracted from different concentrations of sodium bromate and 1 mol/L sodium perchlorate.
  • the invention provides a chaotropic agent which is a compound capable of breaking the non-covalent interaction between histones and genomic DNA, which reversibly denatures histones, thereby releasing genomic DNA To achieve the purpose of genomic DNA extraction.
  • the final concentration of sodium bromate is 0.1-3 mol/L, preferably 0.2-3. Mol/L is more preferably 1-3 mol/L.
  • the present invention also provides a method of extracting DNA, which binds to histones prior to extraction; wherein the method comprises the steps of: (1) dispersing the sample into a single cell suspension, and adding the cell lysis The liquid ruptures the cell, releasing a complex of histones and genomic DNA in the nucleus; (2) mixing the cell lysate with the chaotropic agent to obtain a material separated from the genomic DNA; (3) will (2) The material is uniformly mixed with the protein extract, and centrifuged to separate the supernatant from which the protein is extracted; (4) mixing the supernatant after the protein extraction with the DNA precipitant, and after centrifugation A DNA precipitate is obtained; wherein the chaotropic agent contains sodium bromate.
  • the sample containing histone and genomic DNA contains a cell lysate.
  • the method may further comprise: pretreating the eukaryotic material to obtain a biomaterial containing histone and genomic DNA suitable for direct cleavage.
  • the pretreatment operation includes at least one of washing, drying, shredding, grinding, freezing, and freezing and thawing.
  • the pretreatment operation can be carried out in a conventional manner in the art, for example, according to the contents described in the Guide to Molecular Cloning Experiments.
  • the eukaryotic material may include at least one of a tissue, an organ, an individual, and a symbiont.
  • the biomaterial may include at least one of oral mucosal epithelial cells, blood, and artificial cultured cells.
  • the method further comprises: precipitating, washing and redissolving the DNA.
  • the reagent for genomic DNA precipitation may be 2-2.5 volumes of 95% ethanol or absolute ethanol, an equal volume of isopropanol;
  • the washing liquid used for washing may be an aqueous solution of ethanol having a concentration of 60-80% by volume;
  • the solvent used for redissolution may be water or TE buffer.
  • This example is used to illustrate the extraction of saliva samples using two different methods. DNA Operation.
  • Sample pretreatment centrifuge 2000 g of saliva for 10 min, discard the supernatant, suspend the oral mucosal epithelial cells with 1 mL of normal saline, and centrifuge at 2000 g. Min, discard the supernatant.
  • cell lysate formulation was: 10 mmol/L Tris-HCl (pH 8.0), 30 mmol/L EDTA (pH 8.0), 0.5%. SDS, RNase A (20 ⁇ g/mL).
  • Genomic DNA was extracted using the proteinase K method and the sodium bromate method, respectively.
  • Protease K method Add the proteinase K with a final concentration of 100 ⁇ g/mL to the EP tube of step (2), mix well, and place at 56 °C for 40 min, mixing occasionally.
  • Sodium Bromate Method Add the chaotropic solution sodium bromate with a final concentration of 1 mol/L to the EP tube of step (2) and mix well. The remaining steps are the same.
  • step (4) Add 2.5 volumes of absolute ethanol to the EP tube of step (4).
  • the proteinase K group needs to first add 1/10 volume of NaAc (3 mol/L, pH 5.2), place at -20 ° C for 20 min, and centrifuge. Collecting genomic DNA;
  • TE buffer 100 ⁇ L was added to the EP tube of step (6) to dissolve the genomic DNA pellet.
  • concentration of genomic DNA was determined by densitometry, and the integrity of genomic DNA was detected by agarose gel electrophoresis.
  • This example is intended to illustrate the extraction of oral mucosal epithelial cells collected from buccal swabs using two different methods. DNA Operation.
  • the cell lysate formulation is: 10 mmol/L Tris-HCl (pH 8.0), 30 mmol/L EDTA (pH 8.0), 0.5% SDS, RNase A (20 ⁇ g/mL).
  • Genomic DNA was extracted using the proteinase K method and the sodium bromate method, respectively.
  • Protease K method Add the proteinase K with a final concentration of 100 ⁇ g/mL to the EP tube of step (2), mix well, and place at 56 °C for 40 min, mixing occasionally.
  • Sodium Bromate Method Add the chaotropic solution sodium bromate with a final concentration of 1 mol/L to the EP tube of step (2) and mix well. The remaining steps are the same.
  • step (4) Add 2.5 volumes of absolute ethanol to the EP tube of step (4).
  • the proteinase K group needs to first add 1/10 volume of NaAc (3 mol/L, pH 5.2), place at -20 ° C for 20 min, and centrifuge. Collecting genomic DNA;
  • TE buffer 100 ⁇ L was added to the EP tube of step (6) to dissolve the genomic DNA pellet.
  • concentration of genomic DNA was determined by densitometry, and the integrity of genomic DNA was detected by agarose gel electrophoresis.
  • This embodiment is used to illustrate the utilization Sodium bromate method NaBrO 3 Method and protease K Extraction of oral mucosal epithelial cells in saliva by short-time digestion DNA Operation.
  • Sample pretreatment centrifuge 2000 g of saliva for 10 min, discard the supernatant, suspend the oral mucosal epithelial cells with 1 mL of normal saline, and centrifuge at 2000 g. Min, discard the supernatant.
  • cell lysate formulation was: 10 mmol/L Tris-HCl (pH 8.0), 30 mmol/L EDTA (pH 8.0), 0.5%. SDS, RNase A (20 ⁇ g/mL).
  • Genomic DNA was extracted using the proteinase K short-time digestion method and the sodium bromate method, respectively.
  • Protease K method Add proteinase K at a final concentration of 100 ⁇ g/mL to the EP tube of step (2), mix well, and digest at 56 °C for 5 min.
  • Sodium Bromate Method Add the chaotropic solution sodium bromate with a final concentration of 1 mol/L to the EP tube of step (2) and mix well. The remaining steps are the same.
  • step (4) Add 2.5 volumes of absolute ethanol to the EP tube of step (4).
  • the proteinase K group needs to first add 1/10 volume of NaAc (3 mol/L, pH 5.2), place at -20 ° C for 20 min, and centrifuge. Collecting genomic DNA;
  • TE buffer 100 ⁇ L was added to the EP tube of step (6) to dissolve the genomic DNA pellet.
  • concentration of genomic DNA was determined by densitometry, and the integrity of genomic DNA was detected by agarose gel electrophoresis.
  • This embodiment is used to illustrate the use of different concentrations Sodium bromate NaBrO 3 )with 1 mol/L Sodium perchlorate extracts human blood leukocytes DNA Operation.
  • Sample pretreatment Add 400 ⁇ L of red blood cell lysate to 200 ⁇ L of anticoagulated blood, mix and centrifuge at 2000 g for 10 min, and discard the supernatant. If there is still more red blood cell residue, the lysis can be repeated once.
  • Genomic DNA was extracted using the sodium perchlorate method and the sodium bromate method, respectively.
  • Sodium perchlorate method Add sodium perchlorate with a final concentration of 1 mol/L to the EP tube of step (2) and mix well.
  • Sodium bromate method adding the final concentration to the EP tube of step (2) is 0.1 mol/L, 0.2 Mol/L, 0.5 mol/L, 1 Mix thoroughly with mol/L and 3 mol/L sodium bromate. The remaining steps are the same.
  • step (4) Adding 2.5 volumes of absolute ethanol to the EP tube of step (4), placing at -20 ° C for 20 min, and collecting genomic DNA by centrifugation;
  • TE buffer 100 ⁇ L was added to the EP tube of step (6) to dissolve the genomic DNA pellet.
  • concentration of genomic DNA was determined by densitometry, and the integrity of genomic DNA was detected by agarose gel electrophoresis.
  • Example 1-4 The purity and concentration of the human genomic DNA extracted in Example 1-4 were summarized, and the results are shown in Table 1.
  • the DNA yields of the oral mucosal epithelial cells of the same source, the sodium bromate method and the proteinase K method were observed in the case where the protease K digestion time was sufficient (the digestion time was 40 min in the examples).
  • the purity is equivalent; however, when the digestion time is greatly shortened (the digestion time of 5 min is used in the examples), the protease K cannot sufficiently digest histones, and the DNA yield of the proteinase K method is greatly reduced, and the present invention relates to
  • the chaotropic agent sodium bromate can rapidly separate histones and genomic DNA.
  • the yield of the DNA extracted by this method is about 5 times that of the proteinase K method, and the DNA purity is not affected.
  • 0.1-3 mol/L sodium bromate can extract genomic DNA, and 1-3 mol/L sodium bromate extract has the highest concentration and is relatively stable.
  • the genomic DNA extracted by 1 mol/L sodium bromate and 1 mol/L sodium perchlorate was comparable in yield and purity.
  • Example 1-4 The human genomic DNA extracted in Example 1-4 was subjected to electrophoresis, and the results are shown in Figs. among them:
  • Fig. 1 shows the results of electrophoretic detection of sodium bromate method (NaBrO 3 method) and proteinase K method (digestion for 40 min) for extracting salivary method and buccal swab method for collecting oral mucosal epithelial cells.
  • M is a DNA marker (molecular weight from top to bottom: 2000, 1000, 750, 500, 250, 100, unit is bp)
  • lane 1 is the salivary-derived oral mucosal epithelial DNA extracted by proteinase K method
  • lane 2 is bromine.
  • the saliva derived from the sodium method is derived from the oral mucosal epithelial cell DNA
  • the lane 3 is the oral swab-derived oral mucosal epithelial cell DNA extracted by the protease K method
  • the lane 4 is the oral swab-derived oral mucosal epithelial cell DNA extracted by the sodium bromate method.
  • the protease K digestion time is sufficient, the DNA yield and purity of the oral mucosal epithelial cells of the same source, the sodium bromate method and the proteinase K method are comparable.
  • Fig. 2 shows the results of electrophoretic detection of the extraction of DNA from oral mucosal epithelial cells by saliva extraction by sodium bromate method (NaBrO 3 method) and proteinase K method (digestion only for 5 min).
  • M is a DNA marker (molecular weight from top to bottom: 2000, 1000, 750, 500, 250, 100, unit is bp)
  • lane 1 is the salivary-derived oral mucosal epithelial DNA extracted by proteinase K method
  • lane 2 is bromine.
  • the saliva derived from the sodium method is derived from oral mucosal epithelial DNA.
  • the sodium bromate method used in the present invention has a higher DNA yield than the conventional proteinase K method.
  • Figure 3 shows the results of electrophoretic detection of human leukocyte DNA extracted from different concentrations of sodium bromate (NaBrO 3 ) and 1 mol/L sodium perchlorate.
  • M is a DNA marker (molecular weight from top to bottom: 2000, 1000, 750, 500, 250, 100, bp), and lanes 1-5 are final concentrations of 0.1 mol/L, 0.2 mol/L, and 0.5 mol, respectively.
  • Human blood leukocyte genomic DNA extracted with /L, 1 mol/L and 3 mol/L NaBrO 3 , and lane 6 is human leukocyte genomic DNA extracted with sodium perchlorate at a final concentration of 1 mol/L. According to Fig.
  • 0.1 mol/L-3 mol/L NaBrO 3 can extract cellular genomic DNA, and the genome extracted by 1 mol/L-3 mol/L NaBrO 3 has little difference.
  • the yield of 1 mol/L-3 mol/L NaBrO 3 and 1 mol/L sodium perchlorate method is comparable.

Abstract

A chaotropic agent and a method for extracting genomic DNA by using same. The chaotropic agent contains sodium bromate, and the compound can quickly and effectively separate histone in a nucleosomal structure from genomic DNA. The method for extracting genomic DNA by using the chaotropic agent comprises the following steps: adding a cell lysis solution to a sample to rupture cells; then mixing the sample with a chaotropic agent uniformly to obtain a material in which histone and genomic DNA are separated; adding protein extracting liquid, followed by uniform mixing and centrifugation, to obtain supernatant after protein is extracted; and mixing the supernatant with a DNA precipitant, and centrifuging the mixture to obtain a DNA precipitate. Compared with a method using sodium perchlorate, this method is different in the type of the chaotropic agent, and has lower valence and more appropriate yield and purity of the genomic DNA. Compared with a conventional proteinase K method, this method requires no long-time digestion, saves a great deal of time, overcomes the defect of susceptibility to change in protease K activity and the disadvantage of inadequate digestion, and features good repeatability and high stability.

Description

一种离液剂以及使用该离液剂提取基因组DNA的方法Separating agent and method for extracting genomic DNA using the same 技术领域Technical field
本发明涉及生物技术领域，具体地，涉及一种快速分离组蛋白与基因组DNA的离液剂，以及提取DNA的方法。 The present invention relates to the field of biotechnology, and in particular to a chaotropic agent for rapidly separating histones from genomic DNA, and a method for extracting DNA.
背景技术Background technique
对包括人在内的真核生物而言，一个生物体的基因组DNA是指包含在该生物的DNA中的全部遗传信息。基因组DNA 和组蛋白通过非共价键结合在一起，因此，如何将DNA和组蛋白进行分离，这是基因组DNA提取过程中的关键及难点所在。事实上，不同的基因组DNA提取方法，前期样本预处理（细胞裂解）及后期DNA的沉淀、洗涤等步骤大同小异，不同方法的核心差异在于如何将DNA和组蛋白进行有效分离。For eukaryotes including humans, genomic DNA of an organism refers to all genetic information contained in the DNA of the organism. Genomic DNA and histones are bound together by non-covalent bonds. Therefore, how to separate DNA and histones is the key and difficult point in the process of genomic DNA extraction. In fact, different genomic DNA extraction methods, pre-sample pretreatment (cell lysis) and post-DNA precipitation, washing and other steps are similar, the core difference between the different methods is how to effectively separate DNA and histones.
蛋白酶K法是最经典的方法，该方法利用蛋白酶K来降解组蛋白，从而释放出基因组DNA。该方法的劣势在于：（1）蛋白酶K的孵育温度为50至65℃，需要额外的加热设备。（2）蛋白酶K对组蛋白的消化需要时间，常需0.5h-过夜，实验用时长。（3）随着放置时间的延长和反复冻融次数的增加，蛋白酶K的酶活会下降，实验重复性和稳定性较差。The Protease K method is the most classical method that uses proteinase K to degrade histones, thereby releasing genomic DNA. The disadvantage of this method is that: (1) The incubation temperature of proteinase K is 50 to 65 ° C, requiring additional heating equipment. (2) Proteinase K requires time to digest histones, often 0.5h-overnight, and the duration of the experiment. (3) With the prolongation of the standing time and the increase of the number of repeated freeze-thaw cycles, the enzyme activity of proteinase K will decrease, and the experimental repeatability and stability are poor.
为了规避蛋白酶K法的劣势，实际应用中常采用离液剂来分离组蛋白和基因组DNA。离液剂可以打破组蛋白和基因组DNA之间的非共价作用力（氢键，偶极-偶极相互作用力，疏水相互作用力），使组蛋白发生可逆性变性，从而释放基因组DNA，实现基因组DNA提取的目的。基因组DNA提取中常见的离液剂包括盐酸胍、硫氰酸胍、高氯酸钠、高氯酸锂。其中盐酸胍和硫氰酸胍法所用的试剂浓度高，高氯酸钠和高氯酸锂法所用试剂浓度虽然较低，但高氯酸盐中氯元素的化合价高，为+7价，试剂氧化性强。In order to avoid the disadvantages of the proteinase K method, a chaotropic agent is often used in practical applications to separate histones and genomic DNA. The chaotropic agent can break the non-covalent interaction between histone and genomic DNA (hydrogen bond, dipole-dipole interaction force, hydrophobic interaction force), reversibly denature the histone, and release genomic DNA. The purpose of genomic DNA extraction. The chaotropic agents commonly used in genomic DNA extraction include guanidine hydrochloride, guanidinium thiocyanate, sodium perchlorate, and lithium perchlorate. Among them, the concentration of the reagent used in the guanidine hydrochloride and guanidinium thiocyanate method is high, and the concentration of the reagent used in the sodium perchlorate and the lithium perchlorate method is low, but the valence of the chlorine element in the perchlorate is high, which is +7, the reagent Strong oxidizing.
技术问题technical problem
本发明的目的是克服蛋白酶K法和高氯酸盐法的缺陷，提供一种快速分离组蛋白与基因组DNA的离液剂，以及提取DNA的方法。The object of the present invention is to overcome the deficiencies of the proteinase K method and the perchlorate method, and to provide a chaotropic agent for rapidly separating histones from genomic DNA, and a method for extracting DNA.
离液剂是一类能打破组蛋白和基因组DNA之间的非共价作用力的化合物，其能使组蛋白发生可逆性变性，从而释放基因组DNA，实现基因组DNA提取的目的。Chaotropic agents are a class of compounds that break the non-covalent interaction between histones and genomic DNA, which can reversibly denature histones, thereby releasing genomic DNA and achieving genomic DNA extraction.
技术解决方案Technical solution
本发明提供了一种离液剂，其特征在于，所述离液剂含有溴酸盐。The present invention provides a chaotropic agent, characterized in that the chaotropic agent contains a bromate.
优选的，所述溴酸盐为溴酸钠。Preferably, the bromate is sodium bromate.
将含有组蛋白及基因组DNA的样本与所述离液剂混匀后，所述溴酸钠的终浓度为0.1-3mol/L。After mixing a sample containing histones and genomic DNA with the chaotropic agent, the final concentration of the sodium bromate is 0.1 to 3 mol/L.
优选的，将含有组蛋白及基因组DNA的样本与所述离液剂混匀后，所述溴酸钠的终浓度为0.2-3mol/L。Preferably, after mixing the sample containing histone and genomic DNA with the chaotropic agent, the final concentration of the sodium bromate is 0.2-3 mol/L.
更优选的，将含有组蛋白及基因组DNA的样本与所述离液剂混匀后，所述溴酸钠的终浓度为1-3mol/L。More preferably, after the sample containing histone and genomic DNA is mixed with the chaotropic agent, the final concentration of the sodium bromate is 1-3 mol/L.
本发明还提供了一种使用所述离液剂提取基因组DNA的方法，所述DNA在提取前结合在组蛋白中；其中，该方法包括如下步骤：The present invention also provides a method of extracting genomic DNA using the chaotropic agent, the DNA being bound to histone before extraction; wherein the method comprises the following steps:
（1）将样本分散成细胞悬液，加入细胞裂解液使细胞破裂，释放出细胞核内的组蛋白与基因组DNA的复合物；(1) Dispersing the sample into a cell suspension, adding a cell lysate to rupture the cell, releasing a complex of histones and genomic DNA in the nucleus;
（2）将步骤（1）得到的物料与所述离液剂混合均匀，得到组蛋白与基因组DNA分离后的物料；(2) mixing the material obtained in the step (1) with the chaotropic agent to obtain a material separated from the genomic DNA by the histone protein;
（3）将步骤（2）得到的物料与蛋白抽提液混合均匀，离心，分离得到蛋白被抽提后的上清液；(3) mixing the material obtained in the step (2) with the protein extract, and centrifuging, separating the supernatant from which the protein is extracted;
（4）将所述经蛋白被抽提后的上清液与DNA沉淀剂混合，离心后得到DNA沉淀。(4) The supernatant obtained by extracting the protein is mixed with a DNA precipitating agent, and centrifuged to obtain a DNA precipitate.
该方法还包括，将所述DNA沉淀进行洗涤和再溶解。The method also includes washing and redissolving the DNA pellet.
通过上述技术方案，本发明以化学物质溴酸钠（NaBrO 3）替代蛋白酶K来解离组蛋白和基因组DNA，这种方法具有以下优点：（1）无需使用蛋白酶K，避免加热并需不时混匀等环节，操作简单，且无需加热设备。（2）避免蛋白酶K长时间消化，节省了操作时间。（3）利用化学试剂处理样本，避免蛋白酶K消化不充分的劣势，快速、充分地分离组蛋白和DNA，DNA得率高、纯度高。（4）避免蛋白酶K活性易变化的缺陷，重复性好，稳定性高。与传统蛋白酶K消化法相比，本发明能高效、快速、简便地提取包括人类在内的真核生物基因组DNA。 Through the above technical solution, the present invention replaces the histone and genomic DNA with the chemical substance sodium bromate (NaBrO 3 ) instead of the protease K. This method has the following advantages: (1) no need to use the proteinase K, avoid heating and need to be mixed from time to time. Uniform, easy to operate, and no heating equipment required. (2) Avoid prolonged digestion of proteinase K, saving operating time. (3) Treatment of samples with chemical reagents to avoid insufficient disadvantages of proteinase K digestion, rapid and sufficient separation of histones and DNA, high DNA yield and high purity. (4) It avoids the defect that the activity of proteinase K is easy to change, has good repeatability and high stability. Compared with the conventional proteinase K digestion method, the present invention can extract eukaryotic genomic DNA including humans efficiently, quickly and simply.
有益效果Beneficial effect
与高氯酸钠或高氯酸锂法相比，本发明的方法提取的基因组DNA纯度和浓度相当，但所用离液剂的卤素化合价更低（+5价），试剂氧化性较弱。所提取DNA的纯度和浓度能满足PCR基因扩增、芯片分析、分子克隆、基因（组）测序/检测等分子生物学相关实验需求。The purity and concentration of the genomic DNA extracted by the method of the present invention are comparable to those of the sodium perchlorate or lithium perchlorate method, but the chaotropic agent used has a lower halogenation valence (+5 valence) and the reagent oxidizing property is weaker. The purity and concentration of the extracted DNA can meet the requirements of molecular biology related experiments such as PCR gene amplification, chip analysis, molecular cloning, gene (group) sequencing/detection.
本发明的其他特征和优点将在随后的具体实施方式部分予以详细说明。Other features and advantages of the invention will be described in detail in the detailed description which follows.
附图说明DRAWINGS
附图是用来提供对本发明的进一步理解，并且构成说明书的一部分，与下面的具体实施方式一起用于解释本发明，但并不构成对本发明的限制。在附图中：The drawings are intended to provide a further understanding of the invention, and are intended to be a In the drawing:
图1是溴酸钠法（NaBrO 3法）和蛋白酶K法（消化40min）分别提取唾液法和口腔拭子法采集口腔粘膜上皮细胞DNA的电泳检测结果。 Fig. 1 shows the results of electrophoretic detection of sodium bromate method (NaBrO 3 method) and proteinase K method (digestion for 40 min) for extracting salivary method and buccal swab method for collecting oral mucosal epithelial cells.
图2是溴酸钠法（NaBrO 3法）和蛋白酶K法（仅消化5min）分别提取唾液法采集口腔粘膜上皮细胞DNA的电泳检测结果。 Fig. 2 shows the results of electrophoretic detection of the extraction of DNA from oral mucosal epithelial cells by saliva extraction by sodium bromate method (NaBrO 3 method) and proteinase K method (digestion only for 5 min).
图3是不同浓度的溴酸钠和1 mol/L的高氯酸钠分别提取人血液白细胞DNA的电泳检测结果。Figure 3 shows the results of electrophoretic detection of human leukocyte DNA extracted from different concentrations of sodium bromate and 1 mol/L sodium perchlorate.
本发明的最佳实施方式BEST MODE FOR CARRYING OUT THE INVENTION
在此处键入本发明的最佳实施方式描述段落。The description of the preferred embodiment of the invention is entered here.
本发明的实施方式Embodiments of the invention
以下结合附图对本发明的具体实施方式进行详细说明。应当理解的是，此处所描述的具体实施方式仅用于说明和解释本发明，并不用于限制本发明。The specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative and not restrictive.
一方面，本发明提供了一种离液剂，该离液剂是能打破组蛋白和基因组DNA之间的非共价作用力的化合物，其能使组蛋白发生可逆性变性，从而释放基因组DNA，实现基因组DNA提取的目的。In one aspect, the invention provides a chaotropic agent which is a compound capable of breaking the non-covalent interaction between histones and genomic DNA, which reversibly denatures histones, thereby releasing genomic DNA To achieve the purpose of genomic DNA extraction.
其中，将含有组蛋白及基因组DNA的样本与离液剂混匀后，溴酸钠的终浓度为0.1-3 mol/L，优选为0.2-3 mol/L，更优选为1-3 mol/L。Wherein, after mixing the sample containing histone and genomic DNA with the chaotropic agent, the final concentration of sodium bromate is 0.1-3 mol/L, preferably 0.2-3. Mol/L is more preferably 1-3 mol/L.
另一方面，本发明还提供了一种提取DNA的方法，所述DNA在提取前与组蛋白结合；其中，该方法包括如下步骤：（1）将样本分散成单细胞悬液，加入细胞裂解液使细胞破裂，释放出细胞核内的组蛋白与基因组DNA的复合物；（2）将细胞裂解液与离液剂混合均匀，得到组蛋白与基因组DNA分离后的物料；（3）将（2）所述物料与蛋白抽提液混合均匀，离心，分离得到蛋白被抽提后的上清液；（4）将所述经蛋白被抽提后的上清液与DNA沉淀剂混合，离心后得到DNA沉淀；其中，所述离液剂含有溴酸钠。In another aspect, the present invention also provides a method of extracting DNA, which binds to histones prior to extraction; wherein the method comprises the steps of: (1) dispersing the sample into a single cell suspension, and adding the cell lysis The liquid ruptures the cell, releasing a complex of histones and genomic DNA in the nucleus; (2) mixing the cell lysate with the chaotropic agent to obtain a material separated from the genomic DNA; (3) will (2) The material is uniformly mixed with the protein extract, and centrifuged to separate the supernatant from which the protein is extracted; (4) mixing the supernatant after the protein extraction with the DNA precipitant, and after centrifugation A DNA precipitate is obtained; wherein the chaotropic agent contains sodium bromate.
其中，作为本发明的一种优选实施方式，所述含有组蛋白及基因组DNA的样本中含有细胞裂解液。Among them, as a preferred embodiment of the present invention, the sample containing histone and genomic DNA contains a cell lysate.
其中，该方法还可以进一步包括：将真核生物材料进行预处理，以获得适合直接用于裂解的、含组蛋白和基因组DNA的生物材料。所述预处理的操作包括清洗、干燥、剪碎、研磨、冷冻和冻融中的至少一种。可以按照本领域的常规方式进行预处理操作，例如按照《分子克隆实验指南》中记载的内容进行操作。Wherein, the method may further comprise: pretreating the eukaryotic material to obtain a biomaterial containing histone and genomic DNA suitable for direct cleavage. The pretreatment operation includes at least one of washing, drying, shredding, grinding, freezing, and freezing and thawing. The pretreatment operation can be carried out in a conventional manner in the art, for example, according to the contents described in the Guide to Molecular Cloning Experiments.
其中，所述真核生物材料可以包括组织、器官、个体和共生体中的至少一种。所述生物材料可以包括口腔粘膜上皮细胞、血液和人工培养细胞中的至少一种。Wherein, the eukaryotic material may include at least one of a tissue, an organ, an individual, and a symbiont. The biomaterial may include at least one of oral mucosal epithelial cells, blood, and artificial cultured cells.
其中，该方法还包括：将所述DNA进行沉淀、洗涤和再溶解。其中，基因组DNA沉淀所用的试剂可以为2-2.5倍体积的95%乙醇或无水乙醇、等体积的异丙醇；进行洗涤所用的洗涤液可以为60-80体积%浓度的乙醇水溶液；进行再溶解所用的溶剂可以为水或TE缓冲液。Wherein, the method further comprises: precipitating, washing and redissolving the DNA. Wherein, the reagent for genomic DNA precipitation may be 2-2.5 volumes of 95% ethanol or absolute ethanol, an equal volume of isopropanol; the washing liquid used for washing may be an aqueous solution of ethanol having a concentration of 60-80% by volume; The solvent used for redissolution may be water or TE buffer.
以下通过实施例进一步详细说明本发明。The invention will now be described in further detail by way of examples.
 
实施例Example 11 ：:
本实施例用于说明利用两种不同方法从唾液样品中提取This example is used to illustrate the extraction of saliva samples using two different methods. DNADNA 的操作。Operation.
(1)   样品预处理：将唾液2000 g离心10 min，弃上清，用1 mL的生理盐水悬浮口腔粘膜上皮细胞，再2000 g离心10 min，弃上清。(1) Sample pretreatment: centrifuge 2000 g of saliva for 10 min, discard the supernatant, suspend the oral mucosal epithelial cells with 1 mL of normal saline, and centrifuge at 2000 g. Min, discard the supernatant.
(2)   往Eppendorf（EP）管中加入400 μL的细胞裂解液, 充分混匀至无明显的细胞团。其中细胞裂解液配方为：10 mmol/L Tris-HCl (pH8.0)，30 mmol/L EDTA (pH8.0)，0.5% SDS，RNase A (20 μg/mL)。(2) Add 400 μL of cell lysate to the Eppendorf (EP) tube and mix well until no obvious cell mass. The cell lysate formulation was: 10 mmol/L Tris-HCl (pH 8.0), 30 mmol/L EDTA (pH 8.0), 0.5%. SDS, RNase A (20 μg/mL).
(3)   分别采用蛋白酶K法和溴酸钠法来提取基因组DNA。蛋白酶K法：向步骤（2）EP管中加入终浓度为100 μg/mL的蛋白酶K，充分混匀，56 ℃放置40 min，期间不时混匀。溴酸钠法：向步骤（2）EP管中加入终浓度为1 mol/L的离液剂溴酸钠，充分混匀。其余步骤相同。(3) Genomic DNA was extracted using the proteinase K method and the sodium bromate method, respectively. Protease K method: Add the proteinase K with a final concentration of 100 μg/mL to the EP tube of step (2), mix well, and place at 56 °C for 40 min, mixing occasionally. Sodium Bromate Method: Add the chaotropic solution sodium bromate with a final concentration of 1 mol/L to the EP tube of step (2) and mix well. The remaining steps are the same.
(4)   向步骤(3)的EP管中加入等体积苯酚/氯仿/异戊醇 (25:24:1)，振荡混匀，离心至溶液分层，将上层液体转移至另一洁净的EP管中。可重复本步骤一次，并用氯仿抽提一次上清，将上清转移至另一洁净的EP管中；(4) An equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) was added to the EP tube of step (3), vortexed and mixed, centrifuged until the solution was layered, and the upper layer liquid was transferred to another clean EP tube. This step can be repeated once, and the supernatant is extracted once with chloroform, and the supernatant is transferred to another clean EP tube;
(5)   向步骤(4)的EP管中加入2.5倍体积的无水乙醇，其中蛋白酶K组需要先加1/10体积的NaAc（3 mol/L，pH5.2），-20℃放置20 min，离心收集基因组DNA；(5) Add 2.5 volumes of absolute ethanol to the EP tube of step (4). The proteinase K group needs to first add 1/10 volume of NaAc (3 mol/L, pH 5.2), place at -20 ° C for 20 min, and centrifuge. Collecting genomic DNA;
(6)   向步骤(5)的EP管中加入1 mL的70％乙醇洗涤DNA，去上清，敞口晾干基因组；(6) Wash the DNA into the EP tube of step (5) by adding 1 mL of 70% ethanol, remove the supernatant, and open to dry the genome;
(7)   向步骤(6)的EP管中加入100 µL TE缓冲液，溶解基因组DNA沉淀。利用光密度测定法测定基因组DNA的浓度，利用琼脂糖凝胶电泳检测基因组DNA的完整性。(7) 100 μL of TE buffer was added to the EP tube of step (6) to dissolve the genomic DNA pellet. The concentration of genomic DNA was determined by densitometry, and the integrity of genomic DNA was detected by agarose gel electrophoresis.
 
实施例Example 22 ：:
本实施例用于说明利用两种不同方法从口腔拭子法采集的口腔粘膜上皮细胞中提取This example is intended to illustrate the extraction of oral mucosal epithelial cells collected from buccal swabs using two different methods. DNADNA 的操作。Operation.
(1)   样品预处理：将携带有口腔粘膜上皮细胞的材料剪下，置于Eppendorf（EP）管中。(1) Sample Pretreatment: Materials carrying oral mucosal epithelial cells were cut and placed in Eppendorf (EP) tubes.
(2)   往EP管中加入400 μL的细胞裂解液, 充分混匀至无明显的细胞团。其中细胞裂解液配方为：10 mmol/L Tris-HCl (pH8.0)，30 mmol/L EDTA (pH8.0)，0.5% SDS，RNase A (20 μg/mL)。(2) Add 400 μL of cell lysate to the EP tube and mix well until no obvious cell mass. The cell lysate formulation is: 10 mmol/L Tris-HCl (pH 8.0), 30 mmol/L EDTA (pH 8.0), 0.5% SDS, RNase A (20 μg/mL).
(3)   分别采用蛋白酶K法和溴酸钠法来提取基因组DNA。蛋白酶K法：向步骤（2）EP管中加入终浓度为100 μg/mL的蛋白酶K，充分混匀，56 ℃放置40 min，期间不时混匀。溴酸钠法：向步骤（2）EP管中加入终浓度为1 mol/L的离液剂溴酸钠，充分混匀。其余步骤相同。(3) Genomic DNA was extracted using the proteinase K method and the sodium bromate method, respectively. Protease K method: Add the proteinase K with a final concentration of 100 μg/mL to the EP tube of step (2), mix well, and place at 56 °C for 40 min, mixing occasionally. Sodium Bromate Method: Add the chaotropic solution sodium bromate with a final concentration of 1 mol/L to the EP tube of step (2) and mix well. The remaining steps are the same.
(4)   向步骤(3)的EP管中加入等体积苯酚/氯仿/异戊醇 (25:24:1)，振荡混匀，离心至溶液分层，将上层液体转移至另一洁净的EP管中。可重复本步骤一次，并用氯仿抽提一次上清，将上清转移至另一洁净的EP管中；(4) An equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) was added to the EP tube of step (3), vortexed and mixed, centrifuged until the solution was layered, and the upper layer liquid was transferred to another clean EP tube. This step can be repeated once, and the supernatant is extracted once with chloroform, and the supernatant is transferred to another clean EP tube;
(5)   向步骤(4)的EP管中加入2.5倍体积的无水乙醇，其中蛋白酶K组需要先加1/10体积的NaAc（3 mol/L，pH5.2），-20℃放置20 min，离心收集基因组DNA；(5) Add 2.5 volumes of absolute ethanol to the EP tube of step (4). The proteinase K group needs to first add 1/10 volume of NaAc (3 mol/L, pH 5.2), place at -20 ° C for 20 min, and centrifuge. Collecting genomic DNA;
(6)   向步骤(5)的EP管中加入1 mL的70％乙醇洗涤DNA，去上清，敞口晾干基因组；(6) Wash the DNA into the EP tube of step (5) by adding 1 mL of 70% ethanol, remove the supernatant, and open to dry the genome;
(7)   向步骤(6)的EP管中加入100 µL TE缓冲液，溶解基因组DNA沉淀。利用光密度测定法测定基因组DNA的浓度，利用琼脂糖凝胶电泳检测基因组DNA的完整性。(7) 100 μL of TE buffer was added to the EP tube of step (6) to dissolve the genomic DNA pellet. The concentration of genomic DNA was determined by densitometry, and the integrity of genomic DNA was detected by agarose gel electrophoresis.
 
实施例Example 33 ：:
本实施例用于说明利用This embodiment is used to illustrate the utilization 溴酸钠法（Sodium bromate method NaBrO 3 NaBrO 3 法）和蛋白酶Method and protease KK 短时间消化法分别提取唾液中口腔粘膜上皮细胞Extraction of oral mucosal epithelial cells in saliva by short-time digestion DNADNA 的操作。Operation.
(1)   样品预处理：将唾液2000 g离心10 min，弃上清，用1 mL的生理盐水悬浮口腔粘膜上皮细胞，再2000 g离心10 min，弃上清。(1) Sample pretreatment: centrifuge 2000 g of saliva for 10 min, discard the supernatant, suspend the oral mucosal epithelial cells with 1 mL of normal saline, and centrifuge at 2000 g. Min, discard the supernatant.
(2)   往Eppendorf（EP）管中加入400 μL的细胞裂解液, 充分混匀至无明显的细胞团。其中细胞裂解液配方为：10 mmol/L Tris-HCl (pH8.0)，30 mmol/L EDTA (pH8.0)，0.5% SDS，RNase A (20 μg/mL)。(2) Add 400 μL of cell lysate to the Eppendorf (EP) tube and mix well until no obvious cell mass. The cell lysate formulation was: 10 mmol/L Tris-HCl (pH 8.0), 30 mmol/L EDTA (pH 8.0), 0.5%. SDS, RNase A (20 μg/mL).
(3)   分别采用蛋白酶K短时间消化法和溴酸钠法来提取基因组DNA。蛋白酶K法：向步骤（2）EP管中加入终浓度为100 μg/mL的蛋白酶K，充分混匀，56 ℃消化5 min。溴酸钠法：向步骤（2）EP管中加入终浓度为1 mol/L的离液剂溴酸钠，充分混匀。其余步骤相同。(3) Genomic DNA was extracted using the proteinase K short-time digestion method and the sodium bromate method, respectively. Protease K method: Add proteinase K at a final concentration of 100 μg/mL to the EP tube of step (2), mix well, and digest at 56 °C for 5 min. Sodium Bromate Method: Add the chaotropic solution sodium bromate with a final concentration of 1 mol/L to the EP tube of step (2) and mix well. The remaining steps are the same.
(4)   向步骤(3)的EP管中加入等体积苯酚/氯仿/异戊醇 (25:24:1)，振荡混匀，离心至溶液分层，将上层液体转移至另一洁净的EP管中。可重复本步骤一次，并用氯仿抽提一次上清，将上清转移至另一洁净的EP管中；(4) An equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) was added to the EP tube of step (3), vortexed and mixed, centrifuged until the solution was layered, and the upper layer liquid was transferred to another clean EP tube. This step can be repeated once, and the supernatant is extracted once with chloroform, and the supernatant is transferred to another clean EP tube;
(5)   向步骤(4)的EP管中加入2.5倍体积的无水乙醇，其中蛋白酶K组需要先加1/10体积的NaAc（3 mol/L，pH5.2），-20℃放置20 min，离心收集基因组DNA；(5) Add 2.5 volumes of absolute ethanol to the EP tube of step (4). The proteinase K group needs to first add 1/10 volume of NaAc (3 mol/L, pH 5.2), place at -20 ° C for 20 min, and centrifuge. Collecting genomic DNA;
(6)   向步骤(5)的EP管中加入1 mL的70％乙醇洗涤DNA，去上清，敞口晾干基因组；(6) Wash the DNA into the EP tube of step (5) by adding 1 mL of 70% ethanol, remove the supernatant, and open to dry the genome;
(7)   向步骤(6)的EP管中加入100 µL TE缓冲液，溶解基因组DNA沉淀。利用光密度测定法测定基因组DNA的浓度，利用琼脂糖凝胶电泳检测基因组DNA的完整性。(7) 100 μL of TE buffer was added to the EP tube of step (6) to dissolve the genomic DNA pellet. The concentration of genomic DNA was determined by densitometry, and the integrity of genomic DNA was detected by agarose gel electrophoresis.
 
实施例Example 44 ：:
本实施例用于说明利用不同浓度的This embodiment is used to illustrate the use of different concentrations 溴酸钠（Sodium bromate NaBrO 3 NaBrO 3 ）和)with 1 mol/L1 mol/L 的高氯酸钠分别提取人血液白细胞Sodium perchlorate extracts human blood leukocytes DNADNA 的操作。Operation.
(1)   样品预处理（可选）：往200 μL的抗凝血中加入400 μL的红细胞裂解液，混匀后2000g离心10 min，弃上清。若仍有较多的红细胞残留，也可重复裂解一次。(1) Sample pretreatment (optional): Add 400 μL of red blood cell lysate to 200 μL of anticoagulated blood, mix and centrifuge at 2000 g for 10 min, and discard the supernatant. If there is still more red blood cell residue, the lysis can be repeated once.
(2)   往Eppendorf（EP）管中加入400 μL的上述细胞裂解液, 充分混匀至无明显的细胞团。(2) Add 400 μL of the above cell lysate to an Eppendorf (EP) tube and mix well until no obvious cell mass is present.
(3)   分别采用高氯酸钠法和溴酸钠法来提取基因组DNA。高氯酸钠法：向步骤（2）EP管中加入终浓度为1 mol/L的高氯酸钠，充分混匀。溴酸钠法：向步骤（2）EP管中加入终浓度分别为0.1 mol/L、0.2 mol/L、0.5 mol/L、1 mol/L和3 mol/L的溴酸钠，充分混匀。其余步骤相同。(3) Genomic DNA was extracted using the sodium perchlorate method and the sodium bromate method, respectively. Sodium perchlorate method: Add sodium perchlorate with a final concentration of 1 mol/L to the EP tube of step (2) and mix well. Sodium bromate method: adding the final concentration to the EP tube of step (2) is 0.1 mol/L, 0.2 Mol/L, 0.5 mol/L, 1 Mix thoroughly with mol/L and 3 mol/L sodium bromate. The remaining steps are the same.
(4)   向步骤(3)的EP管中加入等体积苯酚/氯仿/异戊醇 (25:24:1)，振荡混匀，离心至溶液分层，将上层液体转移至另一洁净的EP管中。可重复本步骤一次，并用氯仿抽提一次上清，将上清转移至另一洁净的EP管中；(4) An equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) was added to the EP tube of step (3), vortexed and mixed, centrifuged until the solution was layered, and the upper layer liquid was transferred to another clean EP tube. This step can be repeated once, and the supernatant is extracted once with chloroform, and the supernatant is transferred to another clean EP tube;
(5)   向步骤(4)的EP管中加入2.5倍体积的无水乙醇，-20℃放置20 min，离心收集基因组DNA； (5) Adding 2.5 volumes of absolute ethanol to the EP tube of step (4), placing at -20 ° C for 20 min, and collecting genomic DNA by centrifugation;
(6)   向步骤(5)的EP管中加入1 mL的70％乙醇洗涤DNA，去上清，敞口晾干基因组；(6) Wash the DNA into the EP tube of step (5) by adding 1 mL of 70% ethanol, remove the supernatant, and open to dry the genome;
(7)   向步骤(6)的EP管中加入100 µL TE缓冲液，溶解基因组DNA沉淀。利用光密度测定法测定基因组DNA的浓度，利用琼脂糖凝胶电泳检测基因组DNA的完整性。(7) 100 μL of TE buffer was added to the EP tube of step (6) to dissolve the genomic DNA pellet. The concentration of genomic DNA was determined by densitometry, and the integrity of genomic DNA was detected by agarose gel electrophoresis.
 
测试实施例Test example
将实施例1-4提取的人基因组DNA的纯度和浓度进行汇总，结果如表1所示。The purity and concentration of the human genomic DNA extracted in Example 1-4 were summarized, and the results are shown in Table 1.
表1. 利用不同方法对不同来源的样本提取基因组DNA的参数比较。Table 1. Comparison of parameters for extracting genomic DNA from samples from different sources using different methods.
Figure dest_path_image002
Figure dest_path_image002
 
根据表1的数据可见，在蛋白酶K消化时间充分（实施例中采用了40 min的消化时间）的情况下，相同来源的口腔粘膜上皮细胞，溴酸钠法和蛋白酶K法的DNA得率及纯度相当；然而，当消化时间大幅缩短（实施例中采用了5 min的消化时间）导致蛋白酶K无法充分消化组蛋白的情况下，蛋白酶K法的DNA得率大幅降低，而本发明所涉及的离液剂溴酸钠能快速分离组蛋白和基因组DNA，这种方法所提DNA的得率约为蛋白酶K法的5倍，而DNA纯度未受影响。0.1-3 mol/L的溴酸钠均能提取基因组DNA，1-3 mol/L的溴酸钠所提取的浓度最高且相对稳定。1 mol/L的溴酸钠和1 mol/L的高氯酸钠所提取的基因组DNA在得率和纯度上相当。According to the data in Table 1, the DNA yields of the oral mucosal epithelial cells of the same source, the sodium bromate method and the proteinase K method were observed in the case where the protease K digestion time was sufficient (the digestion time was 40 min in the examples). The purity is equivalent; however, when the digestion time is greatly shortened (the digestion time of 5 min is used in the examples), the protease K cannot sufficiently digest histones, and the DNA yield of the proteinase K method is greatly reduced, and the present invention relates to The chaotropic agent sodium bromate can rapidly separate histones and genomic DNA. The yield of the DNA extracted by this method is about 5 times that of the proteinase K method, and the DNA purity is not affected. 0.1-3 mol/L sodium bromate can extract genomic DNA, and 1-3 mol/L sodium bromate extract has the highest concentration and is relatively stable. The genomic DNA extracted by 1 mol/L sodium bromate and 1 mol/L sodium perchlorate was comparable in yield and purity.
 
将实施例1-4提取的人基因组DNA进行电泳检测，结果如图1-图3所示。其中：The human genomic DNA extracted in Example 1-4 was subjected to electrophoresis, and the results are shown in Figs. among them:
图1是溴酸钠法（NaBrO 3法）和蛋白酶K法（消化40min）分别提取唾液法和口腔拭子法采集口腔粘膜上皮细胞DNA的电泳检测结果。M为DNA marker（分子量自上而下分别为：2000、1000、750、500、250、100，单位为bp），泳道1为蛋白酶K法提取的唾液来源口腔粘膜上皮细胞DNA，泳道2为溴酸钠法提取的唾液来源口腔粘膜上皮细胞DNA，泳道3为蛋白酶K法提取的口腔拭子来源口腔粘膜上皮细胞DNA，泳道4为溴酸钠法提取的口腔拭子来源口腔粘膜上皮细胞DNA。根据图1可见，在蛋白酶K消化时间充分的情况下，相同来源的口腔粘膜上皮细胞，溴酸钠法和蛋白酶K法的DNA得率及纯度相当。 Fig. 1 shows the results of electrophoretic detection of sodium bromate method (NaBrO 3 method) and proteinase K method (digestion for 40 min) for extracting salivary method and buccal swab method for collecting oral mucosal epithelial cells. M is a DNA marker (molecular weight from top to bottom: 2000, 1000, 750, 500, 250, 100, unit is bp), lane 1 is the salivary-derived oral mucosal epithelial DNA extracted by proteinase K method, and lane 2 is bromine. The saliva derived from the sodium method is derived from the oral mucosal epithelial cell DNA, the lane 3 is the oral swab-derived oral mucosal epithelial cell DNA extracted by the protease K method, and the lane 4 is the oral swab-derived oral mucosal epithelial cell DNA extracted by the sodium bromate method. As can be seen from Fig. 1, in the case where the protease K digestion time is sufficient, the DNA yield and purity of the oral mucosal epithelial cells of the same source, the sodium bromate method and the proteinase K method are comparable.
图2是溴酸钠法（NaBrO 3法）和蛋白酶K法（仅消化5min）分别提取唾液法采集口腔粘膜上皮细胞DNA的电泳检测结果。M为DNA marker（分子量自上而下分别为：2000、1000、750、500、250、100，单位为bp），泳道1为蛋白酶K法提取的唾液来源口腔粘膜上皮细胞DNA，泳道2为溴酸钠法提取的唾液来源口腔粘膜上皮细胞DNA。根据图2可见，在消化时间短的情况下，本发明所用的溴酸钠法要比传统的蛋白酶K法有更高的DNA得率。 Fig. 2 shows the results of electrophoretic detection of the extraction of DNA from oral mucosal epithelial cells by saliva extraction by sodium bromate method (NaBrO 3 method) and proteinase K method (digestion only for 5 min). M is a DNA marker (molecular weight from top to bottom: 2000, 1000, 750, 500, 250, 100, unit is bp), lane 1 is the salivary-derived oral mucosal epithelial DNA extracted by proteinase K method, and lane 2 is bromine. The saliva derived from the sodium method is derived from oral mucosal epithelial DNA. As can be seen from Fig. 2, in the case of a short digestion time, the sodium bromate method used in the present invention has a higher DNA yield than the conventional proteinase K method.
图3是不同浓度的溴酸钠（NaBrO 3）和1 mol/L的高氯酸钠分别提取人血液白细胞DNA的电泳检测结果。M为DNA marker（分子量自上而下分别为：2000、1000、750、500、250、100，单位为bp），泳道1-5分别为终浓度0.1 mol/L、0.2 mol/L、0.5 mol/L、1 mol/L和3 mol/L的NaBrO 3提取的人血液白细胞基因组DNA，泳道6为终浓度为1 mol/L的高氯酸钠提取的人血液白细胞基因组DNA。根据图3可见，0.1 mol/L-3 mol/L的NaBrO 3均能提取细胞基因组DNA，其中1 mol/L-3 mol/L的NaBrO 3提取的基因组差别不大。1 mol/L-3 mol/L的NaBrO 3与1 mol/L的高氯酸钠法的得率相当。 Figure 3 shows the results of electrophoretic detection of human leukocyte DNA extracted from different concentrations of sodium bromate (NaBrO 3 ) and 1 mol/L sodium perchlorate. M is a DNA marker (molecular weight from top to bottom: 2000, 1000, 750, 500, 250, 100, bp), and lanes 1-5 are final concentrations of 0.1 mol/L, 0.2 mol/L, and 0.5 mol, respectively. Human blood leukocyte genomic DNA extracted with /L, 1 mol/L and 3 mol/L NaBrO 3 , and lane 6 is human leukocyte genomic DNA extracted with sodium perchlorate at a final concentration of 1 mol/L. According to Fig. 3, 0.1 mol/L-3 mol/L NaBrO 3 can extract cellular genomic DNA, and the genome extracted by 1 mol/L-3 mol/L NaBrO 3 has little difference. The yield of 1 mol/L-3 mol/L NaBrO 3 and 1 mol/L sodium perchlorate method is comparable.
以上结合附图详细描述了本发明的优选实施方式，但是，本发明并不限于上述实施方式中的具体细节，在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，这些简单变型均属于本发明的保护范围。The preferred embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the specific details of the embodiments described above, and various modifications may be made to the technical solutions of the present invention within the scope of the technical idea of the present invention. These simple variations are within the scope of the invention.
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本发明对各种可能的组合方式不再另行说明。It should be further noted that the specific technical features described in the above specific embodiments may be combined in any suitable manner without contradiction. To avoid unnecessary repetition, the present invention has various possibilities. The combination method will not be described separately.
此外，本发明的各种不同的实施方式之间也可以进行任意组合，只要其不违背本发明的思想，其同样应当视为本发明所公开的内容。In addition, any combination of various embodiments of the invention may be made as long as it does not deviate from the idea of the invention, and it should be regarded as the disclosure of the invention.
工业实用性Industrial applicability
在此处键入工业实用性描述段落。Type the industrial usability description paragraph here.
序列表自由内容Sequence table free content
在此处键入序列表自由内容描述段落。Type the sequence table free content description paragraph here.

Claims (7)

  1. 一种离液剂提取基因组DNA的方法，其特征在于，所述离液剂含有溴酸盐。A method for extracting genomic DNA from a chaotropic agent, characterized in that the chaotropic agent contains a bromate.
  2. 如权利要求1所述的一种离液剂提取基因组DNA的方法，其特征在于，所述溴酸盐为溴酸钠。A method of extracting genomic DNA from a chaotropic agent according to claim 1, wherein the bromate is sodium bromate.
  3. 如权利要求2所述的一种离液剂提取基因组DNA的方法，其特征在于，将含有组蛋白及基因组DNA的样本与所述离液剂混匀后，所述溴酸钠的终浓度为0.1-3mol/L。The method for extracting genomic DNA from a chaotropic agent according to claim 2, wherein after the sample containing histone and genomic DNA is mixed with the chaotropic agent, the final concentration of the sodium bromate is 0.1-3 mol/L.
  4. 如权利要求3所述的一种离液剂提取基因组DNA的方法，其特征在于，将含有组蛋白及基因组DNA的样本与所述离液剂混匀后，所述溴酸钠的终浓度为0.2-3mol/L。The method for extracting genomic DNA from a chaotropic agent according to claim 3, wherein after the sample containing histone and genomic DNA is mixed with the chaotropic agent, the final concentration of the sodium bromate is 0.2-3 mol/L.
  5. 如权利要求4所述的一种离液剂提取基因组DNA的方法，其特征在于，将含有组蛋白及基因组DNA的样本与所述离液剂混匀后，所述溴酸钠的终浓度为1-3mol/L。 The method for extracting genomic DNA from a chaotropic agent according to claim 4, wherein after the sample containing histone and genomic DNA is mixed with the chaotropic agent, the final concentration of the sodium bromate is 1-3 mol/L.
  6. 如权利要求1~5中任意一种离液剂提取基因组DNA的方法，其特征在于，所述DNA在提取前结合在组蛋白中；其中，该方法包括如下步骤：The method for extracting genomic DNA from a chaotropic agent according to any one of claims 1 to 5, wherein the DNA is bound to histone before extraction; wherein the method comprises the following steps:
    （1）将样本分散成细胞悬液，加入细胞裂解液使细胞破裂，释放出细胞核内的组蛋白与基因组DNA的复合物；(1) Dispersing the sample into a cell suspension, adding a cell lysate to rupture the cell, releasing a complex of histones and genomic DNA in the nucleus;
    （2）将步骤（1）得到的物料与所述离液剂混合均匀，得到组蛋白与基因组DNA分离后的物料；(2) mixing the material obtained in the step (1) with the chaotropic agent to obtain a material separated from the genomic DNA by the histone protein;
    （3）将步骤（2）得到的物料与蛋白抽提液混合均匀，离心，分离得到蛋白被抽提后的上清液；(3) mixing the material obtained in the step (2) with the protein extract, and centrifuging, separating the supernatant from which the protein is extracted;
    （4）将所述经蛋白被抽提后的上清液与DNA沉淀剂混合，离心后得到DNA沉淀。(4) The supernatant obtained by extracting the protein is mixed with a DNA precipitating agent, and centrifuged to obtain a DNA precipitate.
  7. 如权利要求6所述的方法，其特征在于，该方法还包括，将所述DNA沉淀进行洗涤和再溶解。The method of claim 6 further comprising washing the DNA precipitate and redissolving.
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