WO2021244469A1 - 一种核酸提取方法以及应用 - Google Patents

一种核酸提取方法以及应用 Download PDF

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WO2021244469A1
WO2021244469A1 PCT/CN2021/097213 CN2021097213W WO2021244469A1 WO 2021244469 A1 WO2021244469 A1 WO 2021244469A1 CN 2021097213 W CN2021097213 W CN 2021097213W WO 2021244469 A1 WO2021244469 A1 WO 2021244469A1
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nucleic acid
extraction
solution
quaternary ammonium
ammonium salt
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PCT/CN2021/097213
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English (en)
French (fr)
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伏东科
何文龙
刘连弟
毕万里
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苏州新海生物科技股份有限公司
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Priority to EP21818014.9A priority Critical patent/EP4163371A1/en
Priority to CN202180014572.3A priority patent/CN115103908A/zh
Priority to US18/000,200 priority patent/US20230203476A1/en
Priority to JP2022573630A priority patent/JP2023527475A/ja
Publication of WO2021244469A1 publication Critical patent/WO2021244469A1/zh

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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
    • 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
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
    • C08K5/00—Use of organic ingredients
    • C08K5/16—Nitrogen-containing compounds
    • C08K5/17—Amines; Quaternary ammonium compounds
    • C08K5/19—Quaternary ammonium compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • the present invention relates to the technical field of biotechnology, more specifically, a nucleic acid extraction method and application.
  • Nucleic acids are carriers that carry genetic information and are usually found in various biological samples, such as blood, serum, tissue, cells, saliva, feces, and so on.
  • biological samples contain a large number of proteins, lipids, carbohydrates and other biological macromolecules, as well as inorganic salt ions, so they cannot be directly used for further molecular biology experiments and detection. They need to be separated and purified to remove nucleic acids. For other impurities, this process is also called nucleic acid extraction.
  • the extracted nucleic acid has many uses in molecular biology, biotechnology and clinical research. For example, PCR (polymerase chain reaction), DNA hybridization, restriction enzyme digestion, DNA sequencing and microarray experiments, etc. Use these methods to study SNP (Single Nucleotide Polymorphism) for clinical applications such as genetic disease screening, tumor detection, and individualized medication guidance, and to study STR (Short Tandem Repeat) for individual identification and paternity identification, etc. Forensic applications. In addition, it can also be used for genetic research such as population screening and phylogeny.
  • SNP Single Nucleotide Polymorphism
  • STR Short Tandem Repeat
  • nucleic acid The method of separating and extracting nucleic acid has been reported in the literature (e.g. Chapter 2 (DNA) and Chapter 4 (RNA) of F. Ausubel et al., eds., Current Protocols in Molecular Biology, Wiley-Interscience, New York, 1993). These methods usually require resuspending the sample in a certain solution and rupturing the cells by chemical reagents or enzyme methods to release the nucleic acid. This process is called lysis. The released nucleic acid will reversibly bind to the nucleic acid-adsorbed material in the solution or precipitate under the condition of a polar organic solvent.
  • chaotropic salt will facilitate the binding of nucleic acids to the above-mentioned materials.
  • Polar organic solvents usually include absolute ethanol, isopropanol, polyethylene glycol (PEG) and the like.
  • Common chaotropic salts include sodium chloride, sodium acetate, guanidine hydrochloride, guanidine isothiocyanate, etc.
  • the combination of nucleic acid and the above-mentioned adsorbent materials includes hydrogen bonding, hydrophobic interaction, and electrostatic interaction. This process is called the conjugation of nucleic acids.
  • the eluent here can be a low-salt buffer or deionized ultrapure water.
  • guanidine salt has the function of denaturing protein, which can lyse biological samples and release nucleic acid molecules.
  • guanidine salt Salt can neutralize the negative charge carried by nucleic acid molecules, so that nucleic acid molecules can be reversibly combined with silica gel, magnetic beads and other materials, so that nucleic acid molecules can be adsorbed on these materials, and impurities such as protein and salt ions can be removed by washing. The conditions will elute the target nucleic acid molecules, so as to achieve the effect of separation and purification of nucleic acids.
  • guanidine salt has poor stability, high sensitivity to light, and is easy to change color and decompose.
  • guanidine hydrochloride is easily decomposed into ammonia and urea in water.
  • the types of guanidine salts to choose from are relatively single, and the commonly available guanidine salts are guanidine hydrochloride or guanidine isothiocyanate.
  • ethanol or isopropanol it is usually necessary to add ethanol or isopropanol to help DNA precipitation, and the solubility of guanidine salt in ethanol and isopropanol is low, which often causes the volume of the binding solution to be too large, which is not conducive to the extraction of trace amounts of DNA. .
  • the purpose of the present invention is to provide the application of quaternary ammonium salt in nucleic acid extraction, so that the quaternary ammonium salt can be used as a chaotropic salt for nucleic acid extraction, so that the nucleic acid molecules in the solution can be adsorbed to silicon beads, magnetic beads, and silicon beads.
  • Adsorption materials such as algae earth or silica gel or precipitation can effectively improve the efficiency of nucleic acid purification and recovery, and can be used for plasmid DNA extraction, viral genome extraction, bacterial genome extraction, and animal, plant and human genome DNA extraction;
  • Another object of the present invention is to provide the quaternary ammonium salt as a nucleic acid extraction reagent as a chaotropic salt;
  • Another object of the present invention is to provide a nucleic acid extraction method using the quaternary ammonium salt as a chaotropic salt.
  • An - is an anion
  • the R 1 , R 2 , R 3 , and R 4 are independently selected from alkyl groups.
  • the present invention uses quaternary ammonium salt as chaotropic salt for nucleic acid extraction. Compared with guanidine salt as chaotropic salt, quaternary ammonium salt is more stable, and there are more types of quaternary ammonium salt, and quaternary ammonium salt is used in organic solvents such as ethanol. The solubility is also higher, so the same working concentration has a lower cost.
  • the anion is an anion of a halogen; further preferably, the anion is a chloride ion or a bromide ion.
  • the R 1 , R 2 , R 3 , and R 4 are independently selected from C1-C5 alkyl groups; further preferably, the R 1 , R 2 , R 3 , and R 4 are independently selected from methyl, ethyl Group, propyl, butyl and pentyl.
  • the quaternary ammonium salt is tetramethylammonium bromide, tetramethylammonium chloride, tetraethylammonium bromide, tetraethylammonium chloride, tetrapropylammonium bromide, tetramethylammonium bromide, Propylammonium chloride, tetrabutylammonium bromide, tetrabutylammonium chloride, tetrapentylammonium bromide or tetrapentylammonium chloride.
  • Quaternary ammonium salt is used as a chaotropic salt for nucleic acid extraction.
  • a certain concentration of quaternary ammonium salt can neutralize the negative charge of nucleic acid.
  • nucleic acid Under the condition of polar solvent (ethanol, isopropanol, etc.), nucleic acid is hydrophobic through hydrogen bond The interaction and electrostatic interaction can be combined with the adsorption material or be precipitated out.
  • the quaternary ammonium salt acts on nucleic acid extraction in the form of a solution.
  • the salt concentration of the quaternary ammonium salt solution ranges from 100 mM to 2.5 M, preferably 250 mM to 2.5 M. In the specific embodiment of the present invention, the quaternary ammonium salt concentration is 250 mM, 1.6 M or 2.5M.
  • the nucleic acid in the present invention refers to a biological macromolecule formed by the polymerization of many nucleotides, and it is an extremely important life substance. Nucleic acids are widely found in animal and plant cells and microorganisms. According to chemical composition, nucleic acids are mainly divided into two types: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), and the nucleic acid molecule in the present invention also refers to DNA, RNA or a mixture of both.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • the nucleic acid extraction is divided into extraction methods including phenol-chloroform extraction method, polar solvent (ethanol, isopropanol or polyethylene glycol) precipitation method, and solid-phase carrier adsorption method.
  • the solid-phase carrier adsorption method utilizes the strong affinity and adsorption power of the carrier for nucleic acid to specifically bind the released nucleic acid to a specific carrier.
  • Some commonly used carriers include silica beads, magnetic beads, diatomaceous earth or silica gel.
  • the nucleic acid extraction is divided into nucleic acid types including plasmid DNA extraction, viral genome extraction (such as lambda DNA extraction), bacterial genome extraction (such as E. coli DNA extraction), and the extraction of animal, plant and human genomic DNA. These types of nucleic acid extraction can be Refer to the existing technology to select the appropriate extraction method.
  • the present invention also provides a nucleic acid extraction reagent, wherein the chaotropic salt is a quaternary ammonium salt of the structure represented by formula I of the present invention.
  • the nucleic acid extraction reagent includes the quaternary ammonium salt of the structure shown in formula I of the present invention, and one or more of the lysis solution, the binding solution, the washing solution, the eluent, and the adsorption material;
  • the quaternary ammonium salt can be independently used as a component of the nucleic acid extraction reagent, or added to the lysis solution, or added to the binding solution.
  • the lysis solution, binding solution, washing solution, adsorption material and elution solution are combined in different combinations according to different extraction methods or different types of extracted nucleic acids.
  • the nucleic acid extraction reagent includes a lysis solution, a binding solution, a washing solution, an adsorption material, and an eluate; in viral genome- ⁇ DNA extraction, the nucleic acid extraction reagent includes a lysis solution, a washing solution, an adsorption material, and an eluate; sometimes
  • the binding solution can be directly added to the lysis solution, called the lysis binding solution, for example, in the extraction of viral genome- ⁇ DNA.
  • the lysis solution includes buffer (10-50mM), protease (0.5mg/ml), alkali (200mM), surfactant (1-10%), nucleic acid stabilizer (36-50mM) according to different nucleic acid extraction. ), lysozyme (5mg/ml), one or more of phenol-chloroform-isoamyl alcohol, such as buffer+protease+surfactant+nucleic acid stabilizer, buffer+surfactant, buffer+ Nucleic acid stabilizer+surfactant+alkali, buffer+lysozyme+protease+phenol-chloroform-isoamyl alcohol, buffer+nucleic acid stabilizer+surfactant+protease, etc.; in the specific embodiment of the present invention, the protease is proteinase K ,
  • the surfactant is SDS, PEG or SLS, the base is sodium hydroxide, the buffer is phosphate buffer or Tris-HCl, and the nucleic acid stabilizer is ED
  • the binding liquid is isopropanol, absolute ethanol or polyethylene glycol.
  • the washing solution is a 75-80% ethanol solution
  • the eluent can be a low-salt buffer (10mM, such as Tris-HCl) or deionized ultrapure water, and further the eluent is also It may contain a surfactant or a nucleic acid stabilizer (1 mM).
  • the surfactant is preferably SDS or TritonX-100 with a mass percentage concentration of 0.1-1%; the nucleic acid stabilizer is preferably EDTA with a concentration of 1 mM.
  • the present invention also provides a nucleic acid extraction method.
  • the quaternary ammonium salt of the structure shown in formula I of the present invention is added as a chaotropic salt for extraction, and other reagents can be selected by reference to conventional methods.
  • a lysis solution is added to the sample to be extracted for lysis to release nucleic acid molecules, and then a binding solution is added to bind the nucleic acid to the adsorbent material or precipitate out, and the quaternary ammonium salt is added after lysis or after adding the binding Add it after the solution, or add it to the lysis solution, or add it to the binding solution.
  • the extraction method further includes washing the nucleic acid on the adsorption material or the precipitated nucleic acid using a washing solution.
  • the extraction method further includes using an eluent to elute the nucleic acid on the adsorption material, or using an eluent to dissolve the precipitated nucleic acid.
  • the present invention provides a quaternary ammonium salt with a structure represented by formula I as a chaotropic salt, which is used to replace the currently commonly used guanidine salt for nucleic acid extraction; it can exist in a polar organic solvent binding liquid Under this condition, the nucleic acid molecules in the solution can be adsorbed to materials such as silica beads, magnetic beads, diatomaceous earth or silica gel or precipitated, which can effectively improve the efficiency of nucleic acid purification and recovery. It can be used for plasmid DNA extraction, viral genome extraction, and bacterial genome extraction. Extraction, or extraction of genomic DNA from animals, plants and humans.
  • Figure 1 shows the comparison of fluorescence quantitative PCR amplification curves of DNA extracted from human blood genome
  • Figure 2 shows the detection pattern of ⁇ DNA extracted by agarose gel electrophoresis
  • Figure 3 shows the detection pattern of plasmid DNA extracted by agarose gel electrophoresis
  • Figure 4 shows the detection pattern of E. coli genome extracted by agarose gel electrophoresis
  • Figure 5 shows the STR-capillary electrophoresis detection pattern of trace genomic DNA extraction
  • Figure 6 shows the comparison of the effect of CTAB and TEAB in extracting trace genomes.
  • Figure 7 shows the fluorescence quantitative PCR amplification curve of MERS pseudovirus nucleic acid extraction.
  • the invention discloses a nucleic acid extraction method and application, and those skilled in the art can learn from the content of this article and appropriately improve the process parameters to achieve.
  • all similar replacements and modifications are obvious to those skilled in the art, and they are all deemed to be included in the present invention.
  • the extraction methods and applications of the present invention have been described through preferred embodiments. It is obvious that relevant persons can make changes or appropriate changes and combinations to the extraction methods and applications described herein without departing from the content, spirit and scope of the present invention. Realize and apply the technology of the present invention.
  • the raw materials used in each treatment group of the comparison test provided are the same, and the other test conditions of each group are consistent except for due differences.
  • the raw materials, reagents, etc. involved in the present invention can be obtained through commercial channels unless otherwise specified.
  • the extraction solution containing tetraethylammonium chloride was used to extract nucleic acid by the magnetic bead method, and the other group of extraction solution containing no tetraethylammonium chloride and the remaining components was used as a control to compare the extraction effects of the two groups.
  • the reagent components used are as follows:
  • composition of extraction solution 1 includes Tris-HCl (10mM), EDTA (50mM), SDS (1%), tetraethylammonium chloride (250mM), proteinase K (0.5mg/ml).
  • the composition of the extraction liquid 2 includes 100% isopropanol.
  • the composition of the extract 3 (washing liquid) includes 75% ethanol.
  • the composition of the extract 4 includes ultrapure water.
  • the composition of the magnetic bead suspension includes superparamagnetic nanometer magnetic beads dispersed in ultrapure water.
  • the preparation method includes the following steps:
  • the 25 ⁇ l amplification system includes the following components: 12.5 ⁇ l 2*PCR Mix; 1 ⁇ l 10uM primers each; 3 ⁇ l collected supernatant; 7.5 ⁇ l pure water.
  • the template also contains 1ng human genome as a positive control standard and an extract 4 as a negative control.
  • the thermal cycle is as follows: 95 degrees, 10 minutes; (95 degrees, 15 seconds; 60 degrees, 45 seconds) * 40 cycles
  • This example aims to illustrate the effect of quaternary ammonium salt on nucleic acid adsorption, so lambda DNA is used for simulated purification.
  • An extract containing tetrapropylammonium bromide was used for nucleic acid extraction, while another group of extracts containing no tetrapropylammonium bromide and with the same remaining components was used as a control to compare the extraction effects of the two groups.
  • the reagent components used are as follows:
  • composition of extraction solution 1 (lysis and binding solution) includes Tris-HCl (20mM), tetrapropylammonium bromide (250mM), and 10% PEG8000.
  • the composition of the extract 2 (washing liquid) includes 75% ethanol.
  • the composition of the extract 3 includes ultrapure water.
  • the composition of the magnetic bead suspension includes superparamagnetic nanometer magnetic beads dispersed in ultrapure water.
  • the preparation method includes the following steps:
  • the 20 ⁇ l amplification system includes the following components: 10 ⁇ l 2*PCR Mix; 1 ⁇ l 10uM primers each; 8 ⁇ l of the collected supernatant.
  • the template also contains 10ng ⁇ DNA as a positive control standard and extract 3 as a negative control.
  • the thermal cycle is as follows: 95 degrees, 5 minutes; (95 degrees, 20 seconds; 60 degrees, 3 minutes) * 35 cycles
  • the invention uses the quaternary ammonium salt in the method of ethanol precipitation nucleic acid, which can effectively carry out the plasmid extraction of Escherichia coli, and comprises five parts: extract 1, extract 2, extract 3, extract 4, and extract 5.
  • Extract 1 (lysis solution) contains 10mM Tris-HCl (pH8.0), 50mM EDTA (pH8.0).
  • the composition of the extraction solution 2 includes 200mM NaOH, 1% SDS.
  • composition of the extract 3 includes 2.5M tetrabutylammonium bromide (pH7.0).
  • the composition of the extraction solution 4 includes 100% isopropanol.
  • the composition of the extract 5 (washing liquid) includes 75% ethanol.
  • composition of extract 6 includes 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (pH 8.0).
  • the preparation method includes the following steps:
  • the present invention uses quaternary ammonium salt in the phenol-chloroform nucleic acid extraction method, which can effectively carry out the extraction and purification of Escherichia coli genomic DNA, including four parts: extract 1, extract 2, extract 3, extract 4, and magnetic bead suspension composition.
  • composition of extract 1 includes phosphate buffer (pH7.0, 50mM) and lysozyme (5mg/ml).
  • composition of extract 2 includes proteinase K (0.5 mg/ml).
  • composition of the extract 3 includes tetrabutylammonium bromide (2.5M).
  • composition of extraction solution 4 (lysis solution) includes phenol-chloroform-isoamyl alcohol (25:24:1).
  • composition of the extract 5 includes absolute ethanol.
  • the composition of the extract 6 (washing liquid) includes 75% ethanol.
  • composition of extract 7 includes 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (pH 8.0).
  • the preparation method includes the following steps:
  • the 20 ⁇ l amplification system includes the following components: 10 ⁇ l 2*PCR Mix; 1 ⁇ l 10uM primers each; 4 ⁇ l collected supernatant; 4 ⁇ l pure water.
  • the extraction solution 3 was changed to 5M sodium chloride, and the other reagents were unchanged, as a positive control; the extraction solution 3 was changed to water, and the other reagents were unchanged, as a negative control.
  • the thermal cycle is as follows: 95 degrees, 5 minutes; (95 degrees, 20 seconds; 60 degrees, 3 minutes) * 35 cycles
  • the invention can effectively extract and purify trace genomic DNA, and compare the extraction efficiency of tetraethylammonium bromide and guanidine hydrochloride at the same concentration.
  • the extraction reagent of the present invention is composed of five parts: extraction solution 1, extraction solution 2, extraction solution 3, extraction solution 4, and magnetic bead suspension.
  • composition of extraction solution 1 includes Tris-HCl (10mM), EDTA (50mM), SLS (1%), and proteinase K (0.5mg/ml).
  • composition A includes 40% isopropanol; B includes 40% isopropanol and 1.6M tetraethylammonium bromide; C includes 1.6M guanidine hydrochloride.
  • the composition of the extract 3 (washing liquid) includes 80% ethanol.
  • the composition of the extraction solution 4 includes 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (pH 8.0).
  • the composition of the magnetic bead suspension includes superparamagnetic nanometer magnetic beads dispersed in ultrapure water.
  • the preparation method includes the following steps:
  • the 15 ⁇ l amplification system includes the following components: 6 ⁇ l 2.5*PCR Mix; 3 ⁇ l NH18 primer mixture; 6 ⁇ l of the collected supernatant.
  • the template also contains 0.2ng human genome as a positive control standard and an extract 4 as a negative control.
  • the thermal cycle is as follows: 95 degrees, 10 minutes; (95 degrees, 10 seconds; 59 degrees, 60 seconds)*30 cycles; 60 degrees, 10 minutes. The result is shown in Figure 5.
  • CTAB Cetyltrimethylammonium bromide
  • CTAB is often used in many plant genome extraction methods.
  • CTAB is essentially a quaternary ammonium salt
  • the role of CTAB in this method is a cationic surfactant. It helps to lyse plant cells instead of acting as a chaotropic salt to help the binding of nucleic acids and adsorption materials or nucleic acid precipitation.
  • CTAB has a low solubility in water (less than 0.1M) and cannot be used alone to complete the purification of nucleic acids.
  • This process also requires other chaotropic salts (such as sodium chloride, sodium acetate, etc.) to separate and purify nucleic acids together. Therefore, the CTAB used in plant genome extraction has no correlation with the solution of the present invention.
  • the invention can effectively extract and purify trace genomic DNA, and compare the extraction effects of CTAB and TEAB (tetrabutylammonium bromide).
  • the extraction reagent of the present invention is composed of five parts: extraction solution 1, extraction solution 2, extraction solution 3, extraction solution 4, and magnetic bead suspension.
  • the composition of Extract 1 includes Tris-HCl (final concentration 50 mM), EDTA (final concentration 36 mM), SLS (final concentration 1%), and proteinase K (0.5 mg/ml).
  • Extract 2 (combination solution) has two components (D, E), D includes isopropanol (final concentration 40%) and TEAB (final concentration 1.6M); E includes isopropanol (final concentration 40%) And CTAB (the mother liquor concentration is 2%, the final concentration is 1.2%, which is already the maximum addition amount).
  • the composition of the extract 3 (washing liquid) includes 80% ethanol.
  • the composition of the extraction solution 4 includes 10mM Tris-HCl (pH 8.0) and 1 mM EDTA (pH 8.0).
  • the composition of the magnetic bead suspension includes superparamagnetic nanometer magnetic beads dispersed in ultrapure water.
  • the preparation method includes the following steps:
  • the 15 ⁇ l amplification system includes the following components: 6 ⁇ l 2.5*PCR Mix; 3 ⁇ l NH18 primer mixture; 6 ⁇ l of the collected supernatant.
  • the template also contains 0.2ng human genome as a positive control standard and an extract 4 as a negative control.
  • the thermal cycle is as follows: 95 degrees, 10 minutes; (95 degrees, 10 seconds; 59 degrees, 90 seconds)*28 cycles; 60 degrees, 10 minutes.
  • the purpose of the present invention is to say that the effect of tetramethylammonium bromide on the extraction of MERS pseudovirus nucleic acid includes three parts: extract 1, extract 2, and extract 3.
  • composition of extract 1 includes MOPS (10mM), isopropanol (50%), and tetramethylammonium bromide (1M).
  • the composition of the extract 2 includes 80% ethanol.
  • the composition of the extract 3 includes ultrapure water.
  • the preparation method includes the following steps:
  • the 25 ⁇ l amplification system includes the following components: 12.5 ⁇ l 2*PCR Mix; each 1.5 ⁇ l primer probe; 10 ⁇ l of collected centrifugal liquid; 1 ⁇ l of pure water.
  • MERS pseudovirus RNA there are 1.5ul MERS pseudovirus RNA at the same concentration as a positive control and extract 3 as a negative control.
  • the thermal cycle is as follows: 95 degrees, 10 minutes; (95 degrees, 15 seconds; 60 degrees, 45 seconds) * 40 cycles

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Abstract

公开了一种核酸提取方法，利用季铵盐使溶液中的核酸分子吸附到硅珠、磁珠、硅藻土或硅胶等吸附材料上或者沉淀出来，从而提高核酸纯化和回收的效率。

Description

一种核酸提取方法以及应用 技术领域
本发明涉及生物技术技术领域，更具体的说是一种核酸提取方法以及应用。
背景技术
核酸是携带遗传信息的载体，通常存在于各种生物样本中，例如血液，血清、组织，细胞，唾液、粪便等等。由于生物样品中除核酸外还有大量的蛋白、脂类、糖类等生物大分子，以及无机盐离子，因此无法直接用于进一步的分子生物学实验和检测，需要分离纯化以除去核酸外的其他杂质，这一过程也称为核酸提取。
提取得到的核酸，特别是高分子量DNA(脱氧核糖核酸)，在分子生物学，生物技术和临床研究中具有多种用途。例如，PCR(聚合酶链反应)，DNA杂交，限制酶消化，DNA测序和微阵列实验等。使用这些方法，研究SNP(单核苷酸多态性)来进行遗传疾病筛查、肿瘤检测、个体化用药指导等临床应用，研究STR(短串联重复序列)来进行个体身份识别和亲权鉴定等法医学应用。除此之外，还可以用于群体筛选、系统发育等遗传学研究。
分离提取核酸的方法在文献中多有报道(e.g.Chapter 2(DNA)and Chapter 4(RNA)ofF.Ausubel et al.,eds.,Current Protocols in Molecular Biology,Wiley-Interscience,New York,1993)。这些方法通常需要将样品在一定溶液中重悬并通过化学试剂或者酶的方法将细胞破裂，将核酸释放出来。这一过程称为裂解。释放出的核酸在溶液中会可逆的结合于吸附核酸的材料上或者在极性有机溶剂条件下沉淀。这些材料包括玻璃颗粒，玻璃纤维，磁珠，硅藻土，硅胶等，或者以上各种材料的变化或组合。高浓度的离液盐将有利于核酸结合到上述材料上。极性有机溶剂通常包括无水乙醇、异丙醇、聚乙二醇(PEG)等。常见的离液盐有氯化钠、醋酸钠、盐酸胍、异硫氰酸胍等，核酸与上述吸附材料的结合作用包括氢键、疏水作用以及静电作用等。这一过程称为核酸的结合。然后用特定的洗涤液洗去盐离子，蛋白等杂质，比如75-80％乙醇等，这一过程称为漂洗，最后，在洗 脱液作用下，核酸分子与上述材料发生分离，被收集在洗脱液中。这一过程称为洗脱。这里的洗脱液可以是低盐缓冲液或者去离子超纯水。
在核酸纯化分离过程中，目前市面上大多数核酸提取试剂和方法都使用胍盐作为离液盐，一方面胍盐具有变性蛋白的作用，可以裂解生物样本，释放核酸分子，另一方面，胍盐能够中和核酸分子所带的负电荷，使核酸分子与硅胶、磁珠等材料发生可逆性的结合，从而可以将核酸分子吸附在这些材料上，通过洗涤除去蛋白、盐离子等杂质，一定条件将目标核酸分子洗脱下来，从而达到分离纯化核酸的作用。
但是，胍盐的稳定性较差，对光敏感性较高，容易变色，出现分解的情况，如盐酸胍在水中容易分解为氨和尿素。可供选择的胍盐种类比较单一，通常可用的胍盐有盐酸胍或者异硫氰酸胍。在DNA提取过程中，通常需要加入乙醇或异丙醇等帮助DNA沉淀，而胍盐在乙醇和异丙醇中的溶解度较低，往往会导致结合液体积过大，不利于微量的DNA的提取。
发明内容
有鉴于此，本发明的目的在于提供季铵盐在核酸提取中的应用，使得所述季铵盐作为离液盐用于核酸提取，使溶液中的核酸分子吸附到硅珠、磁珠、硅藻土或硅胶等吸附材料或者沉淀出来，能够有效提高核酸纯化和回收的效率，可以用于质粒DNA提取、病毒基因组提取、细菌基因组提取，以及动植物和人基因组DNA的提取；
本发明的另外一个目的在于提供所述季铵盐作为离液盐的核酸提取试剂；
本发明的另外一个目的在于提供所述季铵盐作为离液盐的核酸提取方法。
为实现上述发明目的，本发明提供如下技术方案：
季铵盐在核酸提取中的应用，所述季铵盐具有式I所示结构，
Figure PCTCN2021097213-appb-000001
其中，An -是阴离子，所述R 1，R 2，R 3，R 4独立选自烷基。
本发明使用季铵盐作为离液盐用于核酸提取，与胍盐作为离液盐相比，季铵盐更加稳定，季铵盐选择种类也更多，并且季铵盐在乙醇等有机溶剂中的溶解度也更高，因此相同工作浓度具有更低的成本。
作为优选，所述阴离子为卤素的阴离子；进一步优选地，所述阴离子为氯离子或溴离子。
作为优选，所述R 1，R 2，R 3，R 4独立选自C1-C5的烷基；进一步优选地，所述R 1，R 2，R 3，R 4独立选自甲基、乙基、丙基、丁基和戊基。
在本发明具体实施方式中，所述季铵盐为四甲基溴化铵，四甲基氯化铵、四乙基溴化铵，四乙基氯化铵、四丙基溴化铵、四丙基氯化铵、四丁基溴化铵、四丁基氯化铵、四戊基溴化铵或四戊基氯化铵。
将季铵盐作为离液盐用于核酸提取，一定浓度的季铵盐能中和核酸所带的负电荷，在极性溶剂(乙醇，异丙醇等)条件下，核酸通过氢键，疏水作用和静电作用能与吸附材料相结合或被沉淀出来。所述季铵盐以溶液形式作用于核酸提取中，季铵盐溶液的盐浓度范围为100mM～2.5M，优选为250mM～2.5M，在本发明具体实施方式中季铵盐浓度为250mM、1.6M或2.5M。
本发明所述核酸是指由许多核苷酸聚合而成的生物大分子，它是一种极为重要的生命物质。核酸广泛存在于动植物细胞，微生物体内。根据化学组成，核酸主要分为脱氧核糖核酸(DNA)与核糖核酸(RNA)两种，而本发明所述核酸分子也指DNA、RNA或两者的混合。
作为优选，所述核酸提取从提取手段划分包括酚-氯仿提取法、极性溶剂(乙醇、异丙醇或聚乙二醇)沉淀法、固相载体吸附法。固相载体吸附法是利用载体对核酸的较强亲和力和吸附力，将释放出来的核酸特异性地结合在特定载体上，一些常用的载体包括硅珠，磁珠，硅藻土或硅胶等。所述核酸提取从核酸类型划分包括质粒DNA提取、病毒基因组提取(如λDNA提取)、细菌基因组提取(如大肠杆菌DNA提取)，以及动植物和人基因组DNA的提取，这些类型的核酸提取均可以参照现有技术选择合适的提取手段。
依据本发明提供的上述应用，本发明还提供了一种核酸提取试剂，其中的离液盐为本发明式I所示结构的季铵盐。
作为优选，所述核酸提取试剂包括本发明的式I所示结构的季铵盐，以及裂解液、结合液、洗涤液、洗脱液、吸附材料中的一种或两种以上组分； 所述季铵盐可以独立作为核酸提取试剂中的一个组分，或添加到裂解液，或添加到结合液。
所述裂解液、结合液、洗涤液、吸附材料以及洗脱液根据不同的提取手段或提取核酸类型不同而以不同的组合形式组合，例如在提取人类基因组、质粒DNA和微量基因组DNA中，所述核酸提取试剂包括裂解液、结合液、洗涤液、吸附材料以及洗脱液；在病毒基因组-λDNA提取中，所述核酸提取试剂包括裂解液、洗涤液、吸附材料以及洗脱液；有些时候，所述结合液可以直接加到裂解液中，称为裂解结合液，比如在病毒基因组-λDNA提取中。
其中，所述裂解液根据不同的核酸提取包括缓冲液(10-50mM)、蛋白酶(0.5mg/ml)、碱(200mM)、表面活性剂(1-10％)、核酸稳定剂(36-50mM)、溶菌酶(5mg/ml)、酚-氯仿-异戊醇中的一种或两种以上，例如缓冲液+蛋白酶+表面活性剂+核酸稳定剂，缓冲液+表面活性剂，缓冲液+核酸稳定剂+表面活性剂+碱，缓冲液+溶菌酶+蛋白酶+酚-氯仿-异戊醇，缓冲液+核酸稳定剂+表面活性剂+蛋白酶等；本发明具体实施方式中蛋白酶为蛋白酶K，表面活性剂为SDS、PEG或SLS，碱为氢氧化钠，缓冲液为磷酸缓冲液或Tris-HCl，核酸稳定剂为EDTA；
作为优选，所述结合液为异丙醇、无水乙醇或聚乙二醇。
作为优选，所述洗涤液为75-80％乙醇溶液；所述洗脱液可以是低盐缓冲液(10mM，如Tris-HCl)或者去离子超纯水，更进一步地所述洗脱液还可以包含表面活性剂或核酸稳定剂(1mM)，表面活性剂优选为SDS或TritonX-100，质量百分比浓度为0.1-1％；核酸稳定剂优选为EDTA，浓度为1mM。
此外本发明还提供了一种核酸提取方法，在核酸提取过程中，添加本发明式I所示结构的季铵盐作为离液盐进行提取，其他试剂可参照常规选择。
更进一步地，所述提取方法向待提取样品中加入裂解液裂解，释放核酸分子，再加入结合液使核酸结合到吸附材料上或者沉淀出来，所述季铵盐在裂解后加入或在加入结合液后加入，或者添加到裂解液中一同加入，或者添加到结合液中一同加入。
作为优选，所述提取方法还包括使用洗涤液对吸附材料上的核酸或者沉淀出来的核酸进行洗涤。
作为优选，所述提取方法还包括使用洗脱液对吸附材料上的核酸进行洗脱，或采用洗脱液溶解沉淀出来的核酸。
由以上技术方案可知，本发明提供了一种以式I所示结构的季铵盐为离液盐，用于替代目前常用的胍盐，进行核酸提取；其能在极性有机溶剂结合液存在下，使溶液中的核酸分子吸附到硅珠、磁珠、硅藻土或硅胶等材料或者沉淀出来，能够有效提高核酸纯化和回收的效率，可以用于质粒DNA提取，病毒基因组提取、细菌基因组提取、或动植物和人基因组DNA的提取。
附图说明
图1所示为人血液基因组提取DNA荧光定量PCR扩增曲线比较
图2所示为λDNA提取琼脂糖凝胶电泳检测图谱；
图3所示为质粒DNA提取琼脂糖凝胶电泳检测图谱；
图4所示为大肠杆菌基因组提取琼脂糖凝胶电泳检测图谱；
图5所示为微量基因组DNA提取STR-毛细管电泳检测图谱；
图6所示为CTAB与TEAB提取微量基因组效果比较。
图7所示为MERS假病毒核酸提取荧光定量PCR扩增曲线。
具体实施方式
本发明公开了一种核酸提取方法以及应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明所述提取方法和应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述提取方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
在本发明具体实施方式中，所提供的对比试验各处理组所采用的原料均相同，且各组除去应有的区别外其他试验条件保持一致。本发明所涉及原料、试剂等，如无特殊说明均可通过市售途径获得。
下面结合实施例，进一步阐述本发明。
实施例1：人类血液DNA的提取纯化
将含有四乙基氯化铵的提取液用于磁珠法提取核酸，同时将另一组不含有四乙基氯化铵，其余组分相同的提取液作为对照，比较两组的提取效果。所用试剂组分如下：
提取液1(裂解液)组成包括Tris-HCl(10mM)，EDTA(50mM)，SDS(1％)，四乙基氯化铵(250mM)，蛋白酶K(0.5mg/ml)。
提取液2(结合液)组成包括100％异丙醇。
提取液3(洗涤液)组成包括75％乙醇。
提取液4(洗脱液)组成包括超纯水。
磁珠混悬液组成包括超顺磁性纳米磁珠，分散在超纯水中。
制备方法包括以下步骤：
1、取5μl人类全血放入离心管中，加入300μl上述提取液1，56度处理15分钟。
2、加入300μl提取液2，400ug磁珠混悬液，800rpm振荡混匀10分钟。
3、将离心管置于磁力架上进行磁分离，吸弃管中液体，加入500μl提取液3，振荡混匀1分钟。磁分离弃上清。重复步骤3一次。
4、待上述磁珠在室温下晾干至无明显乙醇气味后，加入50μl提取液4，置于65度处理10分钟。
5、磁分离，收集上清。
荧光定量PCR扩增上述收集的上清：
25μl扩增体系包括以下组分：12.5μl 2*PCR Mix；各1μl 10uM引物；收集的上清3μl；纯水7.5μl。其中模板除了两组提取的血液，还有作为阳性对照的标准品1ng人基因组及作为阴性对照的提取液4。
热循环如下：95度，10分钟；(95度，15秒；60度，45秒)*40循环
结果如图1所示。结果表明：用磁珠法提取扩增血液DNA时，加四乙基氯化铵比不加四乙基氯化铵提取扩增效果明显变好，说明四乙基氯化铵可以提高DNA的沉淀回收效率。也说明使用含有季铵盐的提取液可以有效进行血液中DNA提取纯化。
实施例2：λDNA提取纯化
本实施例旨在说明季铵盐对核酸吸附的效果，所以采用λDNA进行模拟纯化。使用含有四丙基溴化铵的提取液用于核酸提取，同时将另一组不含有四丙基溴化铵，其余组分相同的提取液作为对照，比较两组的提取效果。所用试剂组分如下：
提取液1(裂解结合液)组成包括Tris-HCl(20mM)，四丙基溴化铵(250mM)，10％PEG8000。
提取液2(洗涤液)组成包括75％乙醇。
提取液3(洗脱液)组成包括超纯水。
磁珠混悬液组成包括超顺磁性纳米磁珠，分散在超纯水中。
制备方法包括以下步骤：
1、取50ngλDNA放入离心管中，加入400μl上述提取液1，100-400μg磁珠混悬液，500rpm-12000rpm振荡混匀10分钟。
2、将离心管置于磁力架上进行磁分离，吸弃管中液体，加入500提取液2，振荡混匀1分钟。磁分离弃上清。重复步骤3一次。
3、待上述磁珠在室温下晾干至无明显乙醇气味后，加入50μl提取液3，置于65度处理10分钟。
4、磁分离，收集上清。
PCR扩增上述收集的上清：
20μl扩增体系包括以下组分：10μl 2*PCR Mix；各1μl 10uM引物；收集的上清8μl。其中模板除了两组提取的血液，还有作为阳性对照的标准品10ngλDNA及作为阴性对照的提取液3。
热循环如下：95度，5分钟；(95度，20秒；60度，3分钟)*35循环
电泳结果如图2所示。
结果表明：在使用上述提取液提取扩增lambdaDNA时，加四丙基溴化铵在6000bp处有一条带，比不加四丙基溴化铵提取扩增效果明显好，说明四丙基溴化铵可以提高DNA的沉淀回收效率。
实施例3：质粒DNA提取纯化
本发明将季铵盐用于乙醇沉淀核酸方法中，可以有效进行大肠杆菌质粒抽提，包括提取液1、提取液2、提取液3、提取液4、提取液5五部分组成。
提取液1(裂解液)成包括10mM Tris-HCl(pH8.0)，50mM EDTA(pH8.0)。
提取液2(裂解液)组成包括200mM NaOH，1％SDS。
提取液3(离液盐)组成包括2.5M四丁基溴化铵(pH7.0)。
提取液4(结合液)组成包括100％异丙醇。
提取液5(洗涤液)组成包括75％乙醇。
提取液6(洗脱液)组成包括10mM Tris-HCl(pH8.0)，1mM EDTA(pH8.0)。
制备方法包括以下步骤：
1、取1ml培养过夜的大肠杆菌放入离心管中，5000rpm 5min，吸弃上清。
2、加入200μl提取液1重悬沉淀，加入1μl RNA酶。
3、加入400μl提取液2，上下轻柔颠倒混匀。
4、加入300μl提取液3，上下颠倒混匀。12000rpm，10分钟。取上清。
5、加入等体积的提取液4上下颠倒混匀，-20度放置半小时。
6、12000rpm，10分钟。弃上清。
7、加入100μl提取液5，漂洗一次，37度晾干。
8、加入30μl提取液6，混匀。
已溶解的DNA，取10μl电泳。同时将提取液3换成3M NaAc(pH5.2)，其他试剂不变，作为阳性对照；将提取液3换成水，其他试剂不变，作为阴性对照。
9、电泳检测，结果如图3所示。
结果表明：在使用上述提取液提取大肠杆菌质粒时，四丁基溴化铵与NaAc提取效果相当，说明四丁基溴化铵可以提高大肠杆菌质粒的沉淀回收效率。
实施例4：大肠杆菌基因组DNA提取纯化
本发明将季铵盐用于酚-氯仿提取核酸方法中，可以有效进行大肠杆菌基因组DNA提取纯化，包括提取液1、提取液2、提取液3、提取液4、磁珠混悬液四部分组成。
提取液1(裂解液)组成包括磷酸缓冲液(pH7.0，50mM)，溶菌酶(5mg/ml)。
提取液2(裂解液)组成包括蛋白酶K(0.5mg/ml)。
提取液3(离液盐)组成包括四丁基溴化铵(2.5M)。
提取液4(裂解液)组成包括酚-氯仿-异戊醇(25:24:1)。
提取液5(结合液)组成包括无水乙醇。
提取液6(洗涤液)组成包括75％乙醇。
提取液7(洗脱液)组成包括10mM Tris-HCl(pH8.0)，1mM EDTA(pH8.0)。
制备方法包括以下步骤：
1、取1ml培养过夜的大肠杆菌感受态细胞放入离心管中，5000rpm5min，吸弃上清。
2、加入200μl提取液1重悬沉淀，放置金属浴上，37度14000rpm，孵育过夜。
3、取100μl消化液，加入10μl提取液2，56度保温处理10分钟。
4、加入100μl提取液3。
5、加入200μl提取液4，上下颠倒混匀。14000rpm，10分钟。取上清。
6、加入200μl提取液5，室温放置半小时。14000rpm，10分钟。
7、弃上清，加入100μl提取液6漂洗一次，37度晾干。
8、加入30μl提取液7，振荡混匀。
PCR扩增上述溶解的DNA：
20μl扩增体系包括以下组分：10μl 2*PCR Mix；各1μl 10uM引物；收集的上清4μl；4μl纯水。同时将提取液3换成5M氯化钠，其他试剂不变，作为阳性对照；将提取液3换成水，其他试剂不变，作为阴性对照。
热循环如下：95度，5分钟；(95度，20秒；60度，3分钟)*35循环
取10μl PCR产物电泳检测，结果如图4所示。
结果表明：在使用上述提取液提取大肠杆菌基因组时，四丁基溴化铵与NaCl提取扩增效果相当，说明四丁基溴化铵可以提高大肠杆菌基因组的沉淀回收效率。
实施例5：微量基因组DNA的提取纯化
本发明可以有效进行微量基因组DNA的提取纯化，并对比同浓度四乙基溴化铵与盐酸胍的提取效率。本发明的提取试剂包括提取液1、提取液2、提取液3、提取液4、磁珠混悬液五部分组成。
提取液1(裂解液)组成包括Tris-HCl(10mM)，EDTA(50mM)，SLS(1％)，蛋白酶K(0.5mg/ml)。
提取液2(结合液)组分为三种，A组成包括40％异丙醇；B包括40％异丙醇和1.6M四乙基溴化铵；C包括1.6M盐酸胍。
提取液3(洗涤液)组成包括80％乙醇。
提取液4(洗涤液)组成包括10mM Tris-HCl(pH8.0)，1mM EDTA(pH8.0)。
磁珠混悬液组成包括超顺磁性纳米磁珠，分散在超纯水中。
制备方法包括以下步骤：
1、取700倍稀释人新鲜血液20μl放入离心管中，加入400μl上述提取液1，56度处理30分钟。
2、加入250μl提取液2，400ug磁珠混悬液，800rpm振荡混匀10分钟。
3、将离心管置于磁力架上进行磁分离，吸弃管中液体，加入500μl提取液3，振荡混匀1分钟。磁分离弃上清。重复步骤3一次。
4、待上述磁珠在室温下晾干至无明显乙醇气味后，加入30μl提取液4，置于65度震荡10分钟。
5、磁分离，收集上清。
取上清6μl，用NH18STR扩增试剂盒检测。
15μl扩增体系包括以下组分：6μl 2.5*PCR Mix；3μlNH18引物混合物；收集的上清6μl。其中模板除了提取的微量样本，还有作为阳性对照的标准品0.2ng人基因组及作为阴性对照的提取液4。
热循环如下：95度，10分钟；(95度，10秒；59度，60秒)*30循环；60度，10分钟。结果如图5所示。
结果表明：加入四乙基溴化铵可以显著提高对于微量DNA的提取效率，同浓度四乙基溴化铵的提取效率远大于盐酸胍。说明使用四乙基溴化铵能有效进行微量基因组DNA的提取纯化。
实施例6：CTAB与TEAB提取微量基因组DNA的比较
在很多植物基因组提取方法中常常用到十六烷基三甲基溴化铵(CTAB)，虽然CTAB本质上也属于季铵盐，但在该方法中CTAB的作用是阳离子表面活性剂，用来辅助裂解植物细胞，而不是作为离液盐帮助核酸与吸附材料的结合或者核酸沉淀。此外，CTAB在水中溶解度很低(小于0.1M)，无法单独使用来完成核酸的纯化，该过程还需要其他离液盐(如氯化钠，醋酸钠等)共同分离和纯化核酸。故植物基因组提取时使用的CTAB与本发明的方案没有相关性。
本发明可以有效进行微量基因组DNA的提取纯化，并对比CTAB与TEAB(四丁基溴化铵)提取效果进行比较。本发明的提取试剂包括提取液1、提取液2、提取液3、提取液4、磁珠混悬液五部分组成。
提取液1(裂解液)组成包括Tris-HCl(终浓度50mM)，EDTA(终浓度36mM)，SLS(终浓度1％)，蛋白酶K(0.5mg/ml)。
提取液2(结合液)组分为两种(D,E)，D组成包括异丙醇(终浓度40％)和TEAB(终浓度1.6M)；E包括异丙醇(终浓度40％)和CTAB(母液浓度2％，终浓度1.2％，已经是最大添加量)。
提取液3(洗涤液)组成包括80％乙醇。
提取液4(洗脱液)组成包括10mM Tris-HCl(pH8.0)，1mM EDTA(pH8.0)。
磁珠混悬液组成包括超顺磁性纳米磁珠，分散在超纯水中。
制备方法包括以下步骤：
1、取700倍稀释人新鲜血液30μl放入离心管中，加入400μl上述提取液1，56度处理30分钟。
2、加入250μl提取液2，400ug磁珠混悬液，800rpm振荡混匀10分钟。
3、将离心管置于磁力架上进行磁分离，吸弃管中液体，加入500μl提取液3，振荡混匀1分钟。磁分离弃上清。重复步骤3一次。
4、待上述磁珠在室温下晾干至无明显乙醇气味后，加入30μl提取液4，置于65度震荡10分钟。
5、磁分离，收集上清。
取上清6μl，用NH18STR扩增试剂盒检测。
15μl扩增体系包括以下组分：6μl 2.5*PCR Mix；3μl NH18引物混合物；收集的上清6μl。其中模板除了提取的微量样本，还有作为阳性对照的标准品0.2ng人基因组及作为阴性对照的提取液4。
热循环如下：95度，10分钟；(95度，10秒；59度，90秒)*28循环；60度，10分钟。
结果如图6所示。结果表明：加入TEAB可以显著提高对于微量DNA的提取效率，而加入CTAB之后几乎提取不到DNA。
实施例7：MERS假病毒核酸提取
本发明旨在说四甲基溴化铵对MERS假病毒核酸提取的影响，包括提取液1、提取液2、提取液3三部分组成。
提取液1组成包括MOPS(10mM),异丙醇(50％)，四甲基溴化铵(1M)。
提取液2组成包括80％乙醇。
提取液3组成包括超纯水。
制备方法包括以下步骤：
1、取10μl MERS假病毒RNA加入到含有390ul0.9％生理盐水的离心管中，再加入400μl上述提取液1，转移至硅膜中，室温静置5min后，8000rpm，离心1min，弃去废液。
2、加入500μl提取液2，室温静置3min。12000rpm离心1min，弃去废液。12000rpm空管离心2min，弃去废液。
3、待上述硅膜在室温下晾干至无明显乙醇气味后，加入70μl提取液3，12000rpm离心1min，收集离心液体。
荧光定量PCR扩增上述收集的离心液体：
25μl扩增体系包括以下组分：12.5μl 2*PCR Mix；各1.5μl引物探针；收集的离心液体10μl；纯水1μl。其中模板除了提取的MERS假病毒RNA，还有作为阳性对照的1.5ul同浓度的MERS假病毒RNA及作为阴性对照的提取液3。
热循环如下：95度，10分钟；(95度，15秒；60度，45秒)*40循环
结果如图7所示。结果表明：提取液1中加入四甲基溴化铵对MERS假病毒核酸提取有效。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (14)

  1. 季铵盐在核酸提取中的应用，所述季铵盐具有式I所示结构，
    Figure PCTCN2021097213-appb-100001
    其中，An -是阴离子，所述R 1，R 2，R 3，R 4独立选自烷基。
  2. 根据权利要求1所述应用，其特征在于，所述阴离子为卤素的阴离子。
  3. 根据权利要求1或2所述应用，其特征在于，所述阴离子为氯离子或溴离子。
  4. 根据权利要求1所述应用，其特征在于，所述R 1，R 2，R 3，R 4独立选自C1-C5的烷基。
  5. 根据权利要求1所述应用，其特征在于，所述R 1，R 2，R 3，R 4独立选自甲基、乙基、丙基、丁基和戊基。
  6. 根据权利要求1所述应用，其特征在于，所述季铵盐在核酸提取中充当离液盐。
  7. 一种核酸提取试剂，其特征在于，核酸提取试剂中的离液盐为权利要求1中的式I所示结构的季铵盐。
  8. 根据权利要求7所述核酸提取试剂，其特征在于，包括权利要求1中的式I所示结构的季铵盐，以及裂解液、结合液、洗涤液、洗脱液、吸附材料中的一种或两种以上组分；所述季铵盐可以独立作为核酸提取试剂中的一个组分，或添加到裂解液，或添加到结合液。
  9. 根据权利要求8所述核酸提取试剂，其特征在于，所述裂解液包括蛋白酶、碱、表面活性剂中的一种或两种以上。
  10. 根据权利要求8所述核酸提取试剂，其特征在于，所述结合液为异丙醇、无水乙醇或聚乙二醇。
  11. 一种核酸提取方法，其特征在于，在核酸提取过程中，添加权利要求1中的式I所示结构的季铵盐作为离液盐进行提取。
  12. 根据权利要求11所述提取方法，其特征在于，向待提取样品中加入裂解液裂解，释放核酸分子，再加入结合液使核酸结合到吸附材料上或者沉淀出来，所述季铵盐在裂解后加入或在加入结合液后加入，或者添加到裂解液中一同加入，或者添加到结合液中一同加入。
  13. 根据权利要求12所述提取方法，其特征在于，还包括使用洗涤液对吸附材料上的核酸或者沉淀出来的核酸进行洗涤。
  14. 根据权利要求12或13所述提取方法，其特征在于，还包括使用洗脱液对吸附材料上的核酸进行洗脱，或采用洗脱液溶解沉淀出来的核酸。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114507662A (zh) * 2022-03-22 2022-05-17 合肥欧创基因生物科技有限公司 一种结合液及基于该结合液的超螺旋质粒大提试剂盒

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115786328A (zh) * 2022-11-29 2023-03-14 青岛大学 基于硅藻壳的快速核酸提取方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3059312A1 (en) * 2015-02-20 2016-08-24 QIAGEN GmbH Nucleic acid extraction method
CN106459965A (zh) * 2014-04-30 2017-02-22 凯杰有限公司 分离多聚(a)核酸的方法
CN108342382A (zh) * 2018-01-30 2018-07-31 广东海大畜牧兽医研究院有限公司 一种核酸快速提取方法及其试剂盒

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105462961B (zh) * 2016-01-14 2019-04-19 苏州市公安局 一种提取核酸的方法及试剂盒

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106459965A (zh) * 2014-04-30 2017-02-22 凯杰有限公司 分离多聚(a)核酸的方法
EP3059312A1 (en) * 2015-02-20 2016-08-24 QIAGEN GmbH Nucleic acid extraction method
CN108342382A (zh) * 2018-01-30 2018-07-31 广东海大畜牧兽医研究院有限公司 一种核酸快速提取方法及其试剂盒

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
"Current Protocols in Molecular Biology", 1993, WILEY-INTERSCIENCE
LIU, HONG-BO ET AL.: "Developments of Nucleic Acid Extraction Method", PROGRESS IN MODERN BIOMEDICINE, vol. 11, no. 16, 31 August 2011 (2011-08-31), pages 3187 - 3190, XP055878068, ISSN: 1673-6273 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114507662A (zh) * 2022-03-22 2022-05-17 合肥欧创基因生物科技有限公司 一种结合液及基于该结合液的超螺旋质粒大提试剂盒

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