WO2007049326A1 - Procede et kit d’extraction d’acide nucleique - Google Patents

Procede et kit d’extraction d’acide nucleique Download PDF

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Publication number
WO2007049326A1
WO2007049326A1 PCT/JP2005/019473 JP2005019473W WO2007049326A1 WO 2007049326 A1 WO2007049326 A1 WO 2007049326A1 JP 2005019473 W JP2005019473 W JP 2005019473W WO 2007049326 A1 WO2007049326 A1 WO 2007049326A1
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agent
nucleic acid
reagent
surfactant
solution
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PCT/JP2005/019473
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English (en)
Japanese (ja)
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Kazunari Hirayasu
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Wako Pure Chemical Industries, Ltd.
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Priority to PCT/JP2005/019473 priority Critical patent/WO2007049326A1/fr
Publication of WO2007049326A1 publication Critical patent/WO2007049326A1/fr

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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 a method for extracting nucleic acid, particularly blood free DNA from blood components, and a kit used therefor.
  • Non-patent Document 6 A method in which protein is denatured to liberate DNA and liberated DNA and glycogen are precipitated with isopropanol (Non-patent Document 6) For example, whole blood is treated with a surfactant to break down and expose the cell membrane of blood cells Cell nuclei are collected and further treated with proteolytic enzymes in the presence of surfactants and salts to destroy the nuclear membrane and nucleoprotein, and then contacted with a chaotropic agent to migrate DNA.
  • Patent Document 1 For example, one kind selected from a reducing agent, a surfactant, a chelating agent, and a protein denaturing agent, if necessary in the presence of a salt and a coprecipitation agent, Treat blood components with proteolytic enzymes to degrade and denature proteins, etc.
  • a method in which a chaotropic agent is liberated and decomposed by the action of a chaotropic agent to dissolve the denatured protein, etc., and the released DNA is precipitated with alcohol in the presence of a salt and a coprecipitation agent Patent Document 2
  • Non-Patent Document 1 Sozzi, G "et al., Clin. Cancer Res., 5, 2689 (1999)
  • Non-Patent Document 2 Sliva, J. M., et al, Cancer Res., 59, 3251 (1999)
  • Non-Patent Document 3 Shao, Z.M., et al "Clin. Cancer Res. 7, 2222 (2001)
  • Non-Patent Document 4 Biochemistry Experiment Course 2, "Nucleic Acid Chemistry 1", pp. 74-80, 262-270, 19
  • Non-Patent Document 5 "Gene Manipulation Manual", pp. 20-23, 1985, Kodansha
  • Non-Patent Document 6 Ishizawa, M., et al "Nucleic Acid Res., 19, 5792 (1991)
  • Patent Document 1 JP-A-6-205676
  • Patent Document 2 Japanese Patent Laid-Open No. 7-236499
  • the present invention relates to a method for easily extracting nucleic acid, particularly blood free DNA, from blood components in a high yield and easily, and a reagent used therein that has a stable and high nucleic acid recovery rate and excellent storage stability.
  • An object is to provide a kit.
  • the present invention has the following configuration.
  • a blood component and a proteolytic enzyme are reacted in the presence of a surfactant and a salt.
  • a surfactant and a salt are reacted in the presence of a surfactant and a salt.
  • a chaotropic agent and a coprecipitation agent are contacted with a chaotropic agent and a coprecipitation agent.
  • a blood component comprising a combination of (1) a reagent containing a proteolytic enzyme, (2) a reagent containing a surfactant and a salt, and (3) a reagent containing an oral picking agent and a coprecipitation agent.
  • Nucleic acid extraction kit comprising a combination of (1) a reagent containing a proteolytic enzyme, (2) a reagent containing a surfactant and a salt, and (3) a reagent containing an oral picking agent and a coprecipitation agent.
  • the present invention provides a method for easily extracting nucleic acid in high yield and a kit used therefor.
  • nucleic acid particularly blood
  • blood components serum, plasma, etc.
  • Medium free DNA can be easily extracted in a high yield, and the storage stability of the reagents used therefor can be improved.
  • FIG. 1 Protein plot assembly was performed on the precipitates (nucleic acids) obtained using the lysates with different surfactant (sodium N-lauroyl sarcosinate) contents obtained in Example 3. It is a result.
  • the upper row shows the case of Case A
  • the lower row shows the case of Case B
  • 1 to 6 show the sample numbers.
  • FIG. 2 Extracting nucleic acid in a sample of hyperamylase by two methods (case 1 and case 2) obtained in Example 5 with different coprecipitation agent (glycogen) addition time, p53 -Shows the results of 3% agarose gel electrophoresis of Exon5 (308bp) region amplified by PCR.
  • lane M is the molecular weight marker DNA Step Ladder Mix (80 bp to 10 kb) (manufactured by Wako Pure Chemical Industries, Ltd.), lane 1 is case 1! /, And sample 1 is the sample, Lane 2 shows the case 2 with Sample 2 as the sample in Case 2.
  • Lane 3 Shows the case where specimen 2 was used as the sample in case 1
  • lane 4 shows the case where specimen 2 was used as the sample in case 2.
  • the arrow indicates the 308 bp p53-Exon5 region.
  • proteolytic enzyme used in the present invention examples include non-specific proteolytic enzymes such as proteinase 1, pronase, trypsin, and subtilisin. Of these, proteinase K is preferred.
  • the amount of proteolytic enzyme used is an amount capable of sufficiently degrading a mixture of proteins in blood components, in other words, the nucleic acid obtained after the method of the present invention is used for various analyses. There is no particular limitation as long as it is an amount that can extract a sufficient amount and quality (purity) of nucleic acid.
  • the lower limit is usually 0.2 mg or more, preferably 1 mg or more, with respect to 1 mL of the blood component.
  • the upper limit is not particularly limited, but considering economics, etc., a concentration of usually not more than lOOmg, preferably not more than 10mg is added to lmL of blood component in the reaction solution when reacting blood component and proteolytic enzyme. .
  • the surfactant used in the present invention has a sufficient protein denaturing action and does not cause precipitation when coexisting with a salt.
  • a surfactant those having the above-mentioned properties are appropriately selected from an anionic surfactant, a cationic surfactant, a nonionic surfactant, and an amphoteric surfactant.
  • preferred anionic surfactants include carboxylic acids, sulfonic acids, sulfate esters, phosphate esters, cholic acids, or salts thereof.
  • carboxylic acids include higher fatty acids such as lauric acid, myristic acid, palmitic acid, stearic acid, and oleic acid, and derivatives thereof. Specific examples include oleic acid, N-lauroyl sarcosine acid, and N-myristoyl. n of N- methyl-13-Ryo alanine, etc. Porioki sheet polyoxyethylene lauryl ether acetic acid Examples of these salts include alkali metal salts such as sodium and potassium, ammonium salts, and the like. Specifically, for example, potassium oleate, sodium N-lauroyl sarcosinate (SLS), N— Examples include myristoyl-N-methyl-13-alanine sodium and polyoxyethylene lauryl ether sodium acetate.
  • SLS sodium N-lauroyl sarcosinate
  • Examples include myristoyl-N-methyl-13-alanine sodium and polyoxyethylene lauryl ether sodium acetate.
  • sulfonic acids examples include alkylbenzene sulfonic acids (for example, laurylbenzene sulfonic acid), naphthalene sulphonic acid (for example, dipropino naphthalene sulphonic acid, dibutino naphthalene sulphonic acid), sulfosuccinic acid (for example, dioctyl sulfosuccinic acid) Etc.
  • alkylbenzene sulfonic acids for example, laurylbenzene sulfonic acid
  • naphthalene sulphonic acid for example, dipropino naphthalene sulphonic acid, dibutino naphthalene sulphonic acid
  • sulfosuccinic acid for example, dioctyl sulfosuccinic acid
  • these salts include alkali metal salts such as sodium and potassium, ammonium salts, and the like.
  • alkali metal salts such as sodium and potassium, ammonium salts, and the like.
  • sodium and sodium dioctylsulfosuccinate examples include sodium and sodium dioctylsulfosuccinate.
  • sulfates examples include higher alcohol sulfates (for example, lauryl sulfate), polyoxyethylene alkyl ether sulfates (for example, polyoxyethylene alkyl ether sulfate, polyoxyethylene lauryl ether sulfate), and the like.
  • these salts include alkali metal salts such as sodium and potassium, ammonium salts, and the like. Specific examples include sodium lauryl sulfate (sodium dodecyl sulfate: SDS), ammonium lauryl sulfate. And sodium polyoxyethylene alkylphenol ether sulfate and sodium polyoxyethylene lauryl ether sulfate.
  • phosphate esters examples include monostearyl phosphate ester, monolauryl phosphate ester, polyoxyethylene lauryl ether phosphate, and the like. Specifically, for example, monostearyl phosphate, monolauryl phosphate, polyester And oxyethylene lauryl ether phosphoric acid.
  • alkali metal salts such as sodium and potassium, ammonium salts, and the like.
  • Specific examples include sodium monostearyl phosphate, sodium monolauryl phosphate, polyoxyethylene lauryl ether. A potassium phosphate etc. are mentioned.
  • cholic acids As cholic acids, cholic acid, cholic acid derivatives (for example, deoxycholic acid, etc.), etc. Is mentioned.
  • examples of these salts include alkali metal salts such as sodium and potassium, ammonium salts, and the like. Specific examples include sodium cholate and sodium deoxycholate.
  • anionic surfactants those having the properties as described above are selected.
  • N-lauroyl sarcosine acid or a salt thereof for example, sodium salt, potassium salt, lithium salt, etc.
  • a salt thereof for example, sodium salt, potassium salt, lithium salt, etc.
  • Sodium sarcosinate is particularly preferred.
  • the amount of the surfactant to be used varies depending on the type of the surfactant used and the like and cannot be generally specified, but may be appropriately selected from the range normally used in this field.
  • Such a use amount is an amount that can denature contaminants such as proteins in blood components and sufficiently improve the function of the protein-degrading enzyme, in other words, obtained after carrying out the method of the present invention.
  • the amount of nucleic acid is sufficient to share nucleic acids for various analyses, and the amount (quality) of the nucleic acid can be extracted from blood components.
  • blood components and proteolytic enzymes are used.
  • the lower limit is usually at least 0.1% (w / v), preferably at least 0.3% (w / v), more preferably at least 1% (w / v). More preferably, it is 2.0% (w / v) or more, and particularly preferably 2.5% (w / v) or more.
  • the upper limit is not particularly limited, but considering economy etc., it is usually 10% (w / v) or less, preferably 5% (w / v) or less, more preferably 3.5% (w / v) or less.
  • the lower limit is usually 0.1% (w / v) or more, for example, as the concentration in the reaction solution when the blood component and the protein degrading enzyme are reacted. Is 0.3% (w / v) or more, more preferably 1% (w / v) or more, and particularly preferably 2.5% (w / v) or more.
  • the upper limit is not particularly limited, but considering economy etc., it is usually 10% (w / v) or less, preferably 5% (w / v) or less, more preferably 3.5% (w / v) or less. is there.
  • alkali metal salt used in the present invention examples include lithium, sodium, and power.
  • examples include those containing an alkali metal ion such as lithium as a cation, and those having an action of activating a proteolytic enzyme are preferable.
  • an alkali metal salt examples include sodium chloride sodium, potassium salt potassium, lithium chloride, sodium acetate, and the like. It should be noted that even if it is an alkali metal salt, a substance having a property as a chaotropic agent such as sodium chloride can not be used for the purpose of the present invention because it deactivates the proteolytic enzyme. Yes.
  • the amount of the alkali metal salt used is an amount that can sufficiently improve the function of a proteolytic enzyme that degrades a contaminant such as a protein in blood components, in other words, obtained after the method of the present invention has been performed.
  • a sufficient amount of nucleic acid can be extracted from blood components to share the nucleic acid for various analyses.
  • a lower limit is usually added at a concentration of 10 mM or more, preferably 30 mM or more in a reaction solution for reacting a blood component with a protease.
  • the upper limit is not particularly limited, but in consideration of economics, etc., it is usually 1.5M or less, preferably 1M or less, more preferably 500mM or less, particularly in the reaction solution when the blood component and the proteolytic enzyme are reacted.
  • a concentration of 10 mM or less is added.
  • the lower limit is usually 10 mM or more, preferably 30 mM or more, added to the reaction solution for reacting blood components with proteolytic enzymes.
  • the upper limit is not particularly limited.
  • the reaction solution for reacting a blood component with a proteolytic enzyme is usually 1.5 M or less, preferably 1 M or less, more preferably 500 mM or less, particularly preferably. Is added at a concentration of lOOmM or less.
  • the chaotropic agent used in the present invention generates a chaotropic ion (a monovalent anion having a large ionic radius) when added to an aqueous solution, which is generally known as a chaotropic agent.
  • a chaotropic agent a monovalent anion having a large ionic radius
  • it is not particularly limited as long as it has an action of increasing the water solubility of, for example, alkali, thiocyanic acid guanidine, alkali metal salt of perchloric acid, trichloric acid.
  • alkali metal salts of acetic acid and alkali metal salts of thiocyanic acid examples include alkali metal salts of acetic acid and alkali metal salts of thiocyanic acid.
  • Examples of the alkali metal in Lucari include lithium, sodium, potassium and the like. Among these, sodium chloride is particularly preferable because sodium alkali is preferred.
  • the concentration of the chaotropic agent used varies depending on the type of chaotropic agent used. Specifically, the concentration of the chaotropic agent in the solution when the nucleic acid (and protein derived from blood components degraded by proteolytic enzymes) is contacted with the chaotropic agent is used.
  • the concentration is usually 2.5M or more as a lower limit, preferably 2.6M or more, more preferably 3.0M or more, more preferably 3.2M or more, particularly preferably 3.5M or more, and the upper limit is usually 7M or less, preferably 5M or less.
  • the lower limit is usually 2.5M or more, preferably 3.2M or more, particularly preferably 3.5M or more
  • the upper limit is usually 7M or less, preferably 5M or less.
  • coprecipitation agent used in the present invention is not particularly limited as long as it is usually used in this field, but high molecular polysaccharides such as glycogen and dextran, such as transfer RNA, Examples include polyacrylamide.
  • glycogen is particularly preferred, which is preferably a high molecular polysaccharide such as glycogen or dextran.
  • the amount of the coprecipitant used is not particularly limited as long as it is appropriately selected within the range power usually used in this field.
  • the lower limit of the concentration in the solution when contacting the nucleic acids and coprecipitate with alcohols is usually 1 ⁇ g / mL or more, preferably 5 ⁇ g / mL or more, more preferably 10 ⁇ g.
  • the upper limit is usually 1 mg / mL or less, preferably 100 ⁇ g / mL or less, more preferably 50 ⁇ g / mL or less.
  • the lower limit is usually 1 g / mL or more, preferably 5 ⁇ g / mL or more, more preferably 10 ⁇ g / mL or more
  • the upper limit is usually 1 mg / mL or less, preferably 100 g.
  • the nucleic acid and the coprecipitation agent are appropriately selected so as to be not more than 50 mL / mL, more preferably not more than 50 g / mL.
  • nucleic acid extraction method of the present invention (1) a blood component and a proteolytic enzyme are reacted in the presence of a surfactant and an alkali metal salt. (2) Then, the obtained reaction solution, a chaotropic agent, and a co-active agent are reacted. (3) A treatment for precipitating nucleic acid is performed after contacting the precipitant. It is.
  • Examples of blood components used in the method of the present invention include serum and plasma.
  • examples of the nucleic acid from which the blood component force as described above is also extracted include blood free DNA, DNA such as viral DNA, RNA such as blood free messenger RNA and viral RNA.
  • the method of the present invention is useful for DNA extraction, and is so-called blood free DNA that is derived from cells damaged or killed in various diseased sites such as cancer and is suspended in serum or plasma. Useful for extraction of (plasma DNA or circulating DNA).
  • proteins in blood components (such as histone proteins forming a complex with nucleic acids) can be denatured and solubilized, and nucleic acids can be released.
  • the recovery rate and quality (purity) of the nucleic acid that is finally obtained (extracted) by performing the reaction between the blood component and the proteolytic enzyme in the presence of an alkali metal salt. ) Is improved.
  • an aqueous solution (reaction solution) containing the blood component as described above, a proteolytic enzyme, a surfactant, and an alkali metal salt is prepared. It can be carried out by allowing a protease to act (react) on the blood component in the presence of a metal salt.
  • the most common methods are (1) a method in which a blood component is added to an aqueous solution (reaction solution) containing a proteolytic enzyme, a surfactant and an alkali metal salt, and (2) a protein in the blood component.
  • a method in which a blood component is added to an aqueous solution (reaction solution) containing a proteolytic enzyme, a surfactant and an alkali metal salt examples thereof include a method of adding an aqueous solution (reaction solution) containing a decomposing enzyme, a surfactant and an alkali metal salt.
  • an aqueous solution containing a proteolytic enzyme, an aqueous solution containing a surfactant, and an aqueous solution containing an alkali metal salt may be separately added to the blood component.
  • aqueous solutions containing one or more of a protease, a surfactant and an alkali metal salt may be separately added to the blood component.
  • an aqueous solution (solution) containing a surfactant and an alkali metal salt is added to a blood component, or a blood component is added to an aqueous solution (solution) containing a surfactant and an alkali metal salt,
  • a method in which a solution containing a blood component, a surfactant and an alkali metal salt is prepared, and then an aqueous solution containing a proteolytic enzyme is added to and mixed with the solution is preferable.
  • the concentration of each component used in each aqueous solution is within the range described above for the concentration in the final reaction solution (solution containing blood components, proteolytic enzymes, surfactants and alkali metal salts). It may be determined as appropriate in consideration of the amount of each solution used so that it is selected.
  • reaction conditions in step (1) of the present invention are as follows.
  • the lower limit of the reaction temperature at the time of reacting a blood component and a proteolytic enzyme in the presence of a surfactant and an alkali metal salt is usually 25 ° C or higher, preferably 37 ° C or higher, more preferably 55
  • the upper limit is usually 70 ° C or lower, preferably 65 ° C or lower, more preferably 60 ° C or lower.
  • the reaction time depends on the reaction temperature and the type of blood component, but the lower limit is generally 1 minute or more, preferably 5 minutes or more, more preferably 10 minutes or more, and the upper limit is usually 36.
  • the time is preferably 12 hours or less, more preferably 5 hours or less.
  • the pH at which the blood component reacts with the proteolytic enzyme is not particularly limited as long as it does not adversely affect the activity of the proteolytic enzyme.
  • the lower limit is usually pH 7 or higher.
  • the pH is 8 or higher, and the lower limit is usually pHIO or lower, preferably pH 9 or lower.
  • a buffer is used to maintain the pH when the blood component and the proteolytic enzyme are reacted in the presence of a surfactant and an alkali metal salt in the above-described range.
  • a buffering agent is not particularly limited as long as it has a buffering capacity in the pH range as described above at the reaction temperature as described above.
  • N- (2-acetamido) -2-amino Ethanesulfonic acid (ACES), ⁇ , ⁇ -bis (2-hydroxyethyl) -2-aminoethanesulfonic acid (BES), ⁇ , ⁇ -bis (2-hydroxyethyl) glycine (Bicine), N-cycloto Xyl-3-aminominosulfonic acid (CAPS), N-cyclohexyl-2-hydroxy-3-aminominosulfonic acid (CAPSO), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), 3 -[ ⁇ , ⁇ -Bis (2-hydroxyethyl) amino] -2-hydroxypropanesulfonic acid (DIPSO), 3- [4- (2-hydroxyethyl) -1-piperaduryl] propanesulfonic acid ( EPPS), 2- [4- (2-hydroxychetyl) -1-piperajuryl] ethanesulf
  • preferable buffering agents are such that the pKa at 60 ° C is usually 6.50 or more, preferably 7.00 or more, more preferably 7.20 or more, more preferably 7.30 or more, and the upper limit is usually
  • the buffer include 13.00 or less, preferably 11.00 or less, more preferably 10.00 or less, and still more preferably 9.50 or less.
  • BES [pKa (60 ° C): 6.51], TES [pKa (60 ° C): 6.70], HEPES [pKa (60 ° C): 6.99], Tris [pKa (60 ° C ): 7.06], glycine amide [pKa (60 ° C): 7.04], HEPPS [pKa (60 ° C): 7.72], Tricine [pKa (60 ° C): 7.31], Bicine [pKa (60 ° C) ): 7.63], CHES [pKa (60 ° C): 9.14], CAPS [pKa (60 ° C): 10.04], glycylglycine [pKa (60 ° C): 7.28], etc.
  • Buffers with a pKa of 6.50 or more are preferred, for example, Tris, glycine amide, HEPPS, Tricine, Bicine, CHES, CAPS, glycylglycine, etc.Buffers with a pKa of 7.00 or more at 60 ° C
  • HEPPS, Tricine, Bicine, CHES, CAPS, glycylglyc Buffers with a pKa of 7.20 or more at 60 ° C such as Shin are particularly preferred.
  • buffers with a pKa of 7.30 or more and 9.50 or less at 60 ° C such as HEPPS, Tricine, Bicine, CHES, etc. Even more preferred. Of these, the most preferred is Tricine.
  • the temperature for carrying out the enzyme reaction from room temperature.
  • the nucleic acid can be released in a high yield by reducing the pH fluctuation accompanying the increase.
  • Examples of such a buffer include HEPPS [ ⁇ pKa / ° C: ⁇ 0.007], Tricine [ ⁇ pKa /. C: —0.021], Bicine [A pKaZ. C: —0.018], CHES [A pKaZ. C: -0.009], CAPS [A pKa / ° C: -0.009], and Tricine is most preferable.
  • the amount of the buffer as described above varies depending on the type of the buffer used and cannot be generally specified, but is an amount that can maintain the pH range as described above at the reaction temperature as described above (see above). As long as the buffering capacity can be obtained within the pH range as described above, and in general, when reacting blood components and proteolytic enzymes in the presence of a surfactant and an alkali metal salt.
  • the concentration in the reaction solution for example, the lower limit is usually ImM or higher, preferably 5 mM or higher, more preferably 9 mM or higher, more preferably 9.5 mM or higher, and the upper limit is usually 500 mM or lower, preferably 250 mM or lower, more preferably lOO mM. In the following, it is more preferably 50 mM or less.
  • the buffer and the alkali metal salt coexist with the buffer. What is necessary is just to carry out according to the method of preparing an aqueous solution containing a blood component as described above and a proteolytic enzyme, a surfactant and an alkali metal salt. That is, any method that can finally obtain a solution containing a blood component, a proteolytic enzyme, a surfactant, an alkali metal salt and a buffer is sufficient.
  • Proteolytic enzyme, surfactant, alkali metal salt Or a blood component added to an aqueous solution (reaction solution) containing all of the buffer and (2) a method of adding a blood component to the reaction solution, (3) a proteolytic enzyme, a surfactant, an alkali metal salt And (4) proteolytic enzymes, surfactants, alkali metal salts, and buffers.
  • a method of adding two or more aqueous solutions containing one or more agents to each blood component can be mentioned.
  • alkali metal salts are added to the blood components.
  • Add an aqueous solution (solution) containing a surfactant and a buffer, or add a blood component to an aqueous solution (solution) containing an alkali metal salt, a surfactant and a buffer It is preferable to prepare a solution containing a blood component and an alkali metal salt, a surfactant and a buffer, and then add and mix an aqueous solution containing a proteolytic enzyme to the solution.
  • the concentration and pH of the buffer used in each solution are determined in the final reaction solution (a solution containing blood components, proteolytic enzymes, surfactants, alkali metal salts and buffers).
  • the concentration and pH should be determined appropriately taking into account the amount of each solution used so that the range force is selected as described above.
  • a reagent that is usually used in this field for example, a nucleic acid decomposing enzyme such as a chelating agent.
  • Inhibitors such as DNase and RNase
  • reducing agents may coexist.
  • an inhibitor of a nucleolytic enzyme such as a chelating agent (for example, DNase, RNase, etc.).
  • a blood component may be reacted with a proteolytic enzyme in the presence of a surfactant and an alkali metal salt, and if necessary, a chelating agent or Z and a reducing agent.
  • step (1) of the present invention is preferably carried out in the absence of a coprecipitation agent.
  • the chelating agent is not particularly limited as long as it is usually used in this field, but preferred is one having the ability to chelate divalent metal ions.
  • an alkyliminopolycarboxylic acid hydroxyethyliminodiacetic acid (HIDA), iminoniacetic acid (IDA), etc.
  • nitrile polycarboxylic acid [utrilo] Triacetic acid (NTA), ditrimethyl tripropionic acid (NTP), etc.
  • hydroxyalkyl group hydroxyaryl group or hydroxyaralkyl group, which may be mono or poly
  • Realkylene polyamine polycarboxylic acid [ethylene diamine tetraacetic acid (EDTA), ethylene diamine diacetic acid (EDDA), ethylene diamine dipropionic dihydrochloride (EDDP), hydroxychetyl ethylene diamine triacetic acid (EDTA-OH), 1, 6-Hexamethylenediamine- N, ⁇ , ⁇ ', ⁇ '-tetraacetic acid (HDTA), triethylenetetramine hexaacetic acid (TTHA), diethylenetriamine- N, N, N', N ", N"
  • the amount of chelating agent used varies depending on the type of chelating agent used. In general, however, the concentration in the reaction solution when a blood component and a proteolytic enzyme are reacted in the presence of a surfactant and an alkali metal salt, for example, usually has a lower limit.
  • ImM or more preferably 3 mM or more, more preferably 5 mM or more, even more preferably 7 mM or more, particularly preferably 9 mM or more, and the upper limit is not particularly limited, but considering economics, the sample and proteinase K are reacted.
  • the concentration in the reaction solution is usually 200 mM or less, preferably 10 mM or less, more preferably 50 mM or less, still more preferably 25 mM or less, and particularly preferably 10 mM or less.
  • a thiol compound is preferable.
  • thiol compounds include dithiothreitol (hereinafter abbreviated as DTT), ⁇ -mercaptoethanol (hereinafter abbreviated as j8 ME), ⁇ -acetylcystine, systemine, reduced dartathione, dithiol.
  • DTT dithiothreitol
  • j8 ME ⁇ -mercaptoethanol
  • ⁇ -acetylcystine ⁇ -acetylcystine
  • systemine reduced dartathione
  • the amount of reducing agent used depends on the type of reducing agent used, so it cannot be generally stated, but the range power normally used in this field can be selected appropriately.
  • the amount used is such an amount that can sufficiently improve the function of a protein-degrading enzyme that degrades a contaminant such as a protein in a blood component, in other words, a nucleic acid obtained after carrying out the method of the present invention.
  • a contaminant such as a protein in a blood component
  • it is not particularly limited as long as it is an amount that can extract a sufficient amount and quality (purity) of nucleic acid for blood analysis, but generally, blood components and proteolytic enzymes are combined.
  • the concentration in the reaction solution when the reaction is carried out in the presence of a surfactant and an alkali metal salt for example, the lower limit is usually 9.5 mM or more, preferably 20 mM or more, more preferably 38 mM or more, still more preferably 45 mM or more.
  • Is usually 1 M or less, preferably 750 mM or less, more preferably 500 mM or less, and still more preferably 300 mM or less.
  • the lower limit is usually 9.5 mM or more, preferably 20 mM or less as the concentration in the reaction solution when the blood component and the protease are reacted.
  • the upper limit is more preferably 38 mM or more, and the upper limit is usually 10 mM or less, preferably 75 mM or less, more preferably 50 mM or less.
  • the lower limit of the concentration in the reaction solution when reacting the blood component with the proteolytic enzyme is usually lOOmM or higher, preferably 150mM or higher, more preferably 200mM or higher, and still more preferably. Is 300 mM or more, and the upper limit is usually 1 M or less, preferably 750 mM or less, more preferably 500 mM or less.
  • the chelating agent or Z and the reducing agent can coexist with the chelating agent or alkali metal salt (if necessary, a buffering agent) simultaneously with the action of proteolytic enzymes on blood components.
  • a proteolytic enzyme, a surfactant, and an alkali metal salt (buffer agent if necessary) as long as Z and a reducing agent coexist. Good.
  • any method that can finally obtain a solution containing a blood component, a proteolytic enzyme, a surfactant, an alkali metal salt (buffer agent if necessary) and a chelating agent or Z and a reducing agent is acceptable ( 1) Add blood components to an aqueous solution (reaction solution) containing all of proteolytic enzymes, surfactants, alkali metal salts (buffering agents if necessary) and chelating agents or Z and reducing agents, or (2) (3) Proteolytic enzyme, surfactant, alkali metal salt (buffer if necessary) and chelating agent or aqueous solution containing Z and reducing agent separately in blood (4) Proteolytic enzyme, surfactant, alkali metal salt (buffering agent if necessary) and chelating agent or aqueous solution containing one or more of Z and reducing agent 2 or more To add each to blood components And the like.
  • two or more aqueous solutions containing one or more of proteolytic enzymes, surfactants, alkali metal salts (if necessary, buffering agents) and chelating agents are separately used as blood components.
  • a surfactant, an alkali metal salt (buffer agent if necessary) and a chelating agent or an aqueous solution (solution) containing Z and a reducing agent are added to the blood component, or the interface is added.
  • alkali metal salt (buffer if necessary) and chelating agent or an aqueous solution (solution) containing Z and a reducing agent blood components and surfactants, alkali metals are added.
  • a method of adding and mixing an aqueous solution containing element is preferred.
  • the concentration of the chelating agent and reducing agent used in each solution is determined according to the final reaction solution (blood component, proteolytic enzyme, surfactant, alkali metal salt (buffer agent if necessary)) and chelating agent.
  • the amount of each solution used may be appropriately determined so that the concentration in the agent or the solution containing Z and the reducing agent is selected from the above range.
  • the reaction solution obtained by the step (1) of the present invention as described above is further subjected to the step (2) of the present invention, whereby a protein in a blood component (forms a complex with a nucleic acid, In addition to degrading and solubilizing histone proteins, etc.) to liberate nucleic acids, and increase the recovery rate of trace amounts of nucleic acids released in the subsequent step (3).
  • a protein in a blood component forms a complex with a nucleic acid, In addition to degrading and solubilizing histone proteins, etc.
  • the nucleic acid recovery rate can be further increased by bringing the coprecipitate into contact with a blood component (that is, the reaction solution obtained by the step (1)) for the first time at this stage. That is, by adding a coprecipitation agent together with the chaotropic agent in this step (2) just before the alcohol subsidence.
  • a blood component that is, the reaction solution obtained by the step (1)
  • the step (2) of the present invention comprises the reaction solution obtained by the step (1) of the present invention as described above, ie, (1) a blood component and a proteolytic enzyme, a surfactant and an alkali metal salt. It was obtained by reacting in the presence of a buffer, a chelating agent and a reducing agent (if necessary) to release nucleic acids by degrading and dissolving proteins in serum components. Prepare an aqueous solution (reaction solution) containing a reaction solution containing free nucleic acid, a chaotropic agent and a coprecipitation agent, and bring the reaction solution containing free nucleic acid into contact with the chaotropic agent and coprecipitation agent. can do.
  • reaction solution containing a reaction solution containing free nucleic acid, a chaotropic agent and a coprecipitation agent, and bring the reaction solution containing free nucleic acid into contact with the chaotropic agent and coprecipitation agent.
  • a chaotropic agent and a coprecipitation agent As described above, as a method for preparing an aqueous solution containing the free nucleic acid-containing reaction solution obtained in step (1), a chaotropic agent and a coprecipitation agent, each of these components is finally included. It is not particularly limited as long as it is a method capable of obtaining a solution.
  • reaction solution a method in which the reaction solution containing free nucleic acid obtained in step (1) is added to an aqueous solution (reaction solution) containing a chaotropic agent and a coprecipitation agent
  • reaction solution a aqueous solution containing a chaotropic agent and a coprecipitation agent
  • reaction solution contains an aqueous solution containing a chaotropic agent and a coprecipitation agent (reaction solution).
  • the concentration of each component in each aqueous solution is determined according to the final reaction solution (step (1
  • the concentration in the reaction solution containing free nucleic acid, the solution containing the chaotropic agent and the coprecipitation agent obtained in step) is selected as appropriate in consideration of the amount of each solution used so that the concentration is selected as described above. That's fine.
  • step (3) of the present invention is subsequently performed.
  • the pH at which the reaction solution containing the free nucleic acid obtained in step (1) is contacted with the chaotropic agent and the coprecipitation agent may be in a range that does not hinder the release of the nucleic acid.
  • the lower limit is usually pH 6 or more, preferably pH 7 or more
  • the upper limit is usually pH 9 or less, preferably pH 8 or less.
  • the lower limit of the reaction temperature when the free nucleic acid-containing reaction solution obtained in step (1) is contacted with the chaotropic agent and the coprecipitation agent is usually 4 ° C or higher, preferably 10 ° C or lower.
  • the upper limit is more preferably 20 ° C or higher, and the upper limit is usually 100 ° C or lower, preferably 70 ° C or lower.
  • the reaction time depends on the reaction temperature and the type of blood component, but the lower limit is generally 1 second or longer, preferably 1 minute or longer, more preferably 5 minutes or longer, and the upper limit is usually 24. Within hours, preferably within 12 hours, more preferably within 5 hours, and even more preferably within 1 hour.
  • step (2) of the present invention the pH at which the free nucleic acid-containing reaction solution obtained in step (1) is contacted with the chaotropic agent and the coprecipitation agent is maintained within the above-described range.
  • a buffering agent can be used.
  • Such a buffering agent is not particularly limited as long as it has a buffering capacity in the pH range as described above at the reaction temperature as described above.
  • the amount of the buffer as described above varies depending on the type of the buffer used and cannot be generally specified, but is an amount that can maintain the pH range as described above at the reaction temperature as described above (see above).
  • the reaction solution containing the free nucleic acid obtained in the step (1), the chaotropic agent and the coprecipitation agent may be used.
  • the lower limit is usually ImM or higher, preferably 5 mM or higher, more preferably 9 mM or higher, more preferably 9.5 mM or higher
  • the upper limit is usually 500 mM or lower, preferably 250 mM or lower. More preferably, it is lOOmM or less, and further preferably 5 OmM or less.
  • the reaction solution containing the free nucleic acid obtained in the step (1) is contacted with the chaotropic agent and the coprecipitation agent.
  • it contains a reaction solution containing free nucleic acid obtained in the step (1) as described above, and a chaotropic agent and a coprecipitation agent.
  • the reaction solution containing the free nucleic acid obtained in step (1), a solution containing a chaotropic agent, a coprecipitation agent, and a buffering agent can be obtained.
  • step (1) Add the free nucleic acid-containing reaction solution obtained in step (1) to the aqueous solution (reaction solution) containing all of the chaotropic agent, coprecipitate and buffer, or (2) step ( A method in which the free nucleic acid-containing reaction solution obtained in 1) is added to the aqueous solution (reaction solution), and (3) an aqueous solution separately containing a chaotropic agent, a coprecipitant and a buffer is obtained in step (1). A method of adding each to the free nucleic acid-containing reaction solution, or (4) a free nucleic acid-containing reaction solution obtained in step (1) using two or more aqueous solutions containing one or more of a chaotropic agent, a coprecipitation agent, and a buffering agent. And the like, respectively. Of these, (1) or (2) is preferred.
  • the concentration and pH of the buffer used in each solution include the final reaction solution (free nucleic acid-containing reaction solution obtained in step (1), chaotropic agent, coprecipitation agent, and buffer agent).
  • the use amount of each solution may be determined as appropriate.
  • nuclease eg, DNase
  • a chelating agent e.g., RNase and the like
  • a chelating agent when used in the step (1) of the present invention, it is sufficient when the free nucleic acid-containing reaction solution (nucleic acid) obtained in the step (1) is contacted with the chaotropic agent and the coprecipitation agent.
  • an amount of a chelating agent capable of obtaining a sufficient nucleolytic enzyme inhibitory action is present, when the free nucleic acid-containing reaction solution obtained in step (1) is contacted with the chaotropic agent and the coprecipitation agent, Especially without adding chelating agents.
  • the alkali metal salt is an essential component in the step (1) of the present invention, it is not necessary to add it again in the step (2) of the present invention. On the contrary, it is preferable not to add an alkali metal salt in the step (2) of the present invention in consideration of the recovery rate of nucleic acid, economic efficiency, and the like. That is, in the method for preparing an aqueous solution containing the free nucleic acid-containing reaction solution obtained in the above step (1) and the chaotropic agent and the coprecipitation agent, the free nucleic acid content obtained in the step (1) is contained.
  • reaction solutions used to contact the reaction solution (for example, aqueous solutions (reaction solution) containing all of the chaotropic agent, coprecipitation agent, and buffering agent) do not contain alkali metal salts. preferable.
  • chelating agent examples include those similar to the above step (1). DTA or its salt (sodium salt etc.) is preferred!
  • the amount of chelating agent used varies depending on the type of chelating agent used, so it cannot be said unconditionally. However, any amount that can inhibit the nucleolytic enzyme is acceptable. Generally, it was obtained in step (1).
  • the lower limit is usually O.lmM or more, preferably ImM or more, more preferably 5 mM or more as the concentration in the reaction solution when the free nucleic acid-containing reaction solution is contacted with the chaotropic agent and the coprecipitation agent.
  • the upper limit is not particularly limited, but considering economics, the concentration in the reaction solution when the free nucleic acid-containing reaction solution obtained in step (1) is contacted with the chaotropic agent and coprecipitation agent is usually 500 mM. In the following, it is preferably 10 mM or less, more preferably 50 mM or less.
  • the chelating agent is allowed to coexist with the chaotropic agent and the coprecipitation agent (required when the chaotropic agent and the coprecipitation agent are brought into contact with the reaction solution containing the free nucleic acid obtained in step (1).
  • the reaction solution containing free nucleic acid obtained in the above step (1), and a chaotropic agent and a coprecipitation agent (a buffering agent if necessary) can be used. What is necessary is just to carry out according to the method of preparing the aqueous solution to contain.
  • any method can be used as long as the solution containing the free nucleic acid-containing reaction solution, chaotropic agent, co-precipitation agent (buffer agent if necessary) and chelating agent obtained in step (1) is finally obtained.
  • (1) Add the free nucleic acid-containing reaction solution obtained in step (1) to an aqueous solution (reaction solution) containing all of the chaotropic agent, coprecipitation agent (buffer agent if necessary) and chelating agent
  • the concentration of the chelating agent used in each solution is determined according to the final reaction solution (free nucleic acid-containing reaction solution obtained in step (1), chaotropic agent, coprecipitation agent (buffer agent if necessary)).
  • the concentration in the solution containing a chelating agent may be appropriately determined in consideration of the amount of each solution used so that the range force as described above is selected.
  • a blood component and a protein-degrading enzyme are combined with a surfactant and an alkali metal salt (buffering agent if necessary, Reaction in the presence of a chelating agent and a reducing agent) to decompose and solubilize proteins in serum components to liberate nucleic acids, and (2) then contain the resulting free nucleic acids After contacting the reaction solution with a chaotropic agent and a coprecipitation agent (if necessary, a buffer or Z and a chelating agent), and further degrading and solubilizing proteins in serum components to liberate nucleic acids, By performing a treatment for precipitating the nucleic acid and collecting the precipitate, the released nucleic acid can be purified and collected.
  • a surfactant and an alkali metal salt buffering agent if necessary, Reaction in the presence of a chelating agent and a reducing agent
  • step (2) since a chaotropic agent is used in step (2), for example, nucleic acid extracted by precipitation with nucleic acid without performing nucleic acid extraction with an organic solvent such as phenol or chloroform is purified and collected. can do. That is, free nucleic acid-containing solution obtained after carrying out the steps (1) and (2) of the present invention without performing nucleic acid extraction with an organic solvent can be subjected to a treatment for precipitating or concentrating the nucleic acid as it is. .
  • the treatment for precipitating or concentrating the nucleic acid in the step (3) of the present invention is not particularly limited as long as it is performed in this field in order to precipitate or concentrate the nucleic acid.
  • examples of such methods include alcohol precipitation, salt-cesium density gradient centrifugation (ultracentrifugation) and the like, and alcohol precipitation is preferred.
  • the alcohol to be used is not particularly limited as long as it has a property capable of specifically precipitating a nucleic acid, and examples thereof include alkyl alcohols having 1 to 10 carbon atoms, preferably 2 to 9 carbon atoms.
  • alkyl alcohols include ethanol, pro Panol (n-propyl alcohol, isopropyl alcohol), butanol (n-butyl alcohol, isobutyl alcohol, sec butyl alcohol, tert butyl alcohol;), amyl alcohol (n-amyl alcohol, sec amyl alcohol, tert amyl alcohol), Isoamyl alcohol, sec-isoamyl alcohol, etc.), hexanol, heptanol, octanol, strong prill alcohol, noral alcohol, decyl alcohol.
  • alkyl alcohols having 3 or less carbon atoms may be used alone or in combination of two.
  • an alkyl alcohol having a carbon number of at least (for example, butanol) is used as an alcohol mixture containing two or more alcohols in combination with an alkyl alcohol having 3 or less carbon atoms (for example, ethanol or Z and isopropanol). It is preferable to use it.
  • alkyl alcohol having a carbon number or more butanol (n-butyl alcohol, isobutyl alcohol, sec butyl alcohol, tert butyl alcohol) is preferable, but n-butyl alcohol (1-butanol) is more preferable.
  • nucleic acid when targeting a sample containing a large amount of lipids such as blood components, lipids can be dissolved and removed from the nucleic acid.
  • nucleic acid is precipitated using a mixture of two or more alcohols in combination with 3 or less alkyl alcohols.
  • ethanol, isopropanol, and butanol strength are preferred.
  • One or more selected ethanol or Z, and a mixture of two or more alcohols in combination of isopropanol and butanol are more preferable, and isopropanol and butanol are combined.
  • a combined mixture is particularly preferred.
  • the concentration of these alcohols is not particularly limited as long as it is a concentration capable of precipitating nucleic acid from a solution, but in general, the final concentration of alcohols (contacting nucleic acids with alcohols) is not limited. Concentration in the solution) is usually 40% or more, preferably 50% or less As described above, it is added to the free nucleic acid-containing reaction solution obtained in step (1) and step (2) of the present invention. Specifically, for example, when ethanol is used alone, the final concentration of ethanol (concentration in the solution when contacting the nucleic acid and ethanol) is usually 60% or more, preferably 70% or more.
  • the final concentration of isopropanol is used.
  • the concentration in the solution is usually 40% or more, preferably 50% or more. Added.
  • the final concentration of all alcohols used is usually It is added to the reaction solution containing free nucleic acid obtained in the step (1) and the step (2) of the present invention so as to be 40% or more, preferably 50% or more.
  • the final concentration of the alcohols used when two or more alkyl alcohols having a carbon number of S4 or more and alkyl alcohols having 3 or less carbon atoms are used in combination, generally the final concentration of the alcohols used (when contacting the nucleic acid and alcohols) Is added to the free nucleic acid-containing reaction solution obtained in steps (1) and (2) of the present invention so that the concentration in the solution is usually 40% or more, preferably 50% or more.
  • the final concentration of isopropanol and butanol (concentration in the solution when contacting nucleic acid with isopropanol and butanol) is usually 40% or more, preferably 50%. As described above, it is added to the free nucleic acid-containing reaction solution obtained in step (1) and step (2) of the present invention.
  • the carbon number power contained in the mixture is more than
  • the ratio of alkyl alcohol (content in the alcohol mixture) is generally 20% or more as a lower limit, preferably 30% or more, more preferably 40% or more, still more preferably 50% or more, particularly Preferably it is 60% or more, and the upper limit is usually 80% or less, preferably 70% or less.
  • the ratio of butanol contained in the mixed liquid is usually 20% or more, preferably 30% as a lower limit.
  • the upper limit is usually 80% or less, preferably 70% or less.
  • the ratio of butanol contained in these mixed solutions is usually 40% or more, more preferably 50% or more, and still more preferably 60% as the lower limit.
  • the upper limit is usually 80% or less, preferably 70% or less.
  • the solution in order to facilitate the precipitation of the nucleic acid, it is possible to cool the solution at a low temperature of 80 ° C. to 4 ° C. after mixing the solution containing the free nucleic acid and the alcohols.
  • nucleic acid extraction method of the present invention that is, steps (1) to (3) of the present invention as described above, for example, the following may be performed.
  • a blood component in a test tube and contain an appropriate amount of a surfactant and an alkali metal salt-containing aqueous solution (if necessary, further containing at least one selected from a buffer, a chelating agent and a reducing agent) Then, an appropriate amount of an aqueous solution containing a proteolytic enzyme is added and mixed, and the solution (reaction solution) is reacted at the temperature as described above.
  • a surfactant and an alkali metal salt-containing aqueous solution if necessary, further containing at least one selected from a buffer, a chelating agent and a reducing agent
  • an appropriate amount of an aqueous solution containing a proteolytic enzyme is added and mixed, and the solution (reaction solution) is reacted at the temperature as described above.
  • aqueous solution containing a proteolytic enzyme and an aqueous solution containing a surfactant and an alkali metal salt (containing at least one selected from a buffer, a chelating agent and a reducing agent if necessary) are used as serum.
  • a surfactant and an alkali metal salt containing at least one selected from a buffer, a chelating agent and a reducing agent if necessary.
  • the solution in order to facilitate the precipitation of the nucleic acid, it is possible to cool the solution at a low temperature of 80 ° C. to 4 ° C. after mixing the solution containing the free nucleic acid and the alcohols. After mixing (after standing if necessary), the mixture is centrifuged to precipitate the nucleic acid, the supernatant is removed, and the nucleic acid is recovered as a precipitate.
  • an appropriate amount of an aqueous solution containing alcohols is added to the collected precipitate, and the precipitate is washed once or more.
  • the nucleic acid precipitate is washed once or more using one or more aqueous solutions containing alcohols.
  • the alcohols used are the same as those in the step (3) of the present invention, and one of these may be used, or two or more may be used in combination.
  • the aqueous alcohol solution used for the first washing includes the alcohols used in step (3) of the present invention. It is preferable to use an aqueous solution containing the same type of alcohol. In the second and subsequent washings, it is preferable to use 65% to 85% ethanol.
  • an aqueous alcohol solution of usually 40% or more, preferably 50% or more, more preferably 70% or more is used as the aqueous alcohol solution used for the first washing.
  • two or more alcohols may be used so that the total amount of alcohols used is within the above range.
  • ethanol is preferably 60% to 80%, but butanol is preferably 5 to 10%.
  • isopropanol is preferred at 40-60%, but butanol is preferred at 5-10% ⁇
  • the supernatant is removed by centrifugation, and the nucleic acid is recovered again as a precipitate.
  • the nucleic acid precipitate recovered again may be dried by a conventional method such as heating or decompression.
  • a method usually used in this field for example, TE
  • a solution redissolved in a buffer solution or the like usually used in this field such as a buffer solution (10 mM Tris-hydrochloric acid buffer solution, ImM EDTA containing, pH 8.0).
  • the nucleic acid can be further purified by a method such as digesting coexisting RNA by adding RNase to the re-dissolved nucleic acid solution and reacting with it.
  • the nucleic acid may be further purified by, for example, digesting coexisting DNA by adding DNase or the like to the re-dissolved nucleic acid solution and reacting.
  • the kit of the present invention is used for effectively carrying out the nucleic acid extraction method of the present invention as described above, particularly the extraction of free DNA in blood from blood components such as serum and plasma.
  • the reagent containing each component may be in a solution state such as an aqueous solution containing each component so that the concentration at the time of use is an amount selected from the concentration range as described above.
  • the reagent form may be in a lyophilized state or a dried state containing each component so as to be an amount selected from the concentration range. If the reagent form is lyophilized or dried, it can be combined with a solution for dissolving it as necessary.
  • the reagent (1) containing a proteolytic enzyme of the present invention contains a proteolytic enzyme as a main component.
  • the proteolytic enzyme is as described above.
  • the proteinase is preferably proteinase K.
  • the concentration used may be appropriately selected so that the concentration at the time of use falls within the concentration range as described above and contained in the reagent.
  • a reagent that is usually used in this field may coexist, especially a stabilizer.
  • stabilizers include saccharides such as glycerol, and alkaline earth metal salts (eg, calcium chloride) containing alkaline earth metal ions such as magnesium and calcium as cations.
  • the concentration of the stabilizer used is not particularly limited as long as it is usually used in this field.
  • the reagent (1) of the present invention contains 10% to 80%.
  • calcium chloride 0.1 to 10 mM is contained in the reagent (1) of the present invention.
  • the reagent form of reagent (1) can be in a lyophilized state or in a dry state even in a solution state such as an aqueous solution. Although it may be in a state, a solution state that does not require labor for preparation is preferable. In addition, when it is in a freeze-dried state or a dried state, a solution for dissolving it may be separately combined as necessary.
  • the reagent (1) of the present invention is used in step (1) in the above-described method of the present invention. That is, the proteolytic enzyme in the step (1) of the present invention is preferably supplied from the reagent (1) of the present invention.
  • the reagent (1) of the present invention may be directly mixed with the blood component or after the blood component and the reagent (2) are mixed. Either of these liquid mixtures and the reagent (1) may be mixed, but after mixing the blood component and the reagent (2), the liquid mixture and the reagent (1) are mixed. Is more preferable.
  • the reagent (2) containing a surfactant and an alkali metal salt according to the present invention contains a surfactant and an alkali metal salt as main components.
  • the surfactant and the alkali metal salt are as described above.
  • N-lauroyl sarcosine acid or a salt thereof for example, sodium salt, potassium salt, lithium salt, etc.
  • the sodium N-lauroyl sarcosinate is particularly preferred.
  • potassium salt is preferred.
  • these use concentrations may be appropriately selected so that the concentration at the time of use falls within the concentration range as described above and contained in the reagent.
  • the pH in the reaction in the presence of the agent and the alkali metal salt is appropriately adjusted so as to be in the pH range as described above.
  • the lower limit is usually pH 7 or more, preferably pH 8 or more.
  • the lower limit is usually pHIO or lower, preferably pH9 or lower.
  • a buffering agent can be used to adjust the pH to the range as described above.
  • such a buffering agent is as described in the above-mentioned step (1) of the present invention, but at 60 ° C such as HEPPS, Tricine, Bicine, CHES, CAPS, glycylglycine and the like. Buffers with a pKa of 7.20 or more are particularly preferred 6 HEPPS, Tricine, Bicine, CHES, etc. Tricine is the most preferable among the buffering agents having a pKa of 7.30 or more and 9.50 or less at 0 ° C.
  • nucleolytic enzymes such as chelating agents (for example, DNase, RNase, etc.) Inhibitors, reducing agents, etc. may coexist.
  • nucleolytic enzymes such as chelating agents (eg DNase, RNase, etc.) Inhibitors, reducing agents, etc.
  • inhibitors of nucleolytic enzymes such as chelating agents (eg DNase, RNase, etc.)!
  • the chelating agent and reducing agent are as described in the above-mentioned step (1) of the present invention.
  • the chelating agent EDTA or a salt thereof (such as sodium salt) is preferable, and the reducing agent is Is preferably DTT,
  • the concentration of these used may be appropriately selected so that the concentration at the time of use falls within the concentration range as described above and contained in the reagent.
  • the reagent form of reagent (2) may be a solution state such as an aqueous solution, a lyophilized state, or a dried state, but a solution state that does not require labor for preparation is preferable.
  • a solution state that does not require labor for preparation is preferable.
  • a solution for dissolving it may be separately combined as necessary.
  • the reagent (2) (and reagent (1)) of the present invention does not contain a coprecipitation agent. If a coprecipitation agent is contained in these reagents, the recovery rate of the finally obtained (extracted) nucleic acid may be reduced.
  • the reagent (2) of the present invention is used in step (1) in the above-described method of the present invention. That is, the surfactant and alkali metal salt in the step (1) of the present invention are supplied from the reagent (2) of the present invention.
  • the reagent (2) of the present invention may be directly mixed with the blood component or after the blood component and the reagent (1) are mixed. It is preferable to mix the mixed solution of the blood component and the reagent (1) and the reagent (2), either of which is mixed with the mixed solution and the reagent (2).
  • the reagent (3) comprising a chaotropic agent and a coprecipitation agent of the present invention comprises a chaotropic agent and a coprecipitation agent as main components.
  • the reagent (3) of the present invention coexists with a chaotropic agent and a coprecipitation agent, the reagent is stored. Stability is improved and the recovery rate of the finally obtained (extracted) nucleic acid is improved.
  • chaotropic agent and the coprecipitation agent are as described above, but glycogen is preferred as a coprecipitation agent in which sodium salt is preferred as a powerful photopicking agent.
  • these use concentrations may be appropriately selected so that the concentration at the time of use is in the concentration range as described above, and contained in the reagent.
  • the reagent (3) of the present invention is the pH when the reaction solution obtained by mixing and reacting the blood component, the reagent (1) and the reagent (2) and the reagent (3), that is, the process of the present invention. What was appropriately adjusted so that the pH at which the free nucleic acid-containing reaction solution obtained in step (1) in step (2) was brought into contact with the chaotropic agent and coprecipitation agent was in the pH range as described above. Is preferred.
  • the reagent (3) preferably has a lower limit of usually PH6 or higher, preferably pH 7 or higher, and an upper limit of usually pH 9 or lower, preferably pH 8 or lower! /.
  • a buffering agent can be used to adjust the pH to the range as described above.
  • reagents usually used in this field for example, nucleolytic enzymes such as chelating agents (for example, DNase, RNase, etc.) It is preferable to coexist the inhibitor.
  • nucleolytic enzymes such as chelating agents (for example, DNase, RNase, etc.)
  • chelating agents for example, DNase, RNase, etc.
  • the reaction solution (nucleic acid) containing the free nucleic acid obtained in step (1) is contacted with the chaotropic agent and the coprecipitation agent.
  • an amount of chelating agent capable of obtaining an enzyme inhibitory action is contained in the reagent (2), it is not necessary to contain a chelating agent in the reagent (3).
  • chelating agent preferred and embodiments of the chelating agent are as described in the above-mentioned step (2) of the present invention.
  • EDTA or a salt thereof (sodium salt or the like) is preferable.
  • the concentration used may be appropriately selected so that the concentration at the time of use falls within the above-mentioned concentration range, and should be contained in the reagent.
  • the reagent form of reagent (3) can be in a lyophilized or dry state even in a solution state such as an aqueous solution. Although it may be in a state, a solution state that does not require labor for preparation is preferable. In addition, when it is in a freeze-dried state or a dried state, a solution for dissolving it may be separately combined as necessary.
  • the reagent (3) of the present invention preferably does not contain an alkali metal salt. Since the alkali metal salt is already contained in the reagent (2), it is not necessary to contain it in the reagent (3). It is preferable not to include it.
  • the reagent (3) of the present invention is used in step (2) in the above-described method of the present invention. That is, the chaotropic agent and the coprecipitation agent in the step (2) of the present invention are supplied from the reagent (3) of the present invention.
  • Reagent aqueous solution containing surfactant, alkali metal salt and buffer (if necessary, chelating agent)
  • the kit of the present invention is further combined with a reagent (reagent for precipitating nucleic acid) used in the step (3) of the present invention as described above, that is, a treatment for precipitating nucleic acid. It's okay.
  • a reagent include alcohols. Specific examples and preferred embodiments of alcohols are the same as those described in the above-mentioned step (3) of the present invention, such as ethanol, Isopropanol and butanol power One or more selected isopropanol is preferred
  • a mixture of two or more alcohols in which ethanol or Z and isopropanol and butanol are combined is more preferable, and a mixture of isopropanol and butanol is particularly preferable.
  • the concentration used is as described above.
  • the kit of the present invention includes one kind of an aqueous solution containing alcohols used for washing the nucleic acid precipitate obtained by the step (3) of the present invention, that is, the nucleic acid precipitation treatment.
  • the above may be combined. Specific examples, preferred embodiments and the like of such alcohols are as described in the above-mentioned 6-5. Specific operating methods, for example, ethanol, isopropanol and butanol forces. One or more selected ones A combination etc. are mentioned.
  • aqueous solutions containing different alcohols even if they are all aqueous solutions containing the same alcohol.
  • the concentration used is as described above.
  • kit of the present invention is specifically shown below.
  • the reagent (4) includes a reagent composed of an alcohol for precipitating nucleic acid and an aqueous solution containing an alcohol used for washing the nucleic acid precipitate. That is, the kit is a combination of one or more selected from these reagents and an aqueous solution, or a combination of Z and an aqueous solution. Among them, it will be the power of alcohol to precipitate nucleic acids.
  • a kit comprising a combination of at least one kind of reagent.
  • a kit comprising a combination of these is more preferred. In particular, it is used for washing one kind of reagent such as alcohols for precipitating nucleic acid and nucleic acid precipitation.
  • V particularly preferred is a kit comprising a combination of two aqueous solutions containing alcohols.
  • kit of the present invention will be specifically shown below.
  • Reagent aqueous solution containing surfactant, alkali metal salt and buffer (if necessary, chelating agent)
  • Reagent aqueous solution containing chaotropic agent, coprecipitation agent and buffer (if necessary, chelating agent)
  • the reagent (4) includes a reagent composed of an alcohol for precipitating nucleic acid and an aqueous solution containing an alcohol used for washing the nucleic acid precipitate. That is, the kit is a combination of one or more selected from these reagents and an aqueous solution, or a combination of Z and an aqueous solution. Among them, a kit comprising a combination of at least one kind of alcohol capable of precipitating nucleic acids is preferred to precipitate one kind of reagent such as alcohols for precipitating nucleic acids and nucleic acid destruction. Therefore, a kit formed by combining at least one aqueous solution containing alcohols used for the purpose is more preferable. In particular, it is used for washing one kind of reagent such as alcohols for precipitating nucleic acid and nucleic acid precipitation.
  • V particularly preferred is a kit comprising a combination of two aqueous solutions containing alcohols.
  • the reagent (4) includes a reagent composed of an alcohol for precipitating nucleic acid and an aqueous solution containing an alcohol used for washing the nucleic acid precipitate. That is, the kit is composed of one or more reagents selected from these reagents and aqueous solutions, or Z and an aqueous solution. It is a combination. Among them, a kit comprising a combination of at least one kind of alcohol capable of precipitating nucleic acids is preferred to precipitate one kind of reagent such as alcohols for precipitating nucleic acids and nucleic acid destruction. Therefore, a kit formed by combining at least one aqueous solution containing alcohols used for the purpose is more preferable. In particular, it is used for washing one kind of reagent such as alcohols for precipitating nucleic acid and nucleic acid precipitation.
  • V particularly preferred is a kit comprising a combination of two aqueous solutions containing alcohols.
  • kit of the present invention will be specifically shown below.
  • Reagents with a pH of 7-10 including surfactants, alkali metal salts, buffers and chelating agents (aqueous solutions)
  • Reagents with pH 6-9 including chaotropic agents, coprecipitates, buffers and chelating agents (aqueous solutions)
  • the reagent (4) includes a reagent composed of an alcohol for precipitating nucleic acid and an aqueous solution containing an alcohol used for washing the nucleic acid precipitate. That is, the kit is a combination of one or more selected from these reagents and an aqueous solution, or a combination of Z and an aqueous solution. Among them, a kit comprising a combination of at least one kind of alcohol capable of precipitating nucleic acids is preferred to precipitate one kind of reagent such as alcohols for precipitating nucleic acids and nucleic acid destruction. Therefore, a kit formed by combining at least one aqueous solution containing alcohols used for the purpose is more preferable. In particular, it is used for washing one kind of reagent such as alcohols for precipitating nucleic acid and nucleic acid precipitation.
  • V particularly preferred is a kit comprising a combination of two aqueous solutions containing alcohols.
  • the kit of the present invention is particularly preferably one having the following constitutional power, for example.
  • Reagents with pH 6-9 including sodium chloride, glycogen, buffering agent and chelating agent (aqueous solution)
  • Nucleic acids obtained using the methods and kits of the present invention are analyzed by genetic information, genetic diseases 'diagnosis of viral diseases, etc.' in the fields of genetic engineering, clinical diagnosis, forensic medicine, etc. It can be used as a sample to be used for various analyzes (such as Southern blotting and PCR methods) for investigation or for personal identification 'parent-child identification' and crime identification.
  • gene diagnosis such as early diagnosis of various diseases such as cancer or monitoring of disease state using free DNA in blood DNA typing method or DNA fingerprinting method (eg PCR-R FLP method, PCR-SSOP method, PCR -LIPA method, PCR-SSCP method, PCR-SSP method, PCR-CFLP method, PCR-RAPD method, PCR-RDA method, RNase protection method, DGGE method, TGGE method).
  • DNA typing method eg PCR-R FLP method, PCR-SSOP method, PCR -LIPA method, PCR-SSCP method, PCR-SSP method, PCR-CFLP method, PCR-RAPD method, PCR-RDA method, RNase protection method, DGGE method, TGGE method.
  • a solution obtained by dissolving the following reagents in 40 mM Tris-HCl buffer (pH 8.0) so as to have the following concentration was used as a sodium iodide solution.
  • Table 1 shows the amount of sodium iodide solution and alcohol mixture used and the concentration after the addition.
  • the precipitate is dissolved in an appropriate amount of distilled water or Tris buffer solution to show water solubility. Judged by no. If there is an insoluble precipitate, the result is shown in Table 2 along with its size. Judgment criteria are as follows.
  • Example 2 In addition, the insoluble precipitate of impurities decreased with increasing sodium concentration, and was very small compared to 4 at 3.46 ⁇ (samples ⁇ .5 and 6). This indicates that a sufficient protein-soluble effect can be obtained if sodium iodide, a chaotropic agent, is used at a concentration of 3% or more, particularly about 3.5% or more.
  • sodium iodide a chaotropic agent
  • Human serum 7 samples and human plasma: 4 samples were used as samples.
  • a solution obtained by dissolving the following reagents in 40 mM Tris-HCl buffer (pH 8.0) so as to have the following concentration was used as a sodium iodide solution.
  • a solution obtained by dissolving the following reagents in 40 mM Tris-HCl buffer (pH 8.0) so as to have the following concentration was used as a sodium iodide solution.
  • the obtained measurement sample was measured at an absorbance of 260 nm, and the DNA content (recovery rate of salmon sperm-derived DNA) was measured.
  • Table 1 shows the results of measuring the DNA content (recovery rate of salmon sperm-derived DNA) in Case 1 and Case 2. still.
  • the value of each sample indicates the relative value of the measured value (absorbance) in case 2 when the measured value (absorbance) in case 1 is 100.
  • salt (KC1) is not contained in the lysate, so the function of proteinase (Proteinase K) is impaired, and as a result, residual proteins and so on are not contained in the DNA sample. Insoluble precipitates, and even if the precipitate is dissolved with TE buffer, DNA cannot be dissolved in the solution. It was inferred that the NA recovery rate was poor. As in Case 2, when the CV (%) is as bad as about 20% and the recovery rate of DNA varies from sample to sample, it becomes difficult to analyze all samples equally. It becomes a fatal wound. Therefore, as in Case 1 (the present invention), salt (KC1) could be judged to have the necessary force S to be added to the lysate in order to recover DNA stably.
  • Tricine buffer 50mM (concentration during warming reaction 6.6mM) Tricine buffer (p.
  • the resulting precipitate was supplemented with 1 mL of 50% aqueous isopropanol, stirred, centrifuged at 20,000 G for 5 minutes, and the supernatant was discarded to obtain a precipitate. Further, 1 mL of 70% ethanol aqueous solution was added to the resulting precipitate, stirred, centrifuged at 20.000 G for 5 minutes, and the supernatant was discarded to obtain a precipitate. The obtained precipitate was dried and then dissolved and suspended in 400 L of 4% SDS to prepare an analytical sample.
  • Figure 1 shows the results of measuring the amount of protein remaining in the precipitate (nucleic acid) for Case A and Case B.
  • Fig. 1 the upper row shows the case A, the lower row shows the case B, and 1 to 6 show the sample numbers.
  • the color development degree of the sample spotted on the PVDF membrane attached to the kit was judged by relative comparison.
  • a sample obtained by adding 10 ⁇ L of intra-fat injection (10% w / v soybean oil: manufactured by Nippon Pharmaceutical) to 90 ⁇ L of human serum was used as a sample.
  • a sample 100 L was placed in a micro centrifuge tube, and 200 L of a lysis solution was added and mixed. Furthermore, 1.0 L of proteinase K solution (distilled water containing 20 g // z L proteinase K (derived from Tritirachium album, manufactured by Wako Pure Chemical Industries, Ltd.)) was added and mixed, and heated at 56 ° C for 10 minutes. . Thereafter, 300 L of sodium iodide solution was added and mixed. Next, 600 L of the alcohol mixture was added and mixed, and allowed to stand at room temperature for 10 minutes. First, the turbidity (white turbidity) of the mixture was observed.
  • proteinase K solution distilled water containing 20 g // z L proteinase K (derived from Tritirachium album, manufactured by Wako Pure Chemical Industries, Ltd.)
  • the mixed solution was centrifuged at 20,000 G for 10 minutes, and the supernatant was discarded to obtain a precipitate.
  • 1 mL of Washing Solution A was added, stirred, and centrifuged again at 20,000 G for 5 minutes, and the supernatant was discarded to obtain a precipitate.
  • Add lmL of 70% ethanol aqueous solution to the resulting precipitate stir, centrifuge at 20,000G for 5 minutes, discard the supernatant, and check whether the inner wall of the centrifuge tube is sticky. Observed.
  • the criteria for turbidity after addition of the alcohol mixture are as follows.
  • Table 6 shows the observation results of turbidity (white turbidity) after addition of the alcohol mixture and sticky observation of the inner wall of the centrifuge tube after centrifugation.
  • a solution obtained by dissolving the following reagents in 40 mM Tris-HCl buffer (pH 8.0) so as to have the following concentration was used as a sodium iodide solution.
  • a solution obtained by dissolving the following reagents in 40 mM Tris-HCl buffer (pH 8.0) so as to have the following concentration was used as a sodium iodide solution.
  • the obtained precipitate was dried and then dissolved in L TE buffer (ImM EDTA-containing 10 mM Tris-HCl buffer, pH 8.0) to prepare a DNA sample.
  • L TE buffer ImM EDTA-containing 10 mM Tris-HCl buffer, pH 8.0
  • the obtained DNA sample L is subjected to PCR under the following conditions, and the p53-Exon5 (308 bp) region known as a tumor suppressor gene is amplified from the nucleic acid obtained from the sample (blood free DNA). Went.
  • PCR was performed according to the protocol attached to the kit, using the reagents for amplification and the reagents including the primer set as shown below.
  • Fig. 2 shows the results of 3% agarose gel electrophoresis for Case 1 and Case 2.
  • lane M is the molecular weight marker DNA Step Ladder Mix (80 bp to 10 kb) (manufactured by Wako Pure Chemical Industries, Ltd.), and lane 1 is the lane when specimen 1 is the sample in case 1.
  • 2 shows the case 2 where Sample 2 was used as the sample.
  • Lane 3 shows the case where specimen 2 was used as the sample in case 1, and
  • lane 4 shows the case where specimen 2 was used as the sample in case 2.
  • the arrow indicates the 308 bp p53-Exon5 region.

Abstract

La présente invention concerne un procédé d’extraction d’acide nucléique, en particulier, afin de libérer l’ADN dans le sang, à partir d’un composant sanguin, dans un rendement élevé et facilement, et un réactif et un kit à utiliser pour le procédé, ayant une excellente stabilité de stockage et avec lesquels le taux de récupération d’acide nucléique est élevé de manière stable. Le procédé d’extraction d’acide nucléique à partir d’un composant sanguin se caractérise par les étapes de (1) faire réagir un composant sanguin avec une protéase en présence d’un surfactif et d’un sel ; (2) ramener la solution de réaction ainsi produite en contact avec un agent chaotropique et un agent de coprécipitation ; et (3) réaliser un traitement de précipitation d’acide nucléique. Le kit pour l’extraction d’acide nucléique à partir d’un composant sanguin comprend, en combinaison, (1) un réactif contenant une protéase ; (2) un réactif contenant un surfactif et un sel ; et (3) un réactif contenant un agent chaotropique et un agent de coprécipitation. Selon l’invention, il devient possible d’extraire un acide nucléique, en particulier de libérer l’ADN dans le sang, à partir d’un composant sanguin (tel que du sérum ou du plasma), dans un rendement élevé et facilement, et il devient possible d’améliorer la stabilité de stockage d’un réactif destiné à être utilisé de manière analogue.
PCT/JP2005/019473 2005-10-24 2005-10-24 Procede et kit d’extraction d’acide nucleique WO2007049326A1 (fr)

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JP2011522529A (ja) * 2008-05-30 2011-08-04 キアゲン ゲゼルシャフト ミット ベシュレンクテル ハフツング 短鎖核酸を単離する方法
US9695465B2 (en) 2011-08-12 2017-07-04 Qiagen Gmbh Method for isolating nucleic acids
EP3216877A1 (fr) * 2011-08-12 2017-09-13 QIAGEN GmbH Procédé d'isolation d'acides nucléiques
EP3789500A1 (fr) * 2011-08-12 2021-03-10 QIAGEN GmbH Procédé d'isolation d'acides nucléiques
US10808276B2 (en) 2011-08-12 2020-10-20 Qiagen Gmbh Method for isolating nucleic acids
JP2014525234A (ja) * 2011-08-12 2014-09-29 キアゲン ゲゼルシャフト ミット ベシュレンクテル ハフツング 核酸を単離するための方法
WO2013024072A1 (fr) * 2011-08-12 2013-02-21 Qiagen Gmbh Procédé d'isolation d'acides nucléiques
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EP2913399A4 (fr) * 2012-10-26 2016-07-13 Kaneka Corp Méthode de préparation d'arn
CN105143449A (zh) * 2012-10-26 2015-12-09 株式会社钟化 Rna制备方法
US10174362B2 (en) 2017-01-16 2019-01-08 Spectrum Solutions L.L.C. Nucleic acid preservation solution and methods of manufacture and use
US10774368B2 (en) 2017-01-16 2020-09-15 Spectrum Solutions L.L.C. Nucleic acid preservation solution and methods of manufacture and use
US11655495B2 (en) 2017-01-16 2023-05-23 Spectrum Solutions L.L.C. Nucleic acid preservation solution and methods of manufacture and use
CN113106148A (zh) * 2021-03-31 2021-07-13 湖南菲思特精准医疗科技有限公司 一种氯吡格雷剂量相关的基因多态性检测试剂盒及其检测方法和应用
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