WO2007049326A1

WO2007049326A1 - Nucleic acid extraction method and nucleic acid extraction kit

Info

Publication number: WO2007049326A1
Application number: PCT/JP2005/019473
Authority: WO
Inventors: Kazunari Hirayasu
Original assignee: Wako Pure Chemical Industries, Ltd.
Priority date: 2005-10-24
Filing date: 2005-10-24
Publication date: 2007-05-03

Abstract

It is intended to provide a method of extracting a nucleic acid, in particular, free DNA in the blood from a blood component in high yield and easily, and a reagent and a kit to be used for the method, which have excellent storage stability and with which the recovery rate of nucleic acid is stably high. The method of extracting a nucleic acid from a blood component is characterized by comprising the steps of (1) reacting a blood component with a protease in the presence of a surfactant and a salt; (2) bringing the resulting reaction solution into contact with a chaotropic agent and a coprecipitating agent; and (3) performing a treatment for precipitating a nucleic acid. The kit for extracting a nucleic acid from a blood component comprises in combination, (1) a reagent containing a protease; (2) a reagent containing a surfactant and a salt; and (3) a reagent containing a chaotropic agent and a coprecipitating agent. According to the invention, it becomes possible to extract a nucleic acid, in particular free DNA in the blood from a blood component (such as serum or plasma) in high yield and easily, and it becomes possible to improve the storage stability of a reagent to be used for the same.

Description

Nucleic acid extraction method and nucleic acid extraction kit

Technical field

[0001] The present invention relates to a method for extracting nucleic acid, particularly blood free DNA from blood components, and a kit used therefor.

Background art

[0002] In the fields of genetic engineering, clinical diagnosis, forensic medicine, etc., various purposes such as analysis of genetic information, diagnosis of genetic diseases, viral diseases, etc., cause investigation, or individual identification, parent-child identification, criminal identification, etc. Thus, the release (extraction) of nucleic acid strands from various samples and their analysis are widely performed.

[0003] Particularly in recent years, DNA fragment strength released due to cell death or tissue damage caused by various diseases such as cancer Floating in blood (serum, plasma, etc.) (blood free DNA: plasma) (DNA or circulating DNA) is known, and it is reported that genetic diagnosis using this free DNA in blood is useful for early diagnosis of various diseases, especially cancer, or monitoring of disease state. Has been made. (Non-patent documents 1 to 3)

[0004] On the other hand, as a method for extracting nucleic acids with various sample forces, for example, after cells are physically or by treatment with a surfactant or the like and destroyed, impurities are used using an organic solvent such as water-saturated phenol or black mouth form. Next, the DNA in the solution is precipitated with alcohol (Non-Patent Document 4, Non-Patent Document 5, etc.), for example, the sodium is added to the serum so as to contain glycogen, and the cell membrane and cell wall are destroyed. 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), etc. Are known.

[0005] However, these methods are intended for human DNA genomic DNA (or RNA), and are not necessarily sufficient in terms of nucleic acid yield, reagent stability, and the like. .

In addition, these methods are not always satisfactory for the extraction of blood free DNA present in trace amounts in blood components.

[0006] Non-Patent Document 1: Sozzi, G "et al., Clin. Cancer Res., 5, 2689 (1999)

[0007] Non-Patent Document 2: Sliva, J. M., et al, Cancer Res., 59, 3251 (1999)

[0008] Non-Patent Document 3: Shao, Z.M., et al "Clin. Cancer Res. 7, 2222 (2001)

[0009] Non-Patent Document 4: Biochemistry Experiment Course 2, "Nucleic Acid Chemistry 1", pp. 74-80, 262-270, 19

1975, Tokyo Chemical Doujin

[0010] Non-Patent Document 5: "Gene Manipulation Manual", pp. 20-23, 1985, Kodansha

[0011] 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

Disclosure of the invention

Problems to be solved by the invention

[0014] 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.

Means for solving the problem

[0015] As a result of intensive studies on a method for easily extracting blood components, particularly serum and plasma nucleic acids, in high yield, the present inventors have determined that blood components and proteolytic enzymes are separated from surfactants and salts. By reacting in the presence, and then contacting the resulting reaction solution with the chaotropic agent and coprecipitation agent, the nucleic acid is precipitated, so that nucleic acids can be easily extracted from blood components in a higher yield. Exist in blood components that were difficult until now It is found that free DNA in blood can be easily extracted in high yield and combined with reagents containing proteolytic enzymes, reagents containing surfactants and salts, and reagents containing chaotropic agents and coprecipitation agents. It has also been found that by using a kit, the nucleic acid recovery rate can be stabilized stably and the storage stability can be improved, and the present invention has been completed.

The present invention has the following configuration.

1. (1) A blood component and a proteolytic enzyme are reacted in the presence of a surfactant and a salt. (2) Next, after the obtained reaction solution is contacted with a chaotropic agent and a coprecipitation agent, (3) A method for extracting a nucleic acid from blood components, comprising performing a treatment for precipitating a nucleic acid.

2. from 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.

The invention's effect

[0017] The present invention provides a method for easily extracting nucleic acid in high yield and a kit used therefor. According to the present invention, nucleic acid, particularly blood, can be obtained from 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. Brief Description of Drawings

[0018] [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. In Fig. 1, the upper row shows the case of Case A, the lower row shows the case of Case B, and 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. In Figure 2, 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, 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. BEST MODE FOR CARRYING OUT THE INVENTION

[0019] 1. Proteolytic enzyme

Examples of the proteolytic enzyme used in the present invention 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.

Specifically, for example, in a reaction solution for reacting a blood component and a proteolytic enzyme, 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. .

[0020] 2. Surfactant

The surfactant used in the present invention has a sufficient protein denaturing action and does not cause precipitation when coexisting with a salt. As such 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.

Specific examples of preferred anionic surfactants include carboxylic acids, sulfonic acids, sulfate esters, phosphate esters, cholic acids, or salts thereof.

Examples of 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.

Examples of the sulfonic acids 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.

Examples of these salts include alkali metal salts such as sodium and potassium, ammonium salts, and the like. Specifically, for example, sodium laurylbenzenesulfonate, sodium dipropylnaphthalenesulfonate, dibutylnaphthalenesulfonic acid. Examples thereof include sodium and sodium dioctylsulfosuccinate.

Examples of the sulfates 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.

Examples of 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.

Examples of the phosphate esters 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.

Examples of these salts include 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.

As cholic acids, cholic acid, cholic acid derivatives (for example, deoxycholic acid, etc.), etc. Is mentioned.

In addition, 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.

Among the anionic surfactants described above, those having the properties as described above are selected.

Among them, N-lauroyl sarcosine acid or a salt thereof (for example, sodium salt, potassium salt, lithium salt, etc.) that has sufficient proteolytic action and does not cause precipitation even when coexisting with KC1 is preferred. 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. There is no particular limitation as long as 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. However, in general, for example, blood components and proteolytic enzymes are used. For example, 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.

In particular, when using sodium N-lauroyl sarcosinate, for example, 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.

3. Alkali metal salt

Examples of the alkali metal salt used in the present invention 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.

Specific examples of such an alkali metal salt 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. There is no particular limitation as long as a sufficient amount of nucleic acid can be extracted from blood components to share the nucleic acid for various analyses.

Specifically, for example, 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. Preferably, a concentration of 10 mM or less is added.

In particular, when KC1 is used, for example, 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. However, in consideration of economics and the like, 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.

4. Chaotropic agent

In the present invention, 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. Although 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. Examples include alkali metal salts of acetic acid and alkali metal salts of thiocyanic acid. These alkali metal salts or iodides 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. . In particular, when using sodium, the lower limit is usually 2.5M or 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.

[0023] 5. Coprecipitant

The 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.

Of these, 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.

Specifically, the lower limit of the concentration in the solution when contacting the nucleic acids and coprecipitate with alcohols is usually 1 μg / mL or more, preferably 5 μg / mL or more, more preferably 10 μg. The upper limit is usually 1 mg / mL or less, preferably 100 μg / mL or less, more preferably 50 μg / mL or less. Particularly when glycogen is used, the lower limit is usually 1 g / mL or more, preferably 5 μg / mL or more, more preferably 10 μg / mL or more, and 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.

[0024] 6. Nucleic acid extraction method of the present invention

In the 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.

[0025] 6— 1. Blood components

Examples of blood components used in the method of the present invention include serum and plasma.

In the method of the present invention, 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. Among them, 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).

[0026] 6- 2. Process (1)

Below, each process of (1)-(3) in the method of this invention is explained in full detail.

[0027] By the step (1) of the present invention, 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.

In the step (1) of the present invention, 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.

[0028] In the step (1) of the present invention, 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.

[0029] As described above, as a method for preparing an aqueous solution containing a blood component and a proteolytic enzyme, a surfactant and an alkali metal salt, a solution containing these components is finally obtained. It is not particularly limited as long as it is a method.

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. Examples thereof include a method of adding an aqueous solution (reaction solution) containing a decomposing enzyme, a surfactant and an alkali metal salt. In addition, (3) 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. Furthermore, (4) such a method that two or more aqueous solutions containing one or more of a protease, a surfactant and an alkali metal salt may be separately added to the blood component, First, 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.

In the above method, 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.

[0030] The reaction conditions in step (1) of the present invention are as follows.

For example, 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. Specifically, the lower limit is usually pH 7 or higher. Preferably, the pH is 8 or higher, and the lower limit is usually pHIO or lower, preferably pH 9 or lower.

[0031] In the step (1) of the present invention, 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. 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. For example, N- (2-acetamido) -2-amino Ethanesulfonic acid (ACES), Ν, Ν-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid (BES), Ν, Ν-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 -[Ν, Ν-Bis (2-hydroxyethyl) amino] -2-hydroxypropanesulfonic acid (DIPSO), 3- [4- (2-hydroxyethyl) -1-piperaduryl] propanesulfonic acid ( EPPS), 2- [4- (2-hydroxychetyl) -1-piperajuryl] ethanesulfonic acid (HEPES), 2-hydride Xyl-3- [4- (2-hydroxyethyl) -1-piperazyl] propanesulfonic acid (HEPPSO), 3-morpholinoporan pansulfonic acid (MOPS), piperazine-1,4-bis (2-ethane Sulfonic acid) (PIPES), Piperazine-1,4-bis (2-hydroxy-3-propanesulfonic acid) (POPSO), N-Tris (hydroxymethyl) methyl-3-aminominosulfonic acid (TAPS), 2-hydroxy-N-tris (hydroxymethyl) methyl-3-aminominosulfonic acid (TAPSO), N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES), N- [tris (hydroxy Good buffers such as (methyl) methyl] glycine (Tricine), Bis-tris propane, collamine chloride, glycine amide, for example, buffers such as tris (hydroxymethyl) aminomethane (tris), glycylglycine, etc. . Among these, 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 Examples of 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.

Specifically, for example, 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. at 60 ° C 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 For example, HEPPS, Tricine, Bicine, CHES, CAPS, glycylglyc Buffers with a pKa of 7.20 or more at 60 ° C such as Shin are particularly preferred.For example, 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.

In addition, among the buffers as described above, if a buffer having the properties as described above and having an ApKaZ ° C of usually 0.030 or less, preferably 0.025 or less is used, 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 [ΔpKa / ° C: −0.007], Tricine [ΔpKa /. 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. As 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.

In order to use the buffer as described above in the step (1) of the present invention, when the protein degrading enzyme is allowed to act on the blood component, 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. (1) 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. For example, a method of adding two or more aqueous solutions containing one or more agents to each blood component can be mentioned. In addition, as a method of separately adding two or more aqueous solutions containing one or more of proteolytic enzymes, surfactants, alkali metal salts, and buffering agents to blood components, first, 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.

In the above method, 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.

[0032] In the step (1) of the present invention, in addition to the proteolytic enzyme, the surfactant and the alkali metal salt 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) and reducing agents may coexist. In particular, it is preferable to coexist an inhibitor of a nucleolytic enzyme such as a chelating agent (for example, DNase, RNase, etc.).

That is, 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.

In addition, since the recovery rate of the finally obtained (extracted) nucleic acid may be lowered, it is preferable not to coexist with a coprecipitation agent in the step (1) of the present invention. That is, step (1) of the present invention is preferably carried out in the absence of a coprecipitation agent.

[0033] 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.

More specifically, for example, 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, Ν, Ν ', Ν'-tetraacetic acid (HDTA), triethylenetetramine hexaacetic acid (TTHA), diethylenetriamine- N, N, N', N ", N"- Pentaacetic acid (DTPA), Ν, Ν-bis (2-hydroxybenzyl) ethylenediamine-Ν, Ν-diacetic acid (HBED), etc., polyaminoalkanepolycarboxylic acid [diaminopropane tetraacetic acid (Methyl-EDTA), trans- l, 2-Diaminocyclohexane-N, N, N ', N'-tetraacetic acid (CyDTA) etc.], polyaminoalkanol polycarboxylic acid [diaminopropanol tetraacetic acid (DPTA-OH) etc.], hydroxyalkyl ether polyamine Polycarboxylic acid [Glycol A Nitrogen-containing polycarboxylic acids having 1 to 4 nitrogen atoms and 2 to 6 carboxyl groups in the molecule such as ludiamin tetraacetic acid (GEDTA) etc., such as aminopoly (alkylphosphonic acid) [aminotris (methylenephosphonic acid) ), Etc.], ditrimethyl poly (alkylphosphonic acid) [nitrilotris (methylenephosphonic acid) (NTPO), etc.], mono- or polyalkylenepolyamine poly (anolequinolephosphonic acid) [ethylenediaminetetrakis (methylenephosphonic acid) ( EDTPO), Ethylenediamine-Ν, Ν'-bis (methylenephosphonic acid) (EDDPO), isopropylenediaminetetrakis (methylenephosphonic acid), diethylenetriamine-Ν, Ν, Ν ', Ν ", Ν" -penta (Methylene phosphonic acid), ethylenediamine bis (methylene phosphonic acid), hexenediaminetetrakis (methylene phosphonic acid), etc.) 1 to 3 nitrogen atoms in a molecule such as anorequinoleaminopoly (anorequinolephosphonic acid) [ethenoreaminobis (methylenephosphonic acid), dodecylaminobis (methylenephosphonic acid), etc.] Nitrogen-containing polyphosphonic acids having 2 to 5 phosphonic acid groups such as methyldiphosphonic acid, ethylidenediphosphonic acid, 1-hydroxyethylidene-1,1'-diphosphonic acid (HEDPO), 1-hydroxypropylidene- 1,1′-diphosphonic acid, 1-hydroxybutylidene-1,1′-diphosphonic acid and the like having a hydroxy group, alkanepolyphosphonic acids, such as dihydroxyethyldaricin (DHEG), and these (For example, alkali metal salts such as sodium salt, potassium salt and lithium salt, alkali earth metal salts such as calcium salt and magnesium salt, etc.), among others, EDTA or its (Sodium salt) is favorable preferable.

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.

As the reducing agent, a thiol compound is preferable.

Examples of thiol compounds include dithiothreitol (hereinafter abbreviated as DTT), β-mercaptoethanol (hereinafter abbreviated as j8 ME), ァ -acetylcystine, systemine, reduced dartathione, dithiol. Erythritol, 2-aminoethylisothiobromide, thioglycerol, thioglycolic acid, thioglucose, 2-mercaptoethanesulfonic acid, mercaptosuccinic acid, 1 thio 13-D-glucose disodium salt dihydrate Examples thereof include 4-amino-6-hydroxy-1-2-mercaptopyrimidine monohydrate, 2-amino-6-mercaptopurine riboside hydrate, chiofnorole, 2-thiouracil and the like. Of these, DTT and j8 ME are preferred.

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. However, 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. As 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. Specifically, for example, when DTT is used, 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.

Also, for example, when j8 ME is used, 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.

In the above method, 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. According to the method for preparing an aqueous solution containing the blood component as described above, a proteolytic enzyme, a surfactant, and an alkali metal salt (buffer agent if necessary), as long as Z and a reducing agent coexist. Good. In other words, 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.

Of these methods, 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. First, 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. By adding blood components to an activator, 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. Prepare a solution containing a salt (buffer if necessary) and a chelating agent or Z and a reducing agent, and then add further proteolytic fermentation to the solution. A method of adding and mixing an aqueous solution containing element is preferred.

In the above method, 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.

[0036] 6- 3. Process (2)

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).

In particular, 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.

Next step (3) Nucleic acid loss during alcohol precipitation concentration can be prevented and the recovery rate can be increased.

[0037] 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.

[0038] 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.

The most common methods are (1) 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, and (2) The reaction solution containing free nucleic acid obtained in step (1) contains an aqueous solution containing a chaotropic agent and a coprecipitation agent (reaction solution). And the like.

In the above method, 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.

[0039] The reaction (contact) in the step (2) of the present invention does not require any special reaction time or reaction temperature (usually carried out at ordinary temperature). That is, after contacting (adding and mixing) the free nucleic acid-containing reaction solution obtained in step (1), the chaotropic agent and the coprecipitation agent, step (3) of the present invention is subsequently performed.

However, for example, 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. Although not particularly limited, specifically, the lower limit is usually pH 6 or more, preferably pH 7 or more, and the upper limit is usually pH 9 or less, preferably pH 8 or less.

For example, 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.

[0040] In 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. For example, N- (2-acetamido) -2-amino Ethanesulfonic acid (ACES), Ν, Ν-bis (2-hydroxyethyl) -2-aminoethanesulfonic acid (BES), Ν, Ν-bis (2-hydroxyethyl) glycine (Bicine), N-cycloto Kisil -3-aminominosulfonic acid (CAPS), N-cyclohexyl-2-hydroxy-3-aminominosulfonic acid (CAPSO), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), 3- [Ν, Ν-bis (2-hydroxyethyl) amino] -2-hydroxypropanesulfonic acid (DIPSO), 3- [4- (2-hydroxyethyl) -1-piperaduryl] propanesulfonic acid (EPPS) ), 2- [4- (2-hydroxychetyl) -1-piperajuryl] ethanesulfonic acid (HEPES), 2-hydroxy-3- [4- (2-hydroxyethyl) -1-piperadyl] Propanesulfonic acid (HEPPSO), 3-morpholinop pan sulfonic acid (MOPS), piperazine-1,4-bis (2-ethanesulfonic acid) (PIPES), piperazine-1,4-bis (2-hydroxy-3) -Propanesulfonic acid (POPSO), N-tris (hydroxymethyl) methyl-3-aminominosulfonic acid (TAPS), 2-hydroxy-N-to Lis (hydroxymethyl) methyl-3-aminominosulfonic acid (TAPSO), N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (TES), N- [tris (hydroxymethyl) methyl] glycine ( Good buffer such as Tricine), Bis-tris propane, collamine chloride, glycine amide and the like, for example, buffer such as tris (hydroxymethyl) aminomethane (tris), glycylglycine and the like. Of these, tris, tricine and MOPS are particularly preferred.

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). In general, the reaction solution containing the free nucleic acid obtained in the step (1), the chaotropic agent and the coprecipitation agent may be used. 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, it is lOOmM or less, and further preferably 5 OmM or less.

In order to use the buffer as described above in the step (2) of the present invention, the reaction solution containing the free nucleic acid obtained in the step (1) is contacted with the chaotropic agent and the coprecipitation agent. In addition, 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. What is necessary is just to follow according to the method of preparing the aqueous solution. That is, in the final method, 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. (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.

In the above method, 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). In order to select the concentration and pH in the solution), the use amount of each solution may be determined as appropriate.

[0041] In the step (2) of the present invention, in addition to the chaotropic agent and the coprecipitation agent as described above, in addition to reagents usually used in this field, for example, nuclease (eg, DNase) such as a chelating agent. , RNase and the like) are preferably present together.

Incidentally, when a chelating agent is 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. When 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.

In the present invention, since 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. Various aqueous solutions (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.

[0042] Specific examples of the chelating agent 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). For example, 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.

In the above method, 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. In other words, 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 (2 ) Method of adding the free nucleic acid-containing reaction solution obtained in step (1) to the aqueous solution (reaction solution), (3) Separately containing chaotropic agent, co-precipitating agent (buffer agent if necessary) and chelating agent Or (4) containing one or more of a chaotropic agent, a coprecipitation agent (a buffering agent if necessary), and a chelating agent, respectively, in the reaction solution containing the free nucleic acid obtained in step (1) A method of adding two or more aqueous solutions to the reaction solution containing free nucleic acid obtained in step (1). I can get lost. Of these, (1) or (2) is preferable.

In the above method, 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)). In addition, 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. [0044] The nucleic acid liberated by the steps (1) and (2) of the present invention as described above can be used as it is as a sample for various nucleic acid chain analysis and the like. It is also possible to prepare a sample for collecting and purifying a nucleic acid by a method usually used.

[0045] 6-4. Step (3)

After carrying out the steps (1) and (2) of the present invention as described above, that is, (1) 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.

In the present invention, 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.

In order to carry out the alcohol decay method, according to a method known per se, a solution containing a free nucleic acid obtained after carrying out the steps (1) and (2) of the present invention and a solution (reagent) containing alcohols If you mix with ヽ.

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. Such 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. Of these, alkyl alcohols having 3 or less carbon atoms (eg, ethanol and isopropanol) may be used alone or in combination of two. In addition, 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.

In addition, as the 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.

In particular, when targeting a sample containing a large amount of lipids such as blood components, lipids can be dissolved and removed from the nucleic acid. Preferably, nucleic acid is precipitated using a mixture of two or more alcohols in combination with 3 or less alkyl alcohols.

Specifically, 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.

In the case of using sodium chloride as a chaotropic agent in the step (2) of the present invention, it is more preferable to precipitate nucleic acid using isopropanol alone or a mixed solution of isopropanol and butanol in particular. .

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. As described above, when isopropanol is used alone in the reaction solution containing free nucleic acid obtained in the steps (1) and (2) of the present invention, the final concentration of isopropanol (nucleic acid and isopropanol) is used. In the reaction solution containing free nucleic acid obtained in step (1) and step (2) of the present invention so that the concentration in the solution is usually 40% or more, preferably 50% or more. Added.

In addition, when two or more alkyl alcohols having 3 or less carbon atoms are used in combination, generally the final concentration of all alcohols used (concentration in the solution when the nucleic acid and alcohols are contacted) 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.

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.

Specifically, for example, when isopropanol and butanol are used in combination, 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. When 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), 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.

Specifically, a combination of ethanol or Z and isopropanol with butanol 2 When using a mixed liquid of two or more kinds of alcohols, the ratio of butanol contained in the mixed liquid (as a content ratio in the mixed liquid of two or more kinds of alcohols) is usually 20% or more, preferably 30% as a lower limit. Above, more preferably 40% or more, still more preferably 50% or more, particularly preferably 60% or more, and the upper limit is usually 80% or less, preferably 70% or less. In particular, when isoprononol and butanol are used in combination, the ratio of butanol contained in these mixed solutions (as content) is usually 40% or more, more preferably 50% or more, and still more preferably 60% as the lower limit. Thus, the upper limit is usually 80% or less, preferably 70% or less.

Here, 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.

[0047] 6-5. Specific operation method

In order to carry out the 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.

[0048] (1) Put 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.

In the above, two solutions of an 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. Each component is added, but before adding to the sample, these solutions may be mixed in advance to form one solution, which may be added to the sample.

(2) Next, an appropriate amount of an aqueous solution containing a chaotropic agent and a coprecipitation agent (additionally containing a buffering agent or Z and a chelating agent if necessary) is added to and mixed with the obtained reaction solution, and the reaction solution and the chaotropic agent are mixed. And a coprecipitate is contacted.

(3) Thereafter, an appropriate amount of alcohol is added to the obtained mixed solution and mixed. After mixing, it may be allowed to stand as necessary.

Here, 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.

Usually, an appropriate amount of an aqueous solution containing alcohols is added to the collected precipitate, and the precipitate is washed once or more. In other words, 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. In particular, the aqueous alcohol solution used for the first washing (alcohols used in the washing operation subsequent to step (3) of the present invention) 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.

As the aqueous alcohol solution used for the first washing, an aqueous alcohol solution of usually 40% or more, preferably 50% or more, more preferably 70% or more is used. When two or more alcohols are used in combination, they may be used so that the total amount of alcohols used is within the above range. Specifically, for example, in a mixed solution of ethanol and butanol, ethanol is preferably 60% to 80%, but butanol is preferably 5 to 10%. In a mixture of isopropanol and butanol, isopropanol is preferred at 40-60%, but butanol is preferred at 5-10% ^

After washing with the above alcohol solution, the supernatant is removed by centrifugation, and the nucleic acid is recovered again as a precipitate.

Furthermore, the nucleic acid precipitate recovered again may be dried by a conventional method such as heating or decompression. In addition, when the nucleic acid obtained by the above method is used as a sample for various nucleic acid chain analysis (eg, gene analysis by PCR method, etc.), it is further used in a method usually used in this field, for example, TE It is desirable to use 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). If the target nucleic acid is DNA, 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. When the nucleic acid is RNA, 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. [0050] 7. Kit of the present invention

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. (1) A combination of a reagent containing a proteolytic enzyme, (2) a reagent containing a surfactant and an alkali metal salt, and (3) a reagent containing a chaotropic agent and a coprecipitation agent. Aspects, specific examples, and the like are as described above. In the kit of the present invention as described above, 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. In addition, there is no particular limitation on the reagent form that 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.

[0051] 7—1. Reagent (1)

The reagent (1) containing a proteolytic enzyme of the present invention contains a proteolytic enzyme as a main component.

Specific examples and preferred embodiments of the proteolytic enzyme are as described above. The proteinase is preferably proteinase K. Further, 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.

In addition, in the reagent (1) of the present invention, in addition to the proteolytic enzyme, it is preferable that a reagent that is usually used in this field may coexist, especially a stabilizer. . Examples of such 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. For example, in the case of glycerol, the reagent (1) of the present invention contains 10% to 80%. For example, in the case of 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. When the reagent (1) of the present invention is used in the step (1), the reagent (1) 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.

7-2.Reagent (2)

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.

Specific examples, preferred and embodiments of the surfactant and the alkali metal salt are as described above. Force As the surfactant, N-lauroyl sarcosine acid or a salt thereof (for example, sodium salt, potassium salt, lithium salt, etc.) is preferred. The sodium N-lauroyl sarcosinate is particularly preferred. As the alkali metal salt, potassium salt is preferred. Further, 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.

Reagents of the present invention (2), the blood component, reagent (1) and reagent (2) _P H when obtained by mixing, i.e., a blood component and a proteolytic enzyme in the step (1) of the present invention surfactant 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. Specifically, 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.

In the reagent (2) of the present invention, a buffering agent can be used to adjust the pH to the range as described above.

Specific examples and preferred embodiments of such a buffering agent are 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.

Further, in the reagent (2) of the present invention, in addition to the surfactant and the alkali metal salt, reagents used in this field, for example, nucleolytic enzymes such as chelating agents (for example, DNase, RNase, etc.) Inhibitors, reducing agents, etc. may coexist. In particular, it is preferable to coexist with inhibitors of nucleolytic enzymes such as chelating agents (eg DNase, RNase, etc.)!

Specific examples, preferred embodiments and the like of the chelating agent and reducing agent are as described in the above-mentioned step (1) of the present invention. As the chelating agent, EDTA or a salt thereof (such as sodium salt) is preferable, and the reducing agent is Is preferably DTT, | 8 ME. Also, 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. 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.

For the reasons described above, it is preferable that 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. When the reagent (2) of the present invention is used in the step (1), the reagent (2) 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).

7- 3. Reagents (3)

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.

Since 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.

Specific examples, preferred embodiments and the like of the 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. In addition, 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. Specifically, 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! /.

In the reagent (3) of the present invention, a buffering agent can be used to adjust the pH to the range as described above.

Specific examples, preferred, embodiments and the like of such a buffer are as described in the above-mentioned step (2) of the present invention, and tris, tris, Tricine and MOPS are particularly preferred.

In the reagent (3) of the present invention, in addition to the chaotropic agent and the coprecipitation agent, 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. It should be noted that when the reagent (2) contains a chelating agent, sufficient nucleic acid degradation is allowed when the reaction solution (nucleic acid) containing the free nucleic acid obtained in step (1) is contacted with the chaotropic agent and the coprecipitation agent. When 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).

Specific examples, preferred and embodiments of the chelating agent are as described in the above-mentioned step (2) of the present invention. As the chelating agent, EDTA or a salt thereof (sodium salt or the like) is preferable. Also, 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.

For the reasons described above, 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.

[0054] 7-4. Specific examples of the kit of the present invention

Below, an example of the kit of this invention is shown concretely.

(1) Reagents containing proteolytic enzymes (aqueous solution)

(2) Reagent containing surfactant and alkali metal salt (aqueous solution)

(3) Reagent containing chaotropic agent and coprecipitation agent (aqueous solution)

[0055] Hereinafter, another example of the kit of the present invention will be specifically described.

(1) Reagents containing proteolytic enzymes (aqueous solution)

(2) Reagent (aqueous solution) containing surfactant, alkali metal salt and buffer (if necessary, chelating agent)

(3) Reagent (aqueous solution) containing a chaotropic agent, a coprecipitation agent and a buffer (if necessary, a chelating agent) [0056] Hereinafter, another example of the kit of the present invention is specifically shown.

(1) Reagents containing proteolytic enzymes (aqueous solution)

(2) Reagent with pH 7 ~ 10 containing surfactant and alkali metal salt (aqueous solution)

(3) Reagents with pH 6-9 including chaotropic agent and coprecipitation agent (aqueous solution)

[0057] 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. Examples of such 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

Or 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.

[0058] Still further, 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. In particular, it is preferable to combine at least one aqueous solution containing alcohols of the same type as the alcohols used as a reagent for precipitating the nucleic acid. When two or more alcohol-containing aqueous solutions are combined, it is preferable to combine aqueous solutions containing different alcohols, even if they are all aqueous solutions containing the same alcohol. Specifically, it is preferable to combine two types of aqueous solutions, an aqueous solution containing ethanol or Z and isopropanol and butanol, and an aqueous solution containing ethanol, and an aqueous solution containing ethanol and an aqueous solution containing ethanol. Better to combine seed water solution.

The concentration used is as described above.

[0059] A preferred example of the kit of the present invention is specifically shown below.

(1) Reagents containing proteolytic enzymes (aqueous solution)

(2) Reagent containing surfactant and alkali metal salt (aqueous solution)

(4) One or more reagents 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 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. One or more aqueous solutions containing one kind of alcohol or other alcohol used for precipitating nucleic acids, and an alcohol used for washing nucleic acid precipitation. 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.

[0060] Another preferred example of the kit of the present invention will be specifically shown below.

(1) Reagents containing proteolytic enzymes (aqueous solution)

(3) Reagent (aqueous solution) containing chaotropic agent, coprecipitation agent and buffer (if necessary, chelating agent)

(4) One or more reagents 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 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.

[0061] In the following, another preferred example of the kit of the present invention is specifically shown.

(1) Reagents containing proteolytic enzymes (aqueous solution)

(4) One or more reagents 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.

[0062] A more preferred example of the kit of the present invention will be specifically shown below.

(1) Reagents containing proteolytic enzymes (aqueous solution)

(2) Reagents with a pH of 7-10, including surfactants, alkali metal salts, buffers and chelating agents (aqueous solutions)

(3) Reagents with pH 6-9, including chaotropic agents, coprecipitates, buffers and chelating agents (aqueous solutions)

(4) One or more reagents containing alcohols

[0063] The kit of the present invention is particularly preferably one having the following constitutional power, for example.

(1) Reagent containing proteinase K (aqueous solution)

(2) Reagents of pH 7 to 10 containing sodium N-lauroyl sarcosinate, potassium chloride salt, buffer and chelating agent (aqueous solution)

(3) Reagents with pH 6-9 including sodium chloride, glycogen, buffering agent and chelating agent (aqueous solution)

(4) Nucleic acid precipitation reagent containing isopropanol and butanol (5) Nucleic acid precipitation washing reagent containing isopropanol and butanol

(6) Nucleic acid precipitation washing reagent containing ethanol

[0064] 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. In particular, 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).

[0065] Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

Example 1

[0066] [Study of protein solubility of chaotropic agent (yoi sodium)]

[0067] (Dissolution)

A solution obtained by dissolving the following reagent in 50 mM (concentration at the time of warming reaction: 33.2 mM) in Tricine buffer (PH8.5) so as to have the following concentration was used.

EDTA 10mM: Concentration during warming reaction 6.6mM

KC1 75mM: Concentration during warming reaction 49.8mM

DTT 45mM: Concentration during warming reaction 29.9mM

SLS 0.2%: Concentration during warming reaction 0.13%

[0068] (Yo's sodium 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.

Nal 7.62M

EDTA 20mM

Glycogen 70 / zg / mL [0069] (Alcohol mixture)

Isopropanol 37.5% (v / v)

2 Butanol 62.5% (v / v)

[0070] (Operation method)

100 L of human serum was placed in a micro centrifuge tube, and 200 L of lysis solution was added and mixed. Sarasuko, Proteinase K solution [20 g / L proteinase K (derived from Tritirachium album, Wako Pure Chemical Industries, Ltd.)-Containing distilled water] 1.0 L was added and mixed, and the mixture was heated at 37 ° C for 10 minutes. Thereafter, a predetermined amount of sodium chloride solution as shown in Table 1 was added and mixed. Next, the alcohol mixture was added and mixed with a predetermined amount shown in Table 1, and allowed to stand at room temperature for 10 minutes, and then centrifuged at 20,000 G for 10 minutes.

Table 1 shows the amount of sodium iodide solution and alcohol mixture used and the concentration after the addition.

[0071] [Table 1]

[0072] (Result)

Regarding the presence of precipitates other than the nucleic acid and the coprecipitate glycogen among the precipitates obtained after the centrifugation, 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.

[Criteria]

+ + + + +: A remarkably large precipitate (diameter of about 5 mm or more) was observed at the bottom of the centrifuge tube. +++++: A very large sediment (diameter of about 3mn! ~ 5mm) was observed at the bottom of the centrifuge tube. +++: Large sediment (diameter of about 2mn! ~ 3mm) was observed at the bottom of the centrifuge tube.

++: Precipitation (diameter about lmn! ~ 2mm) was observed at the bottom of the centrifuge tube.

+: A small precipitate (diameter of about 0.5 mm to 1 mm) was observed at the bottom of the centrifuge tube.

Shi: The deposit (diameter of about 0.5mm or less) was hardly observed at the bottom of the centrifuge tube.

[0073] [Table 2]

[0074] As is clear from Table 2, in the case of samples よう ο.1 to 3 where the sodium iodide concentration is 2.53Μ or less, after adding the alcohol mixture and centrifuging, it settles at the bottom of the centrifuge tube. Insoluble precipitates of impurities other than nucleic acids (and co-precipitant glycogen) such as proteins were very large in mass.

On the other hand, in the case of samples Νο.4 to 6 with a sodium iodide concentration of 3.04Μ or higher, high-quality (purity) nucleic acid with less insoluble precipitate of impurities was obtained.

In addition, the insoluble precipitate of impurities decreased with increasing sodium concentration, and was very small compared to 4 at 3.46 Μ (samples Νο.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. Example 2

[0075] [Study on salting time of salt (KC1)]

[0076] (Sample)

Human serum: 7 samples and human plasma: 4 samples were used as samples.

[0077] 'Case 1

(Solution)

A solution obtained by dissolving the following reagents in 50 mM Tricine buffer (pH 8.5) so as to have the following concentration was used. EDTA lOmM

KC1 75mM

SLS 1.0%

(Sodium iodide solution)

Nal 7.62M

EDTA 20mM

Glycogen 70 / z g / mL

[0078] 'Case 2

(Solution)

A solution obtained by dissolving the following reagents in 50 mM Tricine buffer (pH 8.5) so as to have the following concentration was used.

EDTA 10mM

SLS 0.2%

(Sodium iodide solution)

Nal 7.62M

EDTA 50mM

KC1 75mM

Glycogen 70 / z g / mL

[0079] As shown below, in cases 1 and 2, the concentrations of the constituent reagents after addition of the sodium iodide solution are all the same.

Tricine 16.5mM

EDTA 13.2mM

KC1 24.8mM

SLS 0.33% Glycogen 34.8 / zg / mL

Nal 3.78M

Tris- HCl 19.9mM

[0080] (Operation method)

Case 1 and Case 2 were measured as follows.

100 L of a sample was placed in a micro centrifuge tube, and 200 L of a lysis solution was added and mixed. Furthermore, 2.0 L of proteinase K solution (distilled water containing 20 g // z L proteinase K (derived from Tritirachium album, Wako Pure Chemical Industries, Ltd.)) was added and mixed, and the mixture was heated at 37 ° C for 10 minutes. . Thereafter, 2 _{μL of} 2 μ _· / μL salmon sperm-derived DNA (manufactured by Wako Pure Chemical Industries, Ltd.) was added, and 300 L of sodium chloride solution was further added and mixed. Next, 600 L of 100% isopropanol was added and mixed, and the mixture was allowed to stand at room temperature for 10 minutes, then centrifuged at 20,000 G for 10 minutes, and the supernatant was discarded to obtain a precipitate. To the resulting precipitate, 1 mL of 50% aqueous isopropanol was added, stirred, centrifuged again at 20,000 G for 5 minutes, and the supernatant was discarded to obtain a precipitate. Furthermore, 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 dissolved in 100 L of TE buffer (ImM EDTA-containing 10 mM Tris-HCl buffer, pH 8.0) to obtain a measurement sample.

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.

[0081] (Result)

Table 1 shows the results of measuring the DNA content (recovery rate of salmon sperm-derived DNA) in Case 1 and Case 2. still. In Table 3, 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.

[0082] [Table 3]

From Table 3, it can be seen that both salt (KC1) and coprecipitate (glycogen) are compared to case 1 where salt (KC1) is included in the solution and coprecipitate (glycogen) is included in the sodium solution. In case 2 contained in the sodium chloride solution, the recovery rate of DNA was low for almost all samples, and in particular, the recovery rate of DNA for two samples (serum sample 3 and plasma sample 1) was low. It can be seen that it has declined significantly. In these two samples, there was contamination of insoluble precipitates in the obtained precipitates, resulting in low purity DNA samples. In case 2, unlike case 1, 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.

Example 3

[0084] [Study of deproteinization effect of surfactant (sodium N-lauroyl sarcosinate: SLS)] [0085] (Solution)

• Case A

50mM (concentration during warming reaction 6.6mM) Tricine buffer (p.

A solution dissolved in H8.5) was used as a solution.

EDTA 10mM: Concentration during warming reaction

KC1 75mM: Concentration during warming reaction 49.8mM

DTT 5mM: Concentration during warming reaction 3.3mM

SLS Prescribed concentrations specified in Table 4

'Case B

A solution obtained by dissolving the following reagents in 50 mM (concentration at the time of warming reaction: 6.6 mM) Tricine buffer (pH 8.5) so as to have the following concentration was used.

EDTA 10mM: Concentration during warming reaction

KC1 75mM: Concentration during warming reaction 49.8mM

DTT 45mM: Concentration during warming reaction 29.9mM

SLS Prescribed concentrations specified in Table 4

[0086] [Table 4]

(Yo 匕 sodium solution) A solution obtained by dissolving the following reagents in 40 mM (concentration 20 mM after addition of sodium chloride solution) Tris-HC1 buffer (pH 8.0) so as to obtain the following concentrations was used as a sodium solution.

Nal 7.62M: Concentration after adding sodium iodide solution 3.8M

EDTA 20mM: Concentration after addition of sodium iodide solution 10mM

Glycogen 70 μg / mL: Concentration after addition of alcohol mixture 17 g / mL

[0088] (Alcohol mixture: common to cases A and B): Final alcohol concentration of 50%

Isopropanol 37.5% (v / v)

2 Butanol 62.5% (v / v)

[0089] (Operation method)

Case A and Case B were measured as follows.

100 L of human serum was placed in a micro centrifuge tube, and 200 L of lysis solution was added and mixed. Sarasuko, proteinase K solution [20 g / L proteinase K (derived from Tritirachium album, Wako Pure Chemical Industries, Ltd.)-Containing distilled water] 1.0 L was added and mixed, and the mixture was heated at 56 ° C for 10 minutes. Thereafter, 300 L of sodium iodide solution was added and mixed. Next, 600 μL of the alcohol mixture was added and mixed, allowed to stand at room temperature for 10 minutes, centrifuged at 20,000 G for 10 minutes, and the supernatant was discarded to obtain a precipitate. 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.

For 20 L of the obtained analytical sample, protein quantification was performed with a protein plot assembly kit (manufactured by Wako Pure Chemical Industries), the amount of protein remaining in the precipitate (nucleic acid) was measured, and each was compared. The deproteinization effect of SLS was examined.

[0090] (Result)

Figure 1 shows the results of measuring the amount of protein remaining in the precipitate (nucleic acid) for Case A and Case B.

In Fig. 1, the upper row shows the case A, the lower row shows the case B, and 1 to 6 show the sample numbers. For comparison, the color development degree of the sample spotted on the PVDF membrane attached to the kit was judged by relative comparison.

As is apparent from Fig. 1, when the color development degree of each sample spotted on the PVDF membrane was compared in Case B (containing 45 mM DTT), sample No. 1 with an SLS concentration of 0.1% showed extreme color development. It can be seen that a large amount of residual protein is mixed in the precipitation that is strong at the edges. As a result, it is clear that the higher the SLS concentration in the solution, the lower the degree of color development. In particular, in the case of sample Nos. 5 to 6, that is, the SLS concentration in the solution is 3% to 5%. In the case of% (when SLS has a concentration power of 99% to 3.32% during protein degradation reaction by proteinase K), almost no color development is observed, indicating that the residual protein in the precipitate has almost disappeared.

In Case A (containing 5 mM DTT in the lysate), sample Nos. 1 to 5 (when the concentration power of SLS at the time of proteolytic reaction with proteinase K is less than 99%), a slight color development was observed, When sample No. 6 was obtained (when the SLS concentration during protein degradation with proteinase K was 3.32%), almost no color was seen and the remaining protein in the precipitate almost disappeared. .

From the above, as a result of high concentration of SLS (especially, SLS concentration of 2% or more during protein degradation by proteinase K) as a surfactant or as a result of working with proteolytic enzymes, protein removal It was possible to conclude that the results showed a stronger effect.

Example 4

[0091] [Study of delipidation effect of butanol]

[0092] (Sample)

A sample obtained by adding 10 μL of intra-fat injection (10% w / v soybean oil: manufactured by Nippon Pharmaceutical) to 90 μL of human serum was used as a sample.

[0093] (Solution)

EDTA 10mM: Concentration during warming reaction 6.6mM

KC1 75mM: Concentration during warming reaction 49.8mM SLS 4%: Concentration during warming reaction 2.66%

[0094] (Yo's sodium solution)

A solution obtained by dissolving the following reagents in 40 mM (concentration 20 mM after addition of sodium chloride solution) Tris-HC1 buffer (pH 8.0) so as to obtain the following concentrations was used as a sodium solution.

Nal 7.62M: Concentration after adding sodium iodide solution 3.8M

EDTA 20mM: Concentration after addition of sodium iodide solution 10mM

Glycogen 70 μg / mL: Concentration after addition of alcohol mixture 17 g / mL

[0095] (Alcohol mixture): final concentration of alcohols 50%

The prescribed concentration specified in Table 5 (% (v / v))

[0096] [Table 5]

[0097] (Washing liquid A)

Isopropanol 40% (v / v)

1-butanol 10% (v / v)

[0098] (Operation method)

100 L of a sample 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. Thereafter, the mixed solution was centrifuged at 20,000 G for 10 minutes, and the supernatant was discarded to obtain a precipitate. To the resulting 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.

[Criteria]

+ + + + To +: Shows turbidity. The greater the number of +, the more turbid it is.

-: Indicates that there is no turbidity.

[0099] (Result)

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.

[0100] [Table 6]

[0101] As is clear from Table 5, as the isopropanol concentration increases, the α-butanol concentration decreases), the turbidity after addition of the alcohol mixture increases, that is, lipids that are not soluble in alcohol become cloudy. In addition, it was found that even after centrifugation, it remained as a sticky inner wall of the centrifuge tube. When the concentration of 1-butanol in the alcohol mixture exceeds 58.3%, the lipid derived from the blood sample dissolves in the alcohol mixture and the solution does not become turbid, and no residual lipid is observed after centrifugation. A precipitate (nucleic acid) was obtained. Although not shown here, the same result was obtained even when 2-butanol was used. Therefore, this was judged to be a result showing the delipidating effect of butanol Example 5

[0102] [Study on the timing of coprecipitation (glycogen) addition]

(Sample)

A sample obtained by adding 1 μL of α-amylase (7 units Z μL: manufactured by Nibonbon Gene) and 3 μL of lOOmM calcium chloride to 99 μL of human serum was used as a sample.

[0103] 'Case 1 (Solution)

EDTA 10mM

KC1 75m

SLS 4.0%

Glycogen 105 / z g / mL

(Yo 匕 sodium solution)

Nal 7.62M

EDTA 20mM

'Case 2

(Solution)

EDTA 10mM

KC1 75mM

SLS 4.0%

(Sodium iodide solution)

Nal 7.62M

EDTA 20m

KC1 75m

Glycogen 70 / z g / mL

As shown below, in case 1 and case 2, the concentrations of the constituent reagents after addition of the sodium chloride solution are all the same. Tricine 16.5mM

EDTA 13.2mM

KC1 24.8mM

SLS 1.32%

Glycogen 34.7 / z g / mL

Nal 3.78M

Tris-HCl 19.8mM

[0105] (Alcohol mixture: common to cases 1 and 2): Final concentration of alcohols 50%

Isopropanol 40% (v / v)

1-butanol 60% (v / v)

[0106] (Cleaning liquid A: Common to cases 1 and 2)

Isopropanol 40% (v / v)

1-butanol 10% (v / v)

[0107] (Operation method)

Case 1 and Case 2 were measured as follows.

100 L of a sample was placed in a micro centrifuge tube, and 200 L of a lysis solution was added and mixed. Further, 5.0 L of proteinase K solution [20 g // z L proteinase K (derived from Tritirachium album, Wako Pure Chemical Industries, Ltd.)-Containing distilled water] was added and mixed, and the mixture was heated at 56 ° C. for 10 minutes. Then, add 300 L of sodium iodide solution and mix. Next, 600 L of an alcohol mixture having the following compositional power was added and mixed, allowed to stand for 10 minutes at room temperature, centrifuged at 20,000 G for 10 minutes, and the supernatant was discarded to obtain a precipitate. To the resulting precipitate, ImL of Washing Solution A was added, stirred, centrifuged again at 20,000 G for 5 minutes, and the supernatant was discarded to obtain a precipitate. To the resulting precipitate, ImL of 70% ethanol aqueous solution was added, 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 in L TE buffer (ImM EDTA-containing 10 mM Tris-HCl buffer, pH 8.0) to prepare a DNA sample.

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. In addition, 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.

After completion of PCR, 3% agarose gel electrophoresis was performed, and the amount of amplified fragment of each DNA sample was compared.

[0108] [Reagents]

Exon5 F— primer (manufactured by Futtsubon Gene): 4pmol

Εχοηδ R-primer (Nitsubon Gene): 4pmol

dNTP (manufactured by Tubongene): 200 ^ Μ

1 X Universal Buffer (-Tubongene)

GenTaq-NT (-Tsubongene): 0.5units

[PCR conditions]

Reaction solution: 10 L

DNA sample: 2 μL

Equipment: Thermal cycler (Model number: Iwaki Glass TSR-300: Iwaki Glass)

Temperature 'time: After reacting at 96 ° C for 30 seconds, the reaction was carried out for 38 cycles of 94 ° C for 30 seconds, 65 ° C for 30 seconds and 72 ° C for 1 minute, and then further reacted at 72 ° C for 5 minutes.

[0109] (Result)

Fig. 2 shows the results of 3% agarose gel electrophoresis for Case 1 and Case 2.

In FIG. 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.

[0110] From the results shown in Fig. 2, in case 1 where both the salt (KC1) and the coprecipitate (glycogen) are contained in the solution, the salt (KC1) is contained in the solution and the coprecipitate (glycogen) is added. It can be seen that the amount of amplified fragments after PCR is clearly smaller in both sample 1 and sample 2 than in case 2 contained in the sodium solution. This shows that the DNA recovery rate of Case 1 is better than Case 2 It was thought to be the result of affecting bad PCR. These specimens are special specimens called hyperamylaseemia. The amylase contained in the blood works to break down glycogen, which is a coprecipitate in the lysate, and remove nucleic acid (free DNA in the blood). This may be because the recovery rate has decreased. In fact, in case 1, it was observed that the amount of glycogen decreased and the size of the precipitate after alcohol precipitation was smaller than in case 2.

In the analysis of free DNA in blood, spleen cancer, which has an abnormally high blood amylase concentration, is also examined, and in malignant tumors such as sarcophagus and cancer, calcium that enhances blood amylase activity is examined. It is also known that hypercalcemia, in which the blood concentration of ions reaches an abnormally high level, can occur frequently. Therefore, in order to avoid the risk of such a phenomenon occurring and the coprecipitation agent being decomposed, the coprecipitation agent, which is a polymeric polysaccharide, as in Case 2, is more than added to the solution. However, it has been found that it is preferable to add it to the sodium solution.

Claims

The scope of the claims

[I] (1) The blood component and the proteolytic enzyme are reacted in the presence of a surfactant and an alkali metal salt. (2) Next, the obtained reaction solution is contacted with a chaotropic agent and a coprecipitation agent. Then, (3) a method for extracting nucleic acid from blood components, comprising performing a treatment for precipitating the nucleic acid.

[2] The method according to claim 1, wherein the blood component is serum or plasma.

[3] The method according to claim 1, wherein the nucleic acid is blood free DNA.

[4] (1) The blood component and the proteolytic enzyme are heated at 25 to 70 ° C. in the presence of a surfactant and an alkali metal salt. (2) The resulting reaction solution and chaotropic agent are then obtained. 4. The method according to any one of claims 1 to 3, wherein after the contact with the coprecipitation agent, (3) a process of further mixing an alcohol with the coprecipitation agent to precipitate the nucleic acid is performed.

[5] The method according to claim 4, wherein the alcohol is one or more selected from ethanol, isopropanol and butanol.

[6] The proteolytic enzyme is supplied from a reagent containing a proteolytic enzyme, the surfactant and the alkali metal salt are supplied from a reagent containing a surfactant and an alkali metal salt, and The method according to any one of claims 1 to 5, wherein the chaotropic agent and the coprecipitation agent are also supplied with a reagent force containing the chaotropic agent and the coprecipitation agent.

[7] A reagent containing a proteolytic enzyme contains a proteolytic enzyme as a main component, and a reagent containing a surfactant and an alkali metal salt mainly contains a surfactant, an alkali metal salt, a chelating agent and a buffer. The method according to claim 6, wherein the reagent is contained as a component, and the reagent containing a chaotropic agent and a coprecipitation agent contains a chaotropic agent, a coprecipitation agent, a chelating agent, and a buffer as main components.

[8] A blood component comprising a combination of (1) a reagent containing a proteolytic enzyme, (2) a reagent containing a surfactant and an alkali metal salt, and (3) a reagent containing a chaotropic agent and a coprecipitation agent. Nucleic acid extraction kit.

[9] The kit according to claim 8, wherein the blood component is serum or plasma.

[10] The kit according to claim 8, wherein the nucleic acid is blood free DNA.

[II] Proteolytic enzymes are proteinase 1, pronase, trypsin and subtilisin The kit according to any one of claims 8 to 10, wherein force is also selected.

[12] Surfactant power The kit according to any one of claims 8 to 10, which is an anionic surfactant.

[13] The kit according to any one of claims 8 to 10, wherein the surfactant power is sodium N-lauroyl sarcosinate.

[14] The kit according to any one of [8] to [10], wherein the alkali metal salt is potassium salt.

[15] The kit according to any one of [8] to [10], which is chaotropic agent power sodium iodide.

[16] The kit according to any one of [8] to [10], wherein the coprecipitation agent is glycogen or dextran.

[17] The kit according to any one of [8] to [8], wherein (2) the reagent power is from pH 7 to pH 10 containing a surfactant and an alkali metal salt.

[18] The kit according to any one of [8] to [10], wherein (2) the reagent containing a surfactant and an alkali metal salt further contains a chelating agent or Z and a buffer.

[19] The kit according to claim 18, wherein the chelating agent is ethylenediamine tetraacetic acid or a salt thereof.

[20] The kit according to claim 18, wherein the buffer is N- [tris (hydroxymethyl) methyl] glycine (Tricine).

[21] The kit according to any one of [8] to [10], which has a reagent power of pH 6 to pH 9, comprising (3) a chaotropic agent and a coprecipitation agent.

[22] The kit according to any one of [8] to [10], wherein (3) the reagent containing the chaotropic agent and the coprecipitation agent further contains a chelating agent or Z and a buffer.

[23] The kit according to claim 22, wherein the chelating agent is ethylenediamine tetraacetic acid or a salt thereof.

24. The kit according to claim 22, wherein the buffer is tris (hydroxymethyl) aminomethane.

[25] The kit according to any one of [8] to [10], further comprising a combination of one or more reagents containing alcohols.

[26] The kit according to claim 25, wherein the kit is one or more selected from ethanol, isopropanol, and butanol.

[27] The kit includes (1) a reagent containing a proteolytic enzyme as a main component, (2) a reagent containing a surfactant, an alkali metal salt, a chelating agent and a buffer as a main component, pH7 to LO reagent, and (3 ) Reducing pH 6 to pH 9 containing as a main ingredient an oral picking agent, a coprecipitation agent, a chelating agent and a buffering agent, 9. The kit according to claim 8, which is a kit for extracting free DNA in serum or plasma that is a combination of (4) one or more reagents containing alcohol as a main component.