WO2015146737A1 - 核酸抽出方法及び試薬 - Google Patents
核酸抽出方法及び試薬 Download PDFInfo
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- WO2015146737A1 WO2015146737A1 PCT/JP2015/058009 JP2015058009W WO2015146737A1 WO 2015146737 A1 WO2015146737 A1 WO 2015146737A1 JP 2015058009 W JP2015058009 W JP 2015058009W WO 2015146737 A1 WO2015146737 A1 WO 2015146737A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
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- the present invention relates to a nucleic acid extraction method and reagent, and more particularly to a nucleic acid extraction method and reagent using a carrier.
- Genetic testing has been widely applied in recent years, including identification tests for viruses and pathogenic bacteria, detection of pathogenic microorganisms in the early stages of infection, investigation of infection sources, and diagnosis of leukemia and tumor genes.
- detection of nucleic acids of pathogenic microorganisms for the purpose of infectious disease diagnosis is one of the few techniques for early detection of infection and confirmation of the course after treatment. In this case, it is necessary to extract and collect a very low concentration of the target nucleic acid from a sample such as a body fluid.
- a carrier may be used to extract and collect a small amount of nucleic acid from a sample.
- a nucleic acid or a substance having similar chemical properties such as the charge or molecular size of the nucleic acid is used.
- nucleic acid carriers such as poly (A), poly (dIdC), and tRNA, and biopolymer carriers such as glycogen are known (Non-patent Documents 1 and 2).
- a biomolecule is used as a carrier, there is a concern that natural nucleic acids having a possibility of affecting detection are mixed in the next step. Therefore, it is desirable that the carrier is not a biomolecule.
- Non-patent Document 3 a method for extracting a nucleic acid by utilizing the property that the nucleic acid binds to a carrier such as silica has been reported.
- Polyacrylamide is known as a non-biological substance carrier, but this is not a technique for binding a nucleic acid to a carrier, but a technique using a centrifuge such as ethanol precipitation.
- An object of the present invention is to realize nucleic acid recovery from a liquid containing a very low concentration of nucleic acid that cannot be achieved by polyacrylamide in a nucleic acid recovery method using a carrier.
- the present inventors have found that the amount of recovered nucleic acid can be improved by allowing an anionic polymer, which is a non-biological substance, to coexist with nucleic acid in a nucleic acid extraction process using a carrier, and the present invention has been completed. I came to let you.
- the gist of the present invention is as follows.
- a nucleic acid extraction method comprising adsorbing a nucleic acid to a carrier in the presence of a chaotropic salt and an alcohol in the presence of an anionic polymer.
- the anionic polymer is a polymer having a carboxyl group in a side chain.
- Anionic polymer is polyacrylic acid, polyacrylate, polyacrylic acid derivative, copolymer containing polyacrylic acid, polyacrylamide derivative having carboxyl group, polysilane derivative, carboxymethylcellulose, carboxymethylcellulose salt, polyglutamic acid
- the chaotropic salt is guanidine hydrochloride, guanidine thiocyanate (guanidine thiocyanate), guanidine sulfate, guanidine isothiocyanate, sodium isothiocyanate, sodium iodide, potassium iodide, urea, sodium bromide, potassium bromide, It is at least one chaotropic salt selected from the group consisting of calcium bromide, ammonium bromide, sodium perchlorate, sodium thiocyanate, potassium thiocyanate, ammonium isothiocyanate, sodium chloride, potassium chloride and ammonium chloride [1 ] The nucleic acid extraction method according to any one of the above.
- the carrier is magnetic particles
- the magnetic particle suspension is handled by the disposable chip
- the solid particles are captured by capturing the magnetic particles on the inner wall surface of the chip by applying a magnet to the outer wall surface of the chip.
- the nucleic acid extraction method as described.
- a nucleic acid extraction reagent comprising an anionic polymer, a chaotropic salt, an alcohol and a carrier.
- the nucleic acid extraction reagent according to [11] further comprising a buffer.
- the nucleic acid extraction reagent according to [11] or [12] which is contained in a kit enclosed in a cartridge.
- nucleic acid can be recovered from a liquid containing a very low concentration of nucleic acid.
- PAAc polyacrylic acid
- DW distilled water
- the present invention provides a nucleic acid extraction method characterized by adsorbing a nucleic acid to a carrier in the presence of a chaotropic salt and an alcohol in the presence of an anionic polymer.
- the nucleic acid may be any of double-stranded DNA, single-stranded DNA, and RNA, and may be a natural product derived from a living tissue, a cell, a virus, or the like, or a synthesized product.
- the solution containing nucleic acid is a part of biological tissue or a crushed material thereof, body fluid (for example, blood (whole blood, serum, plasma, etc.), urine, saliva, amniotic fluid, breast milk, spinal fluid, synovial fluid, ascites, pleural effusion, Examples include otorrhea, nasal discharge, pus, bile, sputum, sweat, etc.), mucous membranes, feces, cells and other solutions containing dissolved samples, plants, enzyme-treated solutions, PCR products, etc. There is no limit.
- the method of the present invention enables extraction and recovery of nucleic acid from a liquid containing a very low concentration of nucleic acid.
- the anionic polymer is preferably a polymer that is negatively charged in an aqueous solution, and further, a polymer that does not inhibit the detection and quantification of nucleic acid performed after the nucleic acid extraction step, or that does not aggregate the carrier in the nucleic acid extraction step.
- a polymer having a carboxyl group in the side chain is preferable, and the polymer is preferably a water-soluble polymer.
- anionic polymers include polyacrylic acid, polyacrylate (such as sodium polyacrylate, potassium polyacrylate, and calcium polyacrylate), polyacrylic acid derivatives (such as polyethyl acrylate and polyacrylate), Copolymer containing polyacrylic acid (copolymer of acrylic acid and maleic acid, styrene-acrylic acid copolymer, ethylene-acrylic acid copolymer, polyoxyethylene-polyacrylic acid copolymer, etc.), carboxyl group Polyacrylamide derivatives (polyacrylamide-polyacrylic acid copolymer, etc.), polysilane derivatives (polycarbosilane, polyphenylsilane, etc.), carboxymethylcellulose, carboxymethylcellulose salts (carboxymethylcellulose sodium, carboxymethylcellulose) Rulose potassium, carboxymethylcellulose calcium, etc.)), polyglutamic acid, polyglutamate (sodium polyglutamate, potassium polyglutamate, calcium polyglutamate, etc.), polyglutamic acid derivatives
- the addition amount (concentration) of the anionic polymer is not particularly limited.
- 0.02 to 0.2% (v / v) is suitable as the final concentration of the carrier in the reaction solution, 0.02 to 0.1% by weight (v / v) is preferable.
- the carrier is not particularly limited as long as it can adsorb nucleic acids, and is preferably a solid having a hydrophilic functional group on the surface layer.
- the hydrophilic functional group include a hydroxyl group, a carboxyl group, a carbonyl group, a nitro group, and a silanol group.
- a hydrophilic functional group can be imparted by surface-treating the carrier. For example, due to silica coating, the support surface will have silanol groups.
- the core component when the core is coated, is either a metal oxide (silica, alumina, ferrite, magnetite, etc.) or a polymer (polystyrene, acrylic resin, cellulose, dextran, etc.) Yes, and the coating component is one of metal oxide (silica, alumina, etc.), polymer (cellulose, dextran, polyacrylic acid, etc.), or coupling agent (silane, titanate, aluminate, etc.) Good.
- the coating component may be the same component as the core component.
- the carrier may have any shape such as a sheet, a sieve, a membrane, an irregular particle, a sphere (bead), a thread (strand), or a magnetic bead, but is preferably spherical.
- the carrier is preferably magnetic particles.
- the sphere (bead) is preferably about 1 mm in size and is preferably made of a material such as plastic or ceramic. Such a sphere (bead) is disclosed in JP 2000-346842 A and JP 14-534657 A. It is described in gazettes.
- ⁇ Magnetic beads should have a size of several tens of ⁇ m and are made magnetic by mixing iron powder or the like into plastic or ceramic. Such magnetic beads are described in JP-A-8-62224 (Patent No. 3115501), International Publication WO96 / 29602, International Publication WO97 / 44671, and the like.
- the yarn (strand) has a diameter of about 0.1 mm and is preferably made of a resin-based material.
- Such yarn is disclosed in JP 2006-214759 A, International Publication WO01 / 53831 Pamphlet, International Publication WO2003. It is described in / 7901 pamphlet.
- Nucleic acid can be adsorbed to a carrier by covalent bond, chemical adsorption, physical adsorption, electrical interaction, hydrophobic interaction, van der Waals force, hydrogen bond and the like.
- the nucleic acid is insolubilized by the chaotropic salt and alcohol, and can be adsorbed on the carrier.
- the alcohol is preferably one that can insolubilize nucleic acids, and examples thereof include ethanol, isopropyl alcohol, n-propanol, n-butanol, 2-butanol, and combinations thereof.
- Chaotropic salts include guanidine hydrochloride, guanidine thiocyanate (guanidine thiocyanate), guanidine sulfate, guanidine isothiocyanate, sodium isothiocyanate, sodium iodide, potassium iodide, urea, sodium bromide, potassium bromide, bromide.
- Examples include calcium, ammonium bromide, sodium perchlorate, sodium thiocyanate, potassium thiocyanate, ammonium isothiocyanate, sodium chloride, potassium chloride, ammonium chloride, combinations thereof and the like.
- the nucleic acid adsorbed on the carrier can be washed and then eluted in the eluate and collected.
- various genetic tests such as identification tests for viruses and pathogens, detection of pathogenic microorganisms in the early stages of infection, investigation of infection sources, and diagnosis of leukemia and tumor genes. .
- the nucleic acid extraction method of the present invention may be controlled and used in an automatic apparatus.
- Magtration registered trademark
- System GC 6GC, 12GC and 12GC PLUS are commercially available (Precision System Science Co., Ltd.), and these can be used.
- Magtration is a coined word for “Magnetic Filtration”, meaning “sieving with magnets”, and is a technology developed to automate the reaction of magnetic particles (magnetic particle handling technology).
- the suspension of magnetic particles is handled by a disposable chip, and by applying a magnet to the outer wall surface of the chip, the magnetic particles can be captured on the inner wall surface of the chip and solid-liquid separation can be performed.
- the present invention also provides a nucleic acid extraction reagent containing an anionic polymer, a chaotropic salt, an alcohol and a carrier.
- anionic polymer The anionic polymer, chaotropic salt, alcohol and carrier are described above.
- the nucleic acid extraction reagent of the present invention may further contain a buffer.
- the buffer include Tris-HCl, TE (Tris-HCl, EDTA), phosphate buffer and the like.
- the nucleic acid extraction reagent of the present invention may further contain a proteolytic enzyme (for example, Proteinase K), a surfactant (for example, sodium dodecyl sulfate), water, an antibiotic (for example, ProClin 300), sodium azide and the like.
- a proteolytic enzyme for example, Proteinase K
- a surfactant for example, sodium dodecyl sulfate
- water for example, an antibiotic (for example, ProClin 300), sodium azide and the like.
- an antibiotic for example, ProClin 300
- sodium azide sodium azide and the like.
- These components can be contained as a single component or a mixture in one or more wells to form a prepacked reagent.
- the prepack reagent may be combined with other consumables (for example, a chip, a chip holder, a screw cap tube, etc.) to form a reagent kit.
- the prepack reagent may be automatically mixed with the sample and each reagent using an automatic device. You may include the automatic process which isolate
- the reagent of the present invention may be a kit enclosed in a cartridge.
- Example 1 It has been found that poly (acrylic acid) (hereinafter referred to as PAAc) is effective as a more effective carrier for recovering viral nucleic acid from serum and physiological saline.
- the nucleic acid extraction step is performed according to the following procedure. (1) Treat a sample containing nucleic acid with a lysis solution containing a guanidine salt. (2) The treated product is mixed with a binding buffer containing alcohol and a carrier having hydroxyl group, carboxyl group or silica on the surface layer. At this time, the nucleic acid binds to the carrier. (3) Separate the carrier and liquid components to obtain only the carrier. (4) Wash the carrier with a buffer containing 2-propanol.
- the carrier is mixed with an eluent (distilled water or Tris-HCl) to obtain nucleic acid in the eluate (hereinafter referred to as “recovered solution”).
- the carrier is added in the above step (2), and the nucleic acid recovery efficiency is improved by the function of assisting and promoting the binding between the nucleic acid and the carrier.
- the nucleic acid extraction step was performed using Magtration System 12GC PLUS (Precision System Science Co., Ltd.).
- M13 DNA real time PCR The nucleic acid extraction operation described above was performed using 200 ⁇ l of virus added to consella or physiological saline as a sample. For each of the collected liquids obtained, real time PCR was performed under the following conditions to obtain Ct values and Tm. As template, extracted M13KO7 DNA or M13KO7 was used. Real time PCR reagent SsoAdvanced SYBR Green SuperMix (BIORAD, 172-5261) was used, and the following primers were used.
- PAAc molecular weight distribution and concentration PAAc (Sigma-Aldrich, 16-1858, 416037, 416029, 81132, 81130) with an average molecular weight of 25000, 15000, 8000, 5100, 2100, 1200, final concentration 1/2000 to 0.1% (w / v) ProClin 300 (Sigma-Aldrich, 48912-U) and 10 mM Tris-HCl (pH 7.5). Nucleic acid extraction operation was performed using each PAAc solution. The obtained recovered solution was subjected to real time PCR according to the PCR conditions described above to obtain a Ct value.
- PAAc with an average molecular weight of 15000, 8000, 2100 is 2, 1, 0.4, 0.2, 0.1, 0.05, 0.025, 0.0125% (w / v) so that the final concentration is 1/2000 ProClin 300 and 10 mM Tris-HCl (pH 7.5). Nucleic acid extraction operation was performed using each PAAc solution. The obtained recovered solution was subjected to real time PCR according to the PCR conditions described above to obtain a Ct value.
- PAAc molecular weight distribution and concentration PAAc molecular weight distribution and concentration
- PAAc with average molecular weights of 1.2k, 2.1k, 5.1k, 8k, 15k, and 25k was adjusted to 0.1% (w / v) respectively with 10 mM Tris HCl (pH 7.5) and ProClin 300 (final concentration 0.05% ( x2,000)) and the effect as a carrier was confirmed by recovering M13 (20 copies / ⁇ l) spiked in physiological saline.
- no molecular weight dependency was observed between molecular weights 1200 and 25000, and it can be said that the carrier effect was exhibited at any molecular weight.
- the Ct value was not obtained when DW was used instead of the carrier.
- the concentration dependence was evaluated with three kinds of molecular weights of 2.1 k, 8 k, and 15 k, the carrier effect was obtained at 0.1-1% (w / v) in all (right side of FIG. 1).
- the PAAc concentration was 0.5% (w / v) or less, the Ct value improved as the PAAc concentration decreased.
- the Ct value was 2 to 3 higher than the case of 0.1 to 1% (w / v) where the carrier effect with the highest Ct value was obtained.
- Example 2 In this example, the effect as a nucleic acid carrier in nucleic acid extraction was evaluated using a polymer having a skeleton similar to sodium polyacrylate or a polymer having different properties.
- the three polymers, Polyanetholesulfonic acid sodium salt, Poly (acrylic acid-co-maleic acid) solution, and Poly-L-glutamic acid sodium salt, which have -COOH as a functional group, are all judged as + and are effective as a carrier effect. It showed that.
- Maleic acid polymers such as poly ethylene-co-ethyl acrylate-co-maleic anhydride and polymalon are ⁇ determined, and even if it is a compound with -COOH, it is not dicarboxyl like maleic acid It became clear that the carrier effect was weak.
- Example 3 Regarding the timing of carrier addition, nucleic acid extraction is performed in two steps: the step of adding PAAc to the mixture just before binding the nucleic acid and carrier (Original) and the step of directly administering PAAc to the sample (Carrier in sample).
- a sample was prepared under the same conditions as in Example 1 except for the administration timing of PAAc, and nucleic acid extraction and PCR were performed.
- the PCR results are shown in Table 7. In the original, the Ct value was 25.03 ⁇ 0.14, whereas when the carrier was added to the sample, 26.79 ⁇ 0.04, indicating no effect as a carrier.
- Example 4 In this reagent composition, alcohol is required to bind the nucleic acid to the carrier. The effect of alcohol use on the need for alcohol was examined.
- the Ct value was 25.03 ⁇ 0.14, but when 2-propanol was not included, 31.91 ⁇ 0.31, which was not effective as a carrier. In order to exert the effect of the carrier, it can be said that it is necessary to contain alcohol as a binding buffer.
- nucleic acid extraction efficiency can be improved in a nucleic acid recovery method using a carrier.
- the present invention can be used for identification tests of viruses and pathogenic bacteria, detection of pathogenic microorganisms at the early stage of infection, investigation of infection sources, and genetic tests such as diagnosis of leukemia and tumor gene levels.
- ⁇ SEQ ID NO: 1> The nucleotide sequence of Forward Primer M13KO7-F 4984 is shown. 5'-GCTATCAGTTCGCGCATTAAAGAC-3 ' ⁇ SEQ ID NO: 2> This shows the nucleotide sequence of Reverse Primer M13KO7-R 5110. 5'-CATTGGCAGATTCACCAGTCACA-3 '
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Abstract
Description
〔1〕カオトロピック塩およびアルコールの存在下、アニオン性高分子を共存させて、核酸を担体に吸着させることを特徴とする核酸抽出方法。
〔2〕アニオン性高分子が、側鎖にカルボキシル基を有するポリマーである〔1〕に記載の核酸抽出方法。
〔3〕アニオン性高分子が、ポリアクリル酸、ポリアクリル酸塩、ポリアクリル酸誘導体、ポリアクリル酸を含むコポリマー、カルボキシル基を有するポリアクリルアミド誘導体、ポリシラン誘導体、カルボキシメチルセルロース、カルボキシメチルセルロース塩、ポリグルタミン酸、ポリグルタミン酸塩、ポリグルタミン酸誘導体及びポリグルタミン酸を含むコポリマーからなる群より選択される少なくとも1種類のポリマーである〔1〕に記載の核酸抽出方法。
〔4〕アルコールが、エタノール、イソプロピルアルコール、n-プロパノール、n-ブタノール及び2-ブタノールからなる群より選択される少なくとも1種類のアルコールである〔1〕1に記載の核酸抽出方法。
〔5〕カオトロピック塩が、塩酸グアニジン、グアニジンチオシアン酸塩(チオシアン酸グアニジン)、グアニジン硫酸塩、イソチオシアン酸グアニジン、イソチアン酸ナトリウム、ヨウ化ナトリウム、ヨウ化カリウム、尿素、臭化ナトリウム、臭化カリウム、臭化カルシウム、臭化アンモニウム、過塩素酸ナトリウム、チオシアン酸ナトリウム、チオシアン酸カリウム、アンモニウムイソチオシアネート、塩化ナトリウム、塩化カリウム及び塩化アンモニウムからなる群より選択される少なくとも1種類のカオトロピック塩である〔1〕に記載の核酸抽出方法。
〔6〕担体が、親水性官能基を表層に有する固体である〔1〕に記載の核酸抽出方法。
〔7〕親水性官能基が、ヒドロキシル基、カルボキシル基、カルボニル基、ニトロ基及びシラノール基からなる群より選択される少なくとも1種類の官能基である〔6〕に記載の方法。
〔8〕担体が磁性粒子である〔6〕に記載の核酸抽出方法。
〔9〕自動装置で制御される〔1〕~〔8〕のいずれかに記載の核酸抽出方法。
〔10〕担体が磁性粒子であり、磁性粒子懸濁がディスポーザブルチップによってハンドリングされ、チップの外壁面に磁石を当てることで磁性粒子をチップ内壁面に補足して、固液分離を行う〔9〕記載の核酸抽出方法。
〔11〕アニオン性高分子、カオトロピック塩、アルコールおよび担体を含む核酸抽出試薬。
〔12〕さらに、バッファーを含む〔11〕に記載の核酸抽出試薬。
〔13〕カートリッジに封入されるキットに含まれる〔11〕又は〔12〕記載の核酸抽出試薬。
本明細書は、本願の優先権の基礎である日本国特許出願、特願2014‐063407の明細書および/または図面に記載される内容を包含する。
本発明の方法は、極めて低濃度の核酸を含む液体からの核酸の抽出、回収を可能とする。
[概要]
血清、生理食塩水からウイルス核酸を回収する為のより効果的なキャリアーとして、ポリアクリル酸(Poly (acrylic acid))(以下、PAAc)が有効であることを見出した。
核酸抽出工程は、次の手順による。(1) 核酸を含む検体をグアニジン塩を含むLysis solutionで処理する。(2) 処理物を表層にヒドロキシル基やカルボキシル基またはシリカを有する担体およびアルコールを含むBinding bufferと混合する。このとき、担体に核酸が結合する。(3) 担体と液体成分を分離し、担体のみを得る。(4) 2-propanolを含む緩衝液を用いて担体を洗浄する。(5) 担体と液体成分を分離し、担体のみを得る。(6) 担体を溶出液(蒸留水またはTris-HCl)と混合し、溶出液中に核酸を得る(以下、「回収液」と記す)。
キャリアーは、上述の工程の(2)で添加し、核酸と担体の結合を補助、促進させる機能によって核酸回収効率を向上させる。
核酸抽出工程は、Magtration System 12GC PLUS(プレシジョン・システム・サイエンス株式会社)を用いて行った。
(キャリアーの評価)
コンセーラ(コントロール血清;ニッスイ)および生理食塩水180 μlに200 copies/μlのM13(大腸菌に感染するバクテリオファージ)をそれぞれ20 μl添加し、20 copies/μlとなった試料から、上記核酸抽出工程により、回収液を得た。このとき、キャリアー0.1 %(w/v)を核酸抽出工程(2)で添加した。得られたそれぞれの回収液から、下記 PCR条件によってreal time PCRでCt値を得た。また、対照として、キャリアーの代わりに蒸留水(DW)を用いて核酸抽出を行った。
(M13 DNAのreal time PCR)
コンセーラもしくは生理食塩水に添加されたウイルス200 μlを試料として、上記の核酸抽出操作を行った。得られたそれぞれの回収液につき、以下の条件で、real time PCRを行い、Ct値およびTmを得た。Templateは、抽出したM13KO7 DNAまたはM13KO7を用いた。
Real time PCR試薬SsoAdvanced SYBR Green SuperMix (BIORAD, 172-5261)を用い、次のプライマーを用いた。Forward Primer M13KO7-F 4984: 5’-GCTATCAGTTCGCGCATTAAAGAC-3’(配列番号1)(eurofins operon)、Reverse Primer M13KO7-R 5110: 5’-CATTGGCAGATTCACCAGTCACA-3’ (配列番号2) (eurofins operon)。Thermal cyclerとして、Applied Biosystems 7500 Fast real time PCRsystem : LifetechnologiesおよびThermal Cycler Dice Real Time System: TaKaRa TP800 S/N R-1387を用いた。PCR時の試薬調製および温度コントロールについて以下のように設定した。Thresholdは、Auto設定を用いた。
平均分子量25000, 15000, 8000, 5100, 2100, 1200のPAAc(Sigma-Aldrich,16-1858, 416037, 416029, 81132, 81130)を、0.1 %(w/v) になるように最終濃度1/2000のProClin 300(Sigma-Aldrich, 48912-U)と10 mM Tris-HCl (pH 7.5)で調製した。各PAAc溶液を用いて、核酸抽出操作を行った。得られた回収液を、上述のPCR条件に従ってreal time PCRを行い、Ct値を得た。
平均分子量15000、8000、2100のPAAcは、2, 1, 0.4, 0.2, 0.1, 0.05, 0.025, 0.0125 %(w/v)になるように最終濃度1/2000のProClin 300と10 mM Tris-HCl (pH 7.5)で調製した。各PAAc溶液を用いて、核酸抽出操作を行った。得られた回収液を、上述のPCR条件に従ってreal time PCRを行い、Ct値を得た。
(PAAc分子量分布と濃度)
PAAc分子量について、平均分子量が1.2k, 2.1k, 5.1k, 8k, 15k, 25kのPAAcをそれぞれ0.1 %(w/v)に10 mM Tris HCl (pH 7.5)とProClin 300 (最終濃度0.05 % (x 2,000))で溶解し、生理食塩水中にスパイクしたM13 (20 copies/μl)の回収によってキャリアーとしての効果を確認した。図1左で示したように、分子量1200から25000の間で、分子量依存性は見られず、いずれの分子量においてもキャリアー効果を発揮したといえる。キャリアーの代わりにDWを用いたものはCt値が得られなかった。
また、分子量2.1k, 8k, 15kの三種で濃度依存性を評価したところ、いずれも0.1-1 %(w/v)でキャリアー効果を得られた(図1右)。PAAc濃度0.5 %(w/v)以下では、PAAc濃度の減少に伴ってCt値が向上した。2 %(w/v)ではCt値が最も高いキャリアー効果を得られた0.1~1 %(w/v)の場合よりもCt値が2~3高くなった。
本実施例では、ポリアクリル酸ナトリウムと類似の骨格を持つ高分子や性質が異なる高分子を用いて、核酸抽出における核酸キャリアーとしての効果を評価した。
Table 1に示した各高分子を0.15 %(w/v)に蒸留水で調製し、PAAcの代わりに使用して、実施例1と同様の方法で核酸抽出操作を行った。
Poly (acrylic acid sodium salt)を含む13種類のポリマーをPoly (acrylic acid sodium salt)の代わりに使用し、核酸抽出を行った。Real-time PCRからCt値を得た(Table 2, 図2)。各高分子を用いて得られたCt値とキャリアーを用いずに抽出操作を行って得られたCt値(Ct without carrier)の差を算出した。また、この差が-1.0以下で、キャリアー効果を+、-1.0~-0.5を+w、-0.5~+0.5を±、+0.5以上を-と判定した。
Table 2の結果から、-COOHを官能基とするPoly (acrylic acid sodium salt)とCarboxy methyl cellulose sodium saltで+と判定され、高いCarrier効果が得られた。アミノ基を多く持ち水溶液中で正電荷に帯電するPoly ethylene imineとpoly-L-lysineは、核酸の回収を阻害した。その他の骨格を持つものは核酸の回収に大きく影響しなかった。
さらに、水溶液中で負電荷に帯電するポリマーを用いて、同様にCt値を得た(Table 3, 図3)。同じく-COOHを官能基として持つPolyanetholesulfonic acid sodium salt、Poly(acrylic acid-co-maleic acid) solution、Poly-L-glutamic acid sodium saltの3つの高分子はいずれも+として判定され、キャリアー効果として有効であることを示した。Poly ethylene-co-ethyl acrylate-co-maleic anhydrideやポリマロンのようなマレイン酸ポリマーは、±判定であり、-COOHを有する化合物であったとしてもマレイン酸のようにジカルボキシとなっているものではキャリアー効果が弱いことが明確となった。
キャリアーを添加するタイミングについて、PAAcを核酸と担体を結合させる直前で混合液に加えた工程(Original)、サンプルにPAAcを直接投与した工程(Carrier in sample)の2つの工程で核酸抽出操作を行い、PCRによって標的遺伝子を増幅した(N=3)。PAAcの投与タイミング以外については、実施例1と同じ条件で、試料を準備し、核酸抽出、PCRを行った。
PCR結果を表7に示した。Originalでは、Ct値が25.03±0.14だったのに対し、キャリアーを試料に添加した場合、26.79±0.04と、キャリアーとして効果を示さなかった。
本試薬組成において、担体に核酸を結合させるためにはアルコールを必要とする。キャリアーの使用によって、アルコールの必要性に対する影響を検討した。キャリアーが核酸回収能力をサポートする機能を発揮する条件として、使用するBinding buffer中の2-propanolを含む工程(Original)、含まない工程(Binding buffer without 2-propanol)でそれぞれ核酸抽出操作を行い、PCRによって標的遺伝子を増幅した(N=3)。2-propanolの有無以外については、実施例1と同じ条件で、試料を準備し、核酸抽出、PCRを行った。
PCR結果を表8に示した。Originalでは、Ct値が25.03±0.14だったのに対し、2-propanolを含まない場合、31.91±0.31と、キャリアーとして効果を示さなかった。キャリアーの効果を発揮する為には、Binding bufferとしてアルコールを含むことが必要であるといえる。
Forward Primer M13KO7-F 4984のヌクレオチド配列を示す。
5’-GCTATCAGTTCGCGCATTAAAGAC-3’
<配列番号2>
Reverse Primer M13KO7-R 5110のヌクレオチド配列を示す。
5’-CATTGGCAGATTCACCAGTCACA-3’
Claims (13)
- カオトロピック塩およびアルコールの存在下、アニオン性高分子を共存させて、核酸を担体に吸着させることを特徴とする核酸抽出方法。
- アニオン性高分子が、側鎖にカルボキシル基を有するポリマーである請求項1に記載の核酸抽出方法。
- アニオン性高分子が、ポリアクリル酸、ポリアクリル酸塩、ポリアクリル酸誘導体、ポリアクリル酸を含むコポリマー、カルボキシル基を有するポリアクリルアミド誘導体、ポリシラン誘導体、カルボキシメチルセルロース、カルボキシメチルセルロース塩、ポリグルタミン酸、ポリグルタミン酸塩、ポリグルタミン酸誘導体及びポリグルタミン酸を含むコポリマーからなる群より選択される少なくとも1種類のポリマーである請求項1に記載の核酸抽出方法。
- アルコールが、エタノール、イソプロピルアルコール、n-プロパノール、n-ブタノール及び2-ブタノールからなる群より選択される少なくとも1種類のアルコールである請求項1に記載の核酸抽出方法。
- カオトロピック塩が、塩酸グアニジン、グアニジンチオシアン酸塩(チオシアン酸グアニジン)、グアニジン硫酸塩、イソチオシアン酸グアニジン、イソチアン酸ナトリウム、ヨウ化ナトリウム、ヨウ化カリウム、尿素、臭化ナトリウム、臭化カリウム、臭化カルシウム、臭化アンモニウム、過塩素酸ナトリウム、チオシアン酸ナトリウム、チオシアン酸カリウム、アンモニウムイソチオシアネート、塩化ナトリウム、塩化カリウム及び塩化アンモニウムからなる群より選択される少なくとも1種類のカオトロピック塩である請求項1に記載の核酸抽出方法。
- 担体が、親水性官能基を表層に有する固体である請求項1に記載の核酸抽出方法。
- 親水性官能基が、ヒドロキシル基、カルボキシル基、カルボニル基、ニトロ基及びシラノール基からなる群より選択される少なくとも1種類の官能基である請求項6に記載の方法。
- 担体が磁性粒子である請求項6に記載の核酸抽出方法。
- 自動装置で制御される請求項1~8のいずれかに記載の核酸抽出方法。
- 担体が磁性粒子であり、磁性粒子懸濁がディスポーザブルチップによってハンドリングされ、チップの外壁面に磁石を当てることで磁性粒子をチップ内壁面に補足して、固液分離を行う請求項9記載の核酸抽出方法。
- アニオン性高分子、カオトロピック塩、アルコールおよび担体を含む核酸抽出試薬。
- さらに、バッファーを含む請求項11に記載の核酸抽出試薬。
- カートリッジに封入されるキットに含まれる請求項11又は12記載の核酸抽出試薬。
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