JPWO2016052386A1 - Nucleic acid separation and purification method and solid phase carrier, device and kit - Google Patents
Nucleic acid separation and purification method and solid phase carrier, device and kit Download PDFInfo
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- JPWO2016052386A1 JPWO2016052386A1 JP2016552006A JP2016552006A JPWO2016052386A1 JP WO2016052386 A1 JPWO2016052386 A1 JP WO2016052386A1 JP 2016552006 A JP2016552006 A JP 2016552006A JP 2016552006 A JP2016552006 A JP 2016552006A JP WO2016052386 A1 JPWO2016052386 A1 JP WO2016052386A1
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- Prior art keywords
- nucleic acid
- solid phase
- phase carrier
- separating
- purifying
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- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 175
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- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 description 2
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- ZZTURJAZCMUWEP-UHFFFAOYSA-N diaminomethylideneazanium;hydrogen sulfate Chemical compound NC(N)=N.OS(O)(=O)=O ZZTURJAZCMUWEP-UHFFFAOYSA-N 0.000 description 1
- HNPSIPDUKPIQMN-UHFFFAOYSA-N dioxosilane;oxo(oxoalumanyloxy)alumane Chemical compound O=[Si]=O.O=[Al]O[Al]=O HNPSIPDUKPIQMN-UHFFFAOYSA-N 0.000 description 1
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- 150000002431 hydrogen Chemical class 0.000 description 1
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M1/00—Apparatus for enzymology or microbiology
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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
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Sustainable Development (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
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- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
【課題】核酸の回収率と通液性に優れた核酸の分離精製方法を提供する。【解決手段】少なくとも（１）多孔性無機粒子と（２）有機または無機繊維と（３）有機バインダーとからなる組成物を含む固相担体を用い、少なくとも下記工程（Ａ）から（Ｄ）をこの順に経ることを特徴とする核酸の分離精製方法。（Ａ）核酸を含む試料溶液と結合液とを混合して混合液を得る工程（Ｂ）前記混合液と固相担体とを接触させ、固相担体に核酸を吸着させる工程（Ｃ）固相担体から核酸以外の成分を洗浄する工程（Ｄ）固相担体から核酸を脱着させる工程【選択図】なしProvided is a method for separating and purifying nucleic acid, which is excellent in nucleic acid recovery and liquid permeability. At least the following steps (A) to (D) are performed using a solid phase carrier comprising a composition comprising at least (1) porous inorganic particles, (2) organic or inorganic fibers, and (3) an organic binder. A method for separating and purifying nucleic acid, which is performed in this order. (A) A step of mixing a sample solution containing nucleic acid and a binding solution to obtain a mixed solution (B) A step of bringing the mixed solution into contact with a solid phase carrier and adsorbing the nucleic acid to the solid phase carrier (C) A solid phase Step of washing components other than nucleic acid from carrier (D) Step of desorbing nucleic acid from solid phase carrier [selection figure] None
Description
本発明は、核酸を分離精製する方法に関する。 The present invention relates to a method for separating and purifying nucleic acid.
遺伝子検査はゲノム解析技術の進歩により大きな注目を浴びてきている。特に、近年では個別化医療の普及や感染症領域におけるＰＯＣＴ（臨床現場即時検査）など遺伝子検査に関連する市場が飛躍的に伸長し、遺伝子検査をさらに普及させると期待されている。 Genetic testing has received much attention due to advances in genome analysis technology. In particular, in recent years, the market related to genetic testing such as the spread of personalized medicine and POCT (immediate clinical on-site testing) in the field of infectious diseases is expected to grow dramatically, and it is expected that genetic testing will be further spread.
遺伝子検査を行なうには、初めに核酸を含む試料溶液から核酸を分離精製することが必要である。従来、核酸を含む試料溶液から核酸を得る方法としては、フェノール・クロロホルム法という技術が知られていた。この方法はフェノール・クロロホルムを用いてタンパク質、脂質などの夾雑物を不溶化させ、核酸を分離精製する方法である。かかる従来技術は、大量の核酸を安価に分離精製できるが、操作が煩雑であり、核酸の回収率も低く、フェノール・クロロホルムが最終精製物にコンタミするという問題点があった。また、フェノール・クロロホルム自体も有毒であるため、作業環境に制限があるという問題点があった。 In order to perform a genetic test, it is necessary to first separate and purify nucleic acid from a sample solution containing the nucleic acid. Conventionally, a technique called a phenol / chloroform method has been known as a method for obtaining nucleic acid from a sample solution containing nucleic acid. In this method, phenol / chloroform is used to insolubilize impurities such as proteins and lipids, and nucleic acids are separated and purified. Although this conventional technique can separate and purify a large amount of nucleic acid at low cost, there are problems in that the operation is complicated, the recovery rate of nucleic acid is low, and phenol / chloroform is contaminated in the final purified product. In addition, phenol / chloroform itself is toxic, so there is a problem that the working environment is limited.
一方、かかる問題点を解消すべく所謂Ｂｏｏｍ法という発明がなされた（例えば、特許文献１、非特許文献１参照）。この方法は、カオトロピック物質を利用してタンパク質、脂質などの夾雑物を可溶化し、核酸を固相担体に吸着させて回収後、核酸を再び溶解することで、核酸を分離精製する方法である。前記Ｂｏｏｍ法で用いられる固相担体としては、シリカフィルター（例えば、特許文献２、３参照）、シリカ磁性ビーズ（例えば、特許文献４〜６参照）などの様々な態様が知られている。 On the other hand, an invention called the “Boom method” has been made to solve such problems (see, for example, Patent Document 1 and Non-Patent Document 1). This method is a method for separating and purifying nucleic acids by solubilizing proteins, lipids and other contaminants using a chaotropic substance, adsorbing the nucleic acids to a solid support, recovering them, and then dissolving the nucleic acids again. . As the solid phase carrier used in the boom method, various modes such as a silica filter (see, for example, Patent Documents 2 and 3) and silica magnetic beads (for example, see Patent Documents 4 to 6) are known.
シリカフィルターを用いることで、迅速・簡便に核酸を分離精製できるが、検体（特に、血液などの高粘性液体）によっては、工程中に固相担体の詰まりが発生するという問題点があった。また、固相担体を通液させるために、１０，０００Ｇ以上の高い遠心力が必要となるため、自動化が困難であり、かつ得られる核酸が物理的な外力により断片化し易いという問題点もあった。 By using a silica filter, nucleic acids can be separated and purified quickly and easily. However, depending on the specimen (especially high-viscosity liquid such as blood), there is a problem that clogging of the solid phase carrier occurs during the process. Moreover, since a high centrifugal force of 10,000 G or more is required for passing the solid phase carrier, there is a problem that automation is difficult and the obtained nucleic acid is easily fragmented by physical external force. It was.
一方、シリカ磁性ビーズを用いることで、固相担体の詰まりの問題は解決でき、自動化も容易であるが、核酸の回収率が低下するという問題点があった。また、除去しきれないシリカ磁性粒子が最終精製物にコンタミするという問題点があった。 On the other hand, by using silica magnetic beads, the problem of clogging of the solid phase carrier can be solved and automation is easy, but there is a problem that the recovery rate of nucleic acid is lowered. There is also a problem that silica magnetic particles that cannot be completely removed are contaminated with the final purified product.
本発明は、かかる従来技術の課題を背景になされたものである。すなわち、本発明の目的は、核酸の回収率と通液性に優れた核酸の分離精製方法を提供することにある。 The present invention has been made against the background of such prior art problems. That is, an object of the present invention is to provide a method for separating and purifying nucleic acid, which is excellent in nucleic acid recovery and liquid permeability.
本発明者らは、（２）有機または無機繊維と（３）有機バインダーとを含む組成物から抄紙される固相担体（例えばシリカフィルター）を用いると、核酸の回収率を上げるために高目付けにせざるを得ず、高目付けにすると通液性が低下する、つまり核酸の回収率と通液性はトレードオフの関係にあることを見出した。そこで本発明者らは、（１）多孔性無機粒子と（２）有機または無機繊維と（３）有機バインダーとからなる組成物を含む固相担体を用いることで、低目付けでも核酸の回収率を保持できる、つまり核酸の回収率と通液性の両立が可能であることを見出し、本発明に至った。 The present inventors use a solid phase carrier (for example, a silica filter) made from a composition containing (2) an organic or inorganic fiber and (3) an organic binder to increase the nucleic acid recovery rate. In other words, it was found that the liquid permeability decreases when the basis weight is increased, that is, the nucleic acid recovery rate and the liquid permeability are in a trade-off relationship. Therefore, the present inventors use (1) porous inorganic particles, (2) organic or inorganic fibers, and (3) a solid phase carrier containing a composition comprising an organic binder, so that the nucleic acid recovery rate can be reduced even at low weight. Has been found, that is, it is possible to achieve both recovery of nucleic acid and liquid permeability.
また、本発明者らは、（３）有機バインダーとして、水系のろ過に一般的に用いられる親水性のバインダーを用いると、核酸の回収率が低下することを見出した。そこで本発明者らは、（３）有機バインダーとしてポリエチレン系ポリマーまたはポリエステル系ポリマー等の疎水性バインダーを用いることで、核酸の回収率がさらに向上することを見出し、本発明を完成させた。 In addition, the present inventors have found that when a hydrophilic binder generally used for aqueous filtration is used as the organic binder (3), the nucleic acid recovery rate decreases. Thus, the present inventors have found that the nucleic acid recovery rate is further improved by using a hydrophobic binder such as polyethylene polymer or polyester polymer as the organic binder (3), and completed the present invention.
すなわち、本発明は、以下の構成からなる。
１．少なくとも（１）多孔性無機粒子と（２）有機または無機繊維と（３）有機バインダーとからなる組成物を含む固相担体を用い、少なくとも下記工程（Ａ）から（Ｄ）をこの順に経ることを特徴とする核酸の分離精製方法。
（Ａ）核酸を含む試料溶液と結合液とを混合して混合液を得る工程
（Ｂ）前記混合液と固相担体とを接触させ、固相担体に核酸を吸着させる工程
（Ｃ）固相担体から核酸以外の成分を洗浄する工程
（Ｄ）固相担体から核酸を脱着させる工程
２．前記有機バインダーがポリエチレン系ポリマーまたはポリエステル系ポリマーを含むものである、１に記載の核酸の分離精製方法。
３．前記多孔性無機粒子が多孔性シリカ粒子である、１または２に記載の核酸の分離精製方法。
４．前記多孔性無機粒子の固相担体に含まれる量が１〜６０重量％である、１〜３のいずれかに記載の核酸の分離精製方法。
５．前記多孔性無機粒子の形状が球状である、１〜４のいずれかに記載の核酸の分離精製方法。
６．前記多孔性無機粒子の平均粒子径が１〜５０μｍである、１〜５のいずれかに記載の核酸の分離精製方法。
７．前記有機または無機繊維がセルロース繊維またはガラス繊維である、１〜６のいずれかに記載の核酸の分離精製方法。
８．前記固相担体の目付けが、２５〜２００ｇ／ｍ２である、１〜７のいずれかに記載の核酸の分離精製方法。
９．前記固相担体の厚みが１００〜５００μｍである、１〜８のいずれかに記載の核酸の分離精製方法。
１０．前記固相担体が湿式抄紙法により製造されたものである、１〜９のいずれかに記載の核酸の分離精製方法。
１１．前記核酸を含む試料溶液が血液である、１〜１０のいずれかに記載の核酸の分離精製方法。
１２．核酸の分離精製方法に用いるための固相担体であって、（１）多孔性無機粒子と（２）有機または無機繊維と（３）疎水性の有機バインダーとからなる組成物を含む固相担体。
１３．核酸の分離精製方法に用いるためのデバイスであって、１２に記載の固相担体を保持しているデバイス。
１４．核酸の分離精製方法を行うための核酸の分離精製キットであって、１２に記載の固相担体または１３に記載のデバイス、固相担体に核酸を吸着させるための結合液、固相担体から核酸以外の成分を洗浄するための洗浄液、固相担体から核酸を溶出するための溶出液を含む、核酸の分離精製キット。That is, this invention consists of the following structures.
1. Using at least the following steps (A) to (D) in this order, using a solid phase carrier comprising a composition comprising at least (1) porous inorganic particles, (2) organic or inorganic fibers, and (3) an organic binder. A method for separating and purifying nucleic acid characterized by the above.
(A) A step of mixing a sample solution containing nucleic acid and a binding solution to obtain a mixed solution (B) A step of bringing the mixed solution into contact with a solid phase carrier and adsorbing the nucleic acid to the solid phase carrier (C) A solid phase 1. Step for washing components other than nucleic acid from carrier (D) Step for desorbing nucleic acid from solid phase carrier 2. The method for separating and purifying nucleic acid according to 1, wherein the organic binder contains a polyethylene polymer or a polyester polymer.
3. 3. The method for separating and purifying nucleic acid according to 1 or 2, wherein the porous inorganic particles are porous silica particles.
4). The method for separating and purifying nucleic acid according to any one of 1 to 3, wherein the amount of the porous inorganic particles contained in the solid phase carrier is 1 to 60% by weight.
5. The method for separating and purifying nucleic acid according to any one of 1 to 4, wherein the porous inorganic particles have a spherical shape.
6). The method for separating and purifying nucleic acid according to any one of 1 to 5, wherein the porous inorganic particles have an average particle diameter of 1 to 50 µm.
7). The method for separating and purifying nucleic acid according to any one of 1 to 6, wherein the organic or inorganic fiber is a cellulose fiber or a glass fiber.
8). The method for separating and purifying nucleic acid according to any one of 1 to 7, wherein the basis weight of the solid phase carrier is 25 to 200 g / m 2 .
9. The method for separating and purifying nucleic acid according to any one of 1 to 8, wherein the solid phase carrier has a thickness of 100 to 500 µm.
10. The method for separating and purifying nucleic acid according to any one of 1 to 9, wherein the solid phase carrier is produced by a wet papermaking method.
11. The method for separating and purifying nucleic acid according to any one of 1 to 10, wherein the sample solution containing the nucleic acid is blood.
12 A solid phase carrier for use in a method for separating and purifying nucleic acid, comprising a composition comprising (1) porous inorganic particles, (2) organic or inorganic fibers, and (3) a hydrophobic organic binder .
13. A device for use in a method for separating and purifying nucleic acid, the device holding the solid phase carrier according to 12.
14 A nucleic acid separation and purification kit for performing a method for separating and purifying nucleic acid, the solid phase carrier according to 12, or the device according to 13, a binding solution for adsorbing the nucleic acid to the solid phase carrier, the nucleic acid from the solid phase carrier A kit for separating and purifying nucleic acid, comprising a washing solution for washing components other than those and an elution solution for eluting nucleic acid from a solid phase carrier.
本発明により、核酸を分離精製する方法において、高い核酸の回収率と高い固相担体の通液性を両立することができる。本発明を用いれば、血液などの粘性の高い生体由来物質からも低い遠心力により核酸を分離精製することができる。 According to the present invention, in a method for separating and purifying nucleic acid, it is possible to achieve both high recovery rate of nucleic acid and high liquid permeability of a solid phase carrier. By using the present invention, nucleic acids can be separated and purified from a highly viscous biological material such as blood with a low centrifugal force.
以下、本発明を詳述する。 The present invention is described in detail below.
（分離精製の対象となる核酸）
本発明における分離精製の対象となる核酸の種類としては、特に限定されないが、例えば、ＤＮＡまたはＲＮＡ、１本鎖核酸または２本鎖核酸、直鎖状核酸または環状核酸が挙げられる。これらの中で、本発明は物理的な外力により極めて分解しやすいＲＮＡの分離精製に用いることができ、ｔｏｔａｌ ＲＮＡの分離精製に好適に用いられる。(Nucleic acids to be separated and purified)
The type of nucleic acid to be separated and purified in the present invention is not particularly limited, and examples thereof include DNA or RNA, single-stranded nucleic acid or double-stranded nucleic acid, linear nucleic acid, or circular nucleic acid. Among these, the present invention can be used for separation and purification of RNA that is extremely easily degraded by physical external force, and is preferably used for separation and purification of total RNA.
（核酸の分離精製原理）
本発明における核酸の分離精製の原理は、ＢＯＯＭ法（非特許文献１）として広く知られているシリカを用いた核酸抽出方法と同様の原理で、核酸の固相担体への特異的な結合、洗浄および溶出により核酸を分離精製する。
具体的には、
工程（Ａ）：混合工程において核酸を含む試料と結合液とを混合し、
工程（Ｂ）：結合工程において前記混合液と固相担体とを接触させ、固相担体に核酸を吸着させ、
工程（Ｃ）：洗浄工程において洗浄液と固相担体とを接触させ、固相担体から核酸以外の成分を洗浄し、
工程（Ｄ）：溶出工程において溶出液と固相担体とを接触させ、固相担体から核酸を脱着させることで、核酸を含む試料から核酸を分離精製することができる。(Principle of separation and purification of nucleic acid)
The principle of separation and purification of nucleic acid in the present invention is the same principle as the nucleic acid extraction method using silica widely known as the BOOM method (Non-patent Document 1), and specific binding of nucleic acid to a solid phase carrier, The nucleic acid is separated and purified by washing and elution.
In particular,
Step (A): mixing the sample containing nucleic acid and the binding solution in the mixing step,
Step (B): In the binding step, the mixed solution and the solid phase carrier are contacted, and the nucleic acid is adsorbed on the solid phase carrier,
Step (C): In the washing step, the washing liquid and the solid phase carrier are brought into contact with each other to wash components other than the nucleic acid from the solid phase carrier,
Step (D): In the elution step, the eluate and the solid phase carrier are brought into contact with each other, and the nucleic acid is desorbed from the solid phase carrier, whereby the nucleic acid can be separated and purified from the sample containing the nucleic acid.
前記工程（Ｂ）〜（Ｄ）では、核酸の吸着・洗浄・溶出のため、混合液・洗浄液・溶出液などの各種溶液に物理的な外力を加えて固相担体を通液させる。例えば、代表的な核酸抽出キットであるＱＩＡａｍｐ、ＲＮｅａｓｙ（共にキアゲン社）では、底面に固相担体を固定したスピンカラムを用い、スピンカラムの上部に各種溶液を添加し、遠心分離機（例えば、８０００〜１５０００Ｇ）により上から下へ通液させる方法が用いられる。 In the steps (B) to (D), in order to adsorb, wash, and elute nucleic acids, a solid external carrier is allowed to flow by applying physical external force to various solutions such as a mixed solution, a washing solution, and an eluting solution. For example, QIAamp and RNeasy (both Qiagen), which are typical nucleic acid extraction kits, use a spin column with a solid support fixed to the bottom, add various solutions to the top of the spin column, and centrifuge (for example, 8000 to 15000 G) is used.
本発明の核酸分離精製方法を用いることにより、弱い外力をかけるだけで核酸の分離精製を行うことができる。例えば、外力が遠心力の場合、その程度は特に限定されないが、４０００Ｇ以下の遠心力により核酸の分離精製を行うことが好ましく、３０００Ｇ以下がより好ましく、２０００Ｇ以下がさらに好ましい。４０００Ｇ超の遠心分離操作を行おうとすると、一般的に大型の遠心分離機が必要となり、迅速・簡便な核酸抽出を行なうのが困難となる。また、物理的なダメージにより、核酸が分解する恐れもある。
具体的には、４０００Ｇ以下の遠心力で遠心分離を行うことができる卓上小型遠心機（例えば、チビタン（登録商標））の使用や、シリンジ、ピペットなどの空気の圧力変化により吸引および吐出を行なう装置の使用が好ましい。前記方法において遠心力の下限は特に限定されないが、１０００Ｇ以上が好ましい。By using the nucleic acid separation and purification method of the present invention, nucleic acid can be separated and purified only by applying a weak external force. For example, when the external force is a centrifugal force, the degree is not particularly limited, but the nucleic acid is preferably separated and purified by a centrifugal force of 4000 G or less, more preferably 3000 G or less, and even more preferably 2000 G or less. When a centrifugal operation exceeding 4000 G is to be performed, a large centrifugal separator is generally required, and it is difficult to perform quick and simple nucleic acid extraction. In addition, the nucleic acid may be decomposed due to physical damage.
Specifically, suction and discharge are performed by using a desktop small centrifuge (for example, Chibitan (registered trademark)) capable of performing centrifugation with a centrifugal force of 4000 G or less, or by changing the pressure of air such as a syringe or pipette. The use of a device is preferred. In the above method, the lower limit of the centrifugal force is not particularly limited, but 1000 G or more is preferable.
前記固相担体を固定する容器としては、遠心機を使用する場合はスピンカラムが、シリンジ、ピペットなどの空気の圧力変化により吸引および吐出を行なう装置を使用する場合は、ピペットチップもしくはそれに類似する形状の容器（例えば、ルアーフィッティング）が適している。ピペットチップの形状は、特に限定されないが、一般的に利用されている円錐形で先が切断されており筒状のチップが好ましい。当然ながら、ピペットチップに限らず、吸引吐出機構とのかん合部に密着できる形状であれば良く、所望の形状が選択できる。 The container for fixing the solid phase carrier is a spin column when using a centrifuge, or a pipette tip or similar when using a device that performs suction and discharge by changing the pressure of air, such as a syringe or pipette. Shaped containers (eg, luer fittings) are suitable. The shape of the pipette tip is not particularly limited, but the tip is cut in a generally used conical shape, and a cylindrical tip is preferable. Needless to say, the shape is not limited to the pipette tip, and any shape can be selected as long as it can be in close contact with the mating portion with the suction / discharge mechanism.
本発明において４０００Ｇ以下の遠心力で核酸の分離精製を行うためには、固相担体として少なくとも（１）多孔性無機粒子と（２）有機または無機繊維と（３）有機バインダーとからなる組成物を含む固相担体を用いる。一般的に固相担体として用いられるシリカフィルターを用いると、核酸の回収率には優れるものの、４０００Ｇを超える遠心力での遠心工程が必要不可欠となり、大型の遠心分離機が必要となる。また核酸を含む試料の種類によってはフィルターに詰まりが発生する恐れがある。本発明においては、抄紙が好ましい。 In the present invention, in order to perform separation and purification of nucleic acids with a centrifugal force of 4000 G or less, a composition comprising at least (1) porous inorganic particles, (2) organic or inorganic fibers, and (3) an organic binder as a solid phase carrier. Is used. When a silica filter generally used as a solid support is used, although a nucleic acid recovery rate is excellent, a centrifugation step with a centrifugal force exceeding 4000 G is indispensable, and a large-sized centrifuge is required. In addition, depending on the type of sample containing nucleic acid, the filter may be clogged. In the present invention, papermaking is preferred.
前記多孔性無機粒子の組成は、核酸を吸着し得るものであれば特に限定されず、例えばシリカ、ガラス、アルミナ、ゼオライト、粘土鉱物などが挙げられる。これらの中でも、シリカが好ましく、多孔性シリカ粒子がより好ましい。 The composition of the porous inorganic particles is not particularly limited as long as it can adsorb nucleic acid, and examples thereof include silica, glass, alumina, zeolite, and clay mineral. Among these, silica is preferable and porous silica particles are more preferable.
前記多孔性無機粒子の固相担体に含まれる量の下限は１重量％であることが好ましく、２重量％がより好ましく、３重量％がさらに好ましい。一方、上限は６０重量％であることが好ましく、４０重量％がより好ましく、２０重量％がさらに好ましい。固相担体に含まれる量が１重量％より少ないと、核酸の回収率が低下する恐れがある。一方、固相担体に含まれる量が６０重量％より多いと固相担体の強度が低下し、核酸の分離精製中に破れが発生する恐れがある。また、通液性が低下し、詰まりが発生する恐れがある。 The lower limit of the amount of the porous inorganic particles contained in the solid phase carrier is preferably 1% by weight, more preferably 2% by weight, and even more preferably 3% by weight. On the other hand, the upper limit is preferably 60% by weight, more preferably 40% by weight, and even more preferably 20% by weight. If the amount contained in the solid support is less than 1% by weight, the nucleic acid recovery rate may decrease. On the other hand, when the amount contained in the solid phase carrier is more than 60% by weight, the strength of the solid phase carrier is lowered, and there is a possibility that tearing may occur during the separation and purification of the nucleic acid. In addition, liquid permeability may be reduced and clogging may occur.
前記多孔性無機粒子の形状は、球状であることが好ましい。形状が破砕状やシート状であると、核酸の回収率が低下する恐れがある。また、通液性が低下し、詰まりが発生する恐れがある。 The shape of the porous inorganic particles is preferably spherical. If the shape is crushed or sheet-like, the nucleic acid recovery rate may decrease. In addition, liquid permeability may be reduced and clogging may occur.
前記多孔性無機粒子の平均粒子径の下限は、１μｍであることが好ましく、２μｍがより好ましく、３μｍがさらに好ましい。一方、上限は５０μｍであることが好ましく、３０μｍがより好ましく、２０μｍがさらに好ましい。平均粒子径が１μｍより小さいと、通液性が低下し、詰まりが発生する恐れがある。一方、平均粒子径が５０μｍより大きいと、固相担体の強度が著しく低下し、核酸の分離精製中に破れが発生する恐れがある。 The lower limit of the average particle size of the porous inorganic particles is preferably 1 μm, more preferably 2 μm, and even more preferably 3 μm. On the other hand, the upper limit is preferably 50 μm, more preferably 30 μm, and even more preferably 20 μm. When the average particle size is smaller than 1 μm, liquid permeability is lowered and clogging may occur. On the other hand, when the average particle diameter is larger than 50 μm, the strength of the solid phase carrier is remarkably lowered, and there is a possibility that the breakage may occur during the separation and purification of the nucleic acid.
前記有機または無機繊維は特に限定されないが、例えばセルロースなどのような有機繊維またはガラスなどのような無機繊維からなることが好ましく、ガラス繊維がより好ましい。 The organic or inorganic fibers are not particularly limited, but are preferably made of organic fibers such as cellulose or inorganic fibers such as glass, and glass fibers are more preferable.
前記有機バインダーは特に限定されないが、例えばポリエチレン系ポリマーまたはポリエステル系ポリマー等の疎水性バインダーであることが好ましく、ポリエステル系ポリマーを含むバインダーであることがより好ましい。 The organic binder is not particularly limited, but is preferably a hydrophobic binder such as a polyethylene polymer or a polyester polymer, and more preferably a binder containing a polyester polymer.
前記固相担体の目付けは特に限定されないが、その下限は２５ｇ／ｍ２であることが好ましく、５０ｇ／ｍ２がより好ましい。一方、上限は２００ｇ／ｍ２であることが好ましく、１５０ｇ／ｍ２がより好ましい。目付けが２５ｇ／ｍ２より小さいと、固相担体の強度が低下し、核酸の分離精製中に破れが発生する恐れがある。また、核酸の回収率が低下する恐れがある。一方、目付けが２００ｇ／ｍ２より大きいと、通液性が低下し、詰まりが発生する恐れがある。なお、固相担体は複数枚重ねて使用してもよく、複数枚重ねた際は全体の目付けを前記固相担体の目付けとする。The basis weight of the solid phase carrier is not particularly limited, but the lower limit is preferably 25 g / m 2 and more preferably 50 g / m 2 . On the other hand, the upper limit is preferably 200 g / m 2 and more preferably 150 g / m 2 . When the basis weight is less than 25 g / m 2 , the strength of the solid phase carrier is lowered, and there is a possibility that tearing may occur during the separation and purification of the nucleic acid. In addition, the nucleic acid recovery rate may be reduced. On the other hand, if the basis weight is larger than 200 g / m 2 , the liquid permeability is lowered and clogging may occur. Note that a plurality of solid phase carriers may be used in a stacked manner, and when a plurality of the solid phase carriers are stacked, the overall basis weight is the basis weight of the solid phase carrier.
前記固相担体の厚みは特に限定されないが、その下限は１００μｍであることが好ましく、２００μｍがより好ましい。一方、上限は５００μｍであることが好ましく、４００μｍがより好ましい。厚みが１００μｍより薄いと、固相担体の強度が低下し、核酸の分離精製中に破れが発生する恐れがある。また、核酸の回収率が低下する恐れがある。一方、厚みが５００μｍより厚いと、通液性が低下し、詰まりが発生する恐れがある。なお、固相担体は複数枚重ねて使用してもよく、複数枚重ねた際は全体の厚みを前記固相担体の厚みとする。 The thickness of the solid support is not particularly limited, but the lower limit is preferably 100 μm, and more preferably 200 μm. On the other hand, the upper limit is preferably 500 μm, more preferably 400 μm. When the thickness is less than 100 μm, the strength of the solid phase carrier is lowered, and there is a possibility that tearing may occur during separation and purification of the nucleic acid. In addition, the nucleic acid recovery rate may be reduced. On the other hand, if the thickness is greater than 500 μm, the liquid permeability is lowered and clogging may occur. In addition, a plurality of solid phase carriers may be used in a stacked manner, and when a plurality of solid phase carriers are stacked, the total thickness is set to the thickness of the solid phase carrier.
前記担体の形態の一例を図３に示す。図３では、黒丸で示される「多孔性無機粒子」と、線で示される「有機または無機繊維」とが、有機バインダーで結合した態様を例示している。「多孔性無機粒子」および「有機または無機繊維」等の密度は、製造時のそれぞれの濃度を適宜設定することにより調整することが可能である。したがって当然であるが、前記担体の形態は図３に示されるものに限定されない。 An example of the form of the carrier is shown in FIG. FIG. 3 illustrates an example in which “porous inorganic particles” indicated by black circles and “organic or inorganic fibers” indicated by lines are bonded with an organic binder. The density of “porous inorganic particles” and “organic or inorganic fibers” can be adjusted by appropriately setting the respective concentrations at the time of production. Therefore, as a matter of course, the form of the carrier is not limited to that shown in FIG.
前記固相担体を製造する方法は特に限定されないが、例えば湿式抄紙法により製造することが好ましい。 The method for producing the solid support is not particularly limited, but for example, it is preferably produced by a wet papermaking method.
以下、各工程について詳述する。 Hereinafter, each process is explained in full detail.
（Ａ：混合工程）
本発明の工程（Ａ）：混合工程は、核酸を含む試料と結合液とを混合する工程である。(A: mixing process)
Step (A) of the present invention: The mixing step is a step of mixing a sample containing nucleic acid and a binding solution.
本発明の混合工程で用いる核酸を含む試料としては、特に限定されず、例えば血液、血清、血球、髄液、喀痰、尿、胃液、鼻汁、糞便、精液、唾液、咽頭ぬぐい液、鼻腔ぬぐい液、鼻腔吸引液などの生体試料が挙げられる。また、培養した組織、細胞、細菌、ウイルスなども挙げられる。さらには、別な方法で精製した核酸についても該当する。これらの中でも、本発明は比較的カラム詰まりの発生しやすい血液からの核酸の分離精製に用いるのが好ましい。 The sample containing the nucleic acid used in the mixing step of the present invention is not particularly limited. For example, blood, serum, blood cells, cerebrospinal fluid, sputum, urine, gastric juice, nasal discharge, feces, semen, saliva, throat swab, nasal rinse And biological samples such as nasal aspirate. In addition, cultured tissues, cells, bacteria, viruses and the like can also be mentioned. Furthermore, this also applies to nucleic acids purified by other methods. Among these, the present invention is preferably used for separation and purification of nucleic acid from blood that is relatively easily clogged with a column.
本発明の混合工程で用いる結合液としては、カオトロピック物質を含む水溶液であることが好ましい。ここでカオトロピック物質とは、水溶液中でカオトロピックイオンを生成し、疎水性分子の水溶性を増加させる作用（カオトロピック効果）を有している物質のことである。前記カオトロピック物質としては、核酸の固相担体への吸着に寄与するものであれば特に限定されないが、例えばグアニジンチオシアン酸塩、グアニジン塩酸塩、グアニジン硝酸塩、グアニジン硫酸塩、ヨウ化ナトリウム、ヨウ化カリウム、過塩素酸ナトリウム、尿素が挙げられる。これらの中でも、カオトロピック効果の強さからグアニジンチオシアン酸塩、グアニジン塩酸塩が好ましい。また、前記カオトロピック物質は単独で用いても、二種以上を組み合わせて用いてもよい。前記カオトロピック物質濃度は、十分なカオトロピック効果が得られれば特に限定されないが、グアニジンチオシアン酸塩の場合は１．０〜６．０Ｍ、グアニジン塩酸塩の場合は１．０〜８．０Ｍであることが好ましい。グアニジンチオシアン酸塩の濃度が１．０Ｍより少ないと、十分なカオトロピック効果が得られない恐れがある。一方、グアニジンチオシアン酸塩の濃度が６．０Ｍより多いと保存中にグアニジンチオシアン酸塩が析出する恐れがある。同様にグアニジン塩酸塩の濃度が１．０Ｍより少ないと、十分なカオトロピック効果が得られない恐れがある。一方、グアニジン塩酸塩の濃度８．０Ｍより多いと保存中にグアニジン塩酸塩が析出する恐れがある。 The binding liquid used in the mixing step of the present invention is preferably an aqueous solution containing a chaotropic substance. Here, the chaotropic substance is a substance having a function (chaotropic effect) of generating chaotropic ions in an aqueous solution and increasing the water solubility of the hydrophobic molecule. The chaotropic substance is not particularly limited as long as it contributes to adsorption of nucleic acid to a solid phase carrier. For example, guanidine thiocyanate, guanidine hydrochloride, guanidine nitrate, guanidine sulfate, sodium iodide, potassium iodide. , Sodium perchlorate and urea. Among these, guanidine thiocyanate and guanidine hydrochloride are preferable because of the strong chaotropic effect. The chaotropic substances may be used alone or in combination of two or more. The concentration of the chaotropic substance is not particularly limited as long as a sufficient chaotropic effect is obtained, but is 1.0 to 6.0 M in the case of guanidine thiocyanate and 1.0 to 8.0 M in the case of guanidine hydrochloride. Is preferred. If the concentration of guanidine thiocyanate is less than 1.0M, a sufficient chaotropic effect may not be obtained. On the other hand, if the concentration of guanidine thiocyanate is higher than 6.0M, guanidine thiocyanate may precipitate during storage. Similarly, if the concentration of guanidine hydrochloride is less than 1.0M, there is a possibility that a sufficient chaotropic effect cannot be obtained. On the other hand, if the concentration of guanidine hydrochloride is more than 8.0M, guanidine hydrochloride may be precipitated during storage.
前記結合液には、核酸と固相担体との結合を補助するために、アルコール類を含有させるのが好ましい。前記アルコール類としては、前記効果が得られれば、いかなる種類のアルコールを用いてもよいが、エタノール、イソプロパノールが好ましく、エタノールがより好ましい。また、前記アルコールは単独で用いても、二種以上を組み合わせて用いてもよい。前記アルコール濃度は、特に限定されないが、１０〜７０％が好ましく、１０〜５０％がより好ましい。アルコール濃度が１０％より少ないと、十分な結合補助効果が得られない恐れがある。一方、アルコール濃度が７０％より多くすると、核酸と固相担体との結合性はかえって減少する。 The binding solution preferably contains alcohols to assist the binding between the nucleic acid and the solid phase carrier. As the alcohols, any kind of alcohol may be used as long as the above effects are obtained, but ethanol and isopropanol are preferable, and ethanol is more preferable. Moreover, the said alcohol may be used independently or may be used in combination of 2 or more type. The alcohol concentration is not particularly limited, but is preferably 10 to 70%, and more preferably 10 to 50%. If the alcohol concentration is less than 10%, a sufficient binding assisting effect may not be obtained. On the other hand, when the alcohol concentration is higher than 70%, the binding property between the nucleic acid and the solid phase carrier decreases.
前記結合液には、ｐＨ調整や核酸の吸着効果向上のために緩衝剤を含有させるのが好ましい。前記緩衝剤としては、目的とするｐＨ範囲において充分な緩衝能力を有していれば、いかなる種類の緩衝剤を用いてもよく、例えば、トリス、リン酸、フタル酸、クエン酸、マレイン酸、コハク酸、シュウ酸、ホウ酸、酒石酸、酢酸、炭酸、グッドバッファー（ＭＥＳ、ＡＤＡ、ＰＩＰＥＳ、ＡＣＥＳ、コラミン塩酸、ＢＥＳ、ＴＥＳ、ＨＥＰＥＳ、アセトアミドグリシン、トリシン、グリシンアミド、ビシン）などが挙げられる。これらの中でも、ｐＨ５．０〜９．０（好ましくはｐＨ６．０〜８．０）において充分な緩衝能力を有するなどの理由から、トリス、リン酸、ＭＥＳ、ＰＩＰＥＳ、ＴＥＳ、ＨＥＰＥＳが好ましい。また、前記緩衝剤は単独で用いても、二種以上を組み合わせて用いてもよい。前記緩衝剤の濃度は、特に限定されないが、１０〜１００ｍＭ程度が好ましい。 The binding solution preferably contains a buffering agent for adjusting the pH and improving the nucleic acid adsorption effect. As the buffer, any kind of buffer may be used as long as it has a sufficient buffering capacity in a target pH range. For example, tris, phosphoric acid, phthalic acid, citric acid, maleic acid, Succinic acid, oxalic acid, boric acid, tartaric acid, acetic acid, carbonic acid, good buffer (MES, ADA, PIPES, ACES, collamine hydrochloride, BES, TES, HEPES, acetamidoglycine, tricine, glycinamide, bicine) and the like. Among these, Tris, phosphoric acid, MES, PIPES, TES, and HEPES are preferable because they have sufficient buffer capacity at pH 5.0 to 9.0 (preferably pH 6.0 to 8.0). Moreover, the said buffer may be used independently or may be used in combination of 2 or more type. The concentration of the buffer is not particularly limited, but is preferably about 10 to 100 mM.
前記結合液には、細胞膜の破壊または細胞中に含まれるタンパク質を変性させる目的で界面活性剤を含有させてもよい。前記界面活性剤としては、前記効果が得られれば、いかなる種類の界面活性剤を用いてもよく、例えばポリオキシエチレンアルキルフェニルエーテル（Ｔｒｉｔｏｎ（登録商標）系界面活性剤など）、ポリオキシエチレンアルキルエーテル（Ｂｒｉｊ（登録商標）系界面活性剤など）、ポリオキシエチレンソルビタン脂肪酸エステル（Ｔｗｅｅｎ（登録商標）系界面活性剤など）、ポリオキシエチレン脂肪酸エステル、ソルビタン脂肪酸エステル、アルキルグルコシド、ショ糖脂肪酸エステルなどの非イオン性界面活性剤が挙げられる。また、前記界面活性剤は単独で用いても、二種以上を組み合わせて用いてもよい。前記界面活性剤濃度は、特に限定されないが、０．１〜２０％であることが好ましい。界面活性剤濃度が０．１％より少ないと、十分な細胞膜の破壊またはタンパク質の変性効果が得られない恐れがある。一方、界面活性剤濃度を２０％より多くしても、効果の向上は見られない。 The binding solution may contain a surfactant for the purpose of breaking the cell membrane or denaturing proteins contained in the cells. As the surfactant, any type of surfactant may be used as long as the above effects are obtained. For example, polyoxyethylene alkylphenyl ether (such as Triton (registered trademark) surfactant), polyoxyethylene alkyl Ether (such as Brij (registered trademark) surfactant), polyoxyethylene sorbitan fatty acid ester (such as Tween (registered trademark) surfactant), polyoxyethylene fatty acid ester, sorbitan fatty acid ester, alkyl glucoside, sucrose fatty acid ester Nonionic surfactants such as Moreover, the said surfactant may be used independently or may be used in combination of 2 or more type. The surfactant concentration is not particularly limited, but is preferably 0.1 to 20%. If the surfactant concentration is less than 0.1%, sufficient cell membrane destruction or protein denaturation effects may not be obtained. On the other hand, even if the surfactant concentration is higher than 20%, no improvement in the effect is observed.
また、前記結合液には、核酸を含む試料中に含まれるタンパク質、特にヌクレアーゼを変性させる目的で、還元剤を含有させてもよい。前記還元剤としては、前記効果が得られれば、いかなる種類の還元剤を用いてもよく、例えば水素、ヨウ化水素、硫化水素、水素化アルミニウムリチウム、水素化ホウ素ナトリウムなどの水素化化合物、アルカリ金属、マグネシウム、カルシウム、アルミニウム、亜鉛などの電気的陽性の大きい金属、またはそれのアマルガム、アルデヒド類、糖類、ギ酸、シュウ酸などの有機酸化物、メルカプト化合物などが挙げられる。これらの中でも、２−メルカプトエタノール、ジチオスレイトールが好ましい。前記還元剤濃度は、特に限定されないが、１．０〜１００ｍＭであることが好ましい。還元剤濃度が１．０ｍＭより少ないと、十分なタンパク質の変性効果が得られない恐れがある。一方、還元剤濃度が１００ｍＭより多くしても、効果の向上は見られない。 The binding solution may contain a reducing agent for the purpose of denaturing proteins, particularly nucleases, contained in a sample containing nucleic acid. As the reducing agent, any kind of reducing agent may be used as long as the above effects are obtained. For example, hydrogen, hydrogen iodide, hydrogen sulfide, lithium aluminum hydride, sodium borohydride and other hydrogenated compounds, alkali Examples thereof include metals, metals, magnesium, calcium, aluminum, zinc and the like, or amalgams thereof, aldehydes, saccharides, organic acids such as formic acid and oxalic acid, mercapto compounds, and the like. Among these, 2-mercaptoethanol and dithiothreitol are preferable. The reducing agent concentration is not particularly limited, but is preferably 1.0 to 100 mM. If the reducing agent concentration is less than 1.0 mM, a sufficient protein denaturation effect may not be obtained. On the other hand, even if the reducing agent concentration is higher than 100 mM, the effect is not improved.
前記核酸を含む試料と結合液とを混合する方法は、特に限定されず、例えばボルテックスミキサーによる混和、転倒混和、ピペッティングなどが挙げられる。 A method of mixing the sample containing the nucleic acid and the binding solution is not particularly limited, and examples thereof include mixing with a vortex mixer, inversion mixing, and pipetting.
（Ｂ：結合工程）
本発明の工程（Ｂ）：結合工程は、前記混合液と固相担体とを接触させ、固相担体に核酸を吸着させる工程である。(B: bonding step)
Step (B) of the present invention: The binding step is a step of bringing the mixed solution into contact with the solid phase carrier and adsorbing the nucleic acid to the solid phase carrier.
前記混合液と固相担体とを接触させ、固相担体に核酸を吸着させる方法は、特に限定されず、例えば
（１）底面に固相担体を固定したスピンカラムを用い、スピンカラムの上部に混合液を添加し、遠心分離機により上から下へ通液させることで、固相担体に核酸を吸着させる方法
（２）ルアーフィッテングにより先端に固相担体を固定したシリンジを用い、混合液をシリンジの吸引吐出により繰り返し通液させることで、固相担体に核酸を吸着させる方法
（３）内部に固相担体を固定したピペットチップを用い、混合液をピペットの吸引吐出により繰り返し通液させることで、固相担体に核酸を吸着させる方法
が挙げられる。
なお、本発明の核酸分離精製方法において、通液方法は特に限定されない。例えば、重力を利用して液体を上から下へ流すことができる。あるいは、遠心分離機で遠心力をかけたり、シリンジ・ピペットなどの空気の圧力変化により吸引吐出を行うなど、何らかの外力をかけることによっても行うことができる。The method of bringing the mixed solution into contact with the solid phase carrier and adsorbing the nucleic acid to the solid phase carrier is not particularly limited. For example, (1) a spin column having a solid phase carrier fixed on the bottom surface is used, Method of adsorbing nucleic acid to solid phase carrier by adding mixed solution and letting it flow from top to bottom with a centrifuge (2) Using a syringe with a solid phase carrier fixed to the tip by luer fitting, Method of adsorbing nucleic acid to solid phase carrier by repeatedly sucking and discharging by syringe (3) Using a pipette tip with a solid phase carrier fixed inside, and repeatedly passing the mixed solution by pipette suction and discharging And a method of adsorbing nucleic acid to a solid phase carrier.
In the method for separating and purifying nucleic acid of the present invention, the liquid passing method is not particularly limited. For example, the liquid can flow from top to bottom using gravity. Alternatively, it can also be performed by applying some external force such as applying centrifugal force with a centrifuge or performing suction and discharge by changing the pressure of air such as a syringe or pipette.
（Ｃ：洗浄工程）
本発明の工程（Ｃ）：洗浄工程は、洗浄液と固相担体とを接触させ、固相担体から核酸以外の成分（例えば、タンパク質、脂質など）を洗浄する工程である。なお、洗浄工程は、１回の洗浄で済ませてもよいし、複数回洗浄を繰り返してもよい。(C: Cleaning process)
Step (C) of the present invention: The washing step is a step in which a washing solution and a solid phase carrier are brought into contact with each other to wash components other than nucleic acids (for example, proteins, lipids, etc.) from the solid phase carrier. Note that the cleaning step may be performed by one cleaning, or the cleaning may be repeated a plurality of times.
本発明の洗浄工程で用いる洗浄液としては、固相担体に吸着している核酸を脱離させず、かつ核酸以外の成分を脱離させるものであれば、特に限定されないが、アルコール類を含む水溶液であることが好ましい。前記アルコール類としては、前記効果が得られれば、いかなる種類のアルコールを用いてもよいが、エタノール、イソプロパノールが好ましく、エタノールがより好ましい。また、前記アルコールは単独で用いても、二種以上を組み合わせて用いてもよい。前記アルコール濃度は、特に限定されないが、２０〜１００％が好ましく、３０〜９０％がより好ましい。アルコール濃度が２０％より少ないと、核酸が脱離する恐れがある。 The washing solution used in the washing step of the present invention is not particularly limited as long as it does not desorb the nucleic acid adsorbed on the solid phase carrier and desorbs components other than the nucleic acid, but an aqueous solution containing alcohols It is preferable that As the alcohols, any kind of alcohol may be used as long as the above effects are obtained, but ethanol and isopropanol are preferable, and ethanol is more preferable. Moreover, the said alcohol may be used independently or may be used in combination of 2 or more type. The alcohol concentration is not particularly limited, but is preferably 20 to 100%, and more preferably 30 to 90%. If the alcohol concentration is less than 20%, the nucleic acid may be detached.
前記洗浄液には、ｐＨ調整や核酸の吸着効果向上のために緩衝剤を含有させるのが好ましい。前記緩衝剤としては、目的とするｐＨ範囲において充分な緩衝能力を有していれば、いかなる種類の緩衝剤を用いてもよく、例えば、トリス、リン酸、フタル酸、クエン酸、マレイン酸、コハク酸、シュウ酸、ホウ酸、酒石酸、酢酸、炭酸、グッドバッファー（ＭＥＳ、ＡＤＡ、ＰＩＰＥＳ、ＡＣＥＳ、コラミン塩酸、ＢＥＳ、ＴＥＳ、ＨＥＰＥＳ、アセトアミドグリシン、トリシン、グリシンアミド、ビシン）などが挙げられる。これらの中でも、ｐＨ５．０〜９．０（好ましくはｐＨ６．０〜８．０）において充分な緩衝能力を有するなどの理由から、トリス、リン酸、ＭＥＳ、ＰＩＰＥＳ、ＴＥＳ、ＨＥＰＥＳが好ましい。また、前記緩衝剤は単独で用いても、二種以上を組み合わせて用いてもよい。前記緩衝剤濃度は、特に限定されないが、１０〜１００ｍＭ程度が好ましい。さらに、最終精製物へのコンタミを低減する目的から、洗浄液中の緩衝剤濃度は結合液中の緩衝剤濃度よりも低いことがより好ましい。 The washing solution preferably contains a buffering agent for adjusting the pH and improving the nucleic acid adsorption effect. As the buffer, any kind of buffer may be used as long as it has a sufficient buffering capacity in a target pH range. For example, tris, phosphoric acid, phthalic acid, citric acid, maleic acid, Succinic acid, oxalic acid, boric acid, tartaric acid, acetic acid, carbonic acid, good buffer (MES, ADA, PIPES, ACES, collamine hydrochloride, BES, TES, HEPES, acetamidoglycine, tricine, glycinamide, bicine) and the like. Among these, Tris, phosphoric acid, MES, PIPES, TES, and HEPES are preferable because they have sufficient buffer capacity at pH 5.0 to 9.0 (preferably pH 6.0 to 8.0). Moreover, the said buffer may be used independently or may be used in combination of 2 or more type. The buffer concentration is not particularly limited, but is preferably about 10 to 100 mM. Furthermore, for the purpose of reducing contamination to the final purified product, the concentration of the buffer in the washing solution is more preferably lower than the concentration of the buffer in the binding solution.
前記洗浄液と固相担体とを接触させ、固相担体から核酸以外の成分を洗浄する方法は、特に限定されず、例えば
（１）底面に固相担体を固定したスピンカラムを用い、スピンカラムの上部に洗浄液を添加し、遠心分離機により上から下へ通液させることで、固相担体から核酸以外の成分を洗浄する方法
（２）ルアーフィッテングにより先端に固相担体を固定したシリンジを用い、洗浄液をシリンジの吸引吐出により繰り返し通液させることで、固相担体から核酸以外の成分を洗浄する方法
（３）内部に固相担体を固定したピペットチップを用い、洗浄液をピペットの吸引吐出により繰り返し通液させることで、固相担体から核酸以外の成分を洗浄する方法
が挙げられる。The method for bringing the washing solution into contact with the solid phase carrier and washing components other than the nucleic acid from the solid phase carrier is not particularly limited. For example, (1) using a spin column having a solid phase carrier fixed on the bottom surface, A method of washing components other than nucleic acid from the solid phase carrier by adding a washing solution to the top and letting it flow from top to bottom with a centrifuge (2) Using a syringe with a solid phase carrier fixed at the tip by luer fitting , A method of washing components other than nucleic acid from the solid phase carrier by repeatedly passing the washing solution by suction and discharge of a syringe (3) Using a pipette tip with a solid phase carrier fixed inside, and using a pipette tip A method of washing components other than the nucleic acid from the solid phase carrier by repeatedly passing the solution is used.
前記洗浄工程後に、必要に応じて、固相担体に残留した洗浄液を、遠心分離や加熱により除去することができる。加熱温度は５０〜９０℃が好ましく、６０〜８０℃がより好ましい。加熱温度が５０℃より低いと、十分な洗浄液の除去効果が得られない。一方、加熱温度が９０℃より高いと、核酸が分解・変性する恐れがある。 After the washing step, the washing liquid remaining on the solid phase carrier can be removed by centrifugation or heating, if necessary. The heating temperature is preferably 50 to 90 ° C, more preferably 60 to 80 ° C. When the heating temperature is lower than 50 ° C., a sufficient cleaning liquid removal effect cannot be obtained. On the other hand, if the heating temperature is higher than 90 ° C., the nucleic acid may be decomposed or denatured.
（Ｄ：溶出工程）
本発明の工程（Ｄ）：溶出工程は、溶出液と固相担体とを接触させ、固相担体から核酸を脱着させる工程である。(D: elution process)
Step (D) of the present invention: The elution step is a step in which the eluate is brought into contact with a solid phase carrier to desorb nucleic acid from the solid phase carrier.
本発明の溶出工程で用いる溶出液としては、固相担体に吸着している核酸を脱離させ、かつ核酸抽出後の反応、例えば逆転写、ＰＣＲに代表される核酸増幅反応を阻害しない溶液組成であれば、特に限定されないが、水、トリス−ＥＤＴＡ緩衝液［１０ｍＭ トリス塩酸緩衝液、１ｍＭ ＥＤＴＡ、ｐＨ８．０］が好ましい。 The eluate used in the elution step of the present invention is a solution composition that desorbs nucleic acid adsorbed on a solid phase carrier and does not inhibit reactions after nucleic acid extraction, such as reverse transcription and nucleic acid amplification reactions typified by PCR. If it is, it will not specifically limit, However, Water and a Tris-EDTA buffer [10 mM Tris-HCl buffer, 1 mM EDTA, pH 8.0] are preferable.
前記溶出液は、溶出効率を上げるために必要に応じて加熱することができる。加熱温度は５０〜９０℃が好ましく、６０〜８０℃がより好ましい。加熱温度が５０℃より低いと、十分な溶出率向上効果が得られない。一方、加熱温度が９０℃より高いと、核酸が分解・変性する恐れがある。 The eluate can be heated as necessary to increase elution efficiency. The heating temperature is preferably 50 to 90 ° C, more preferably 60 to 80 ° C. When the heating temperature is lower than 50 ° C., a sufficient elution rate improvement effect cannot be obtained. On the other hand, if the heating temperature is higher than 90 ° C., the nucleic acid may be decomposed or denatured.
前記溶出液と固相担体とを接触させ、固相担体から核酸を脱着させる方法は、特に限定されず、例えば
（１）底面に固相担体を固定したスピンカラムを用い、スピンカラムの上部に溶出液を添加し、遠心分離機により上から下へ通液させることで、固相担体から核酸を脱着させる方法
（２）ルアーフィッテングにより先端に固相担体を固定したシリンジを用い、溶出液をシリンジの吸引吐出により繰り返し通液させることで、固相担体から核酸を脱着させる方法
（３）内部に固相担体を固定したピペットチップを用い、溶出液をピペットの吸引吐出により繰り返し通液させることで、固相担体から核酸を脱着させる方法
が挙げられる。The method of bringing the eluate into contact with the solid phase carrier and desorbing the nucleic acid from the solid phase carrier is not particularly limited. For example, (1) a spin column having a solid phase carrier fixed to the bottom surface is used, Method of desorbing nucleic acid from solid phase carrier by adding eluate and letting it flow from top to bottom with a centrifuge (2) Using a syringe with a solid phase carrier fixed to the tip by luer fitting, Method of desorbing nucleic acid from solid phase carrier by repeatedly sucking and discharging by syringe (3) Using a pipette tip with a solid phase carrier fixed inside, and repeatedly passing eluate by sucking and discharging the pipette And a method of desorbing nucleic acids from a solid phase carrier.
本発明はまた、前記の核酸分離精製方法に用いるための固相担体であって、（１）多孔性無機粒子と（２）有機または無機繊維と（３）有機バインダーとからなる組成物を含む固相担体である。 The present invention also includes a solid phase carrier for use in the above-described method for separating and purifying nucleic acid, the composition comprising (1) porous inorganic particles, (2) organic or inorganic fibers, and (3) an organic binder. It is a solid support.
本発明はまた、前記の核酸分離精製方法に用いるためのデバイスであって、前記の固相担体を保持しているデバイスである。前記デバイスの形態は特に限定されないが、スピンカラムやシリンジが好ましい。その具体的な形態は後述の実施例などで例示される。 The present invention is also a device for use in the nucleic acid separation and purification method, which holds the solid phase carrier. The form of the device is not particularly limited, but a spin column or a syringe is preferable. The specific form is illustrated by the Example etc. which are mentioned later.
本発明はまた、前記の核酸分離精製方法を行うための核酸の分離精製キットであって、前記の固相担体または前記のデバイス、固相担体に核酸を吸着させるための結合液、固相担体から核酸以外の成分を洗浄するための洗浄液、固相担体から核酸を溶出するための溶出液を含む、核酸の分離精製キットである。本発明のキットにおいて、前記の固相担体、結合液、洗浄液および溶出液以外の構成は特に限定されない。たとえばＢｏｏｍ法で用いられる公知の構成を含んでいてもよい。 The present invention also provides a nucleic acid separation and purification kit for performing the nucleic acid separation and purification method, the solid phase carrier or the device, a binding solution for adsorbing nucleic acid to the solid phase carrier, and a solid phase carrier. A kit for separating and purifying nucleic acid, comprising a washing solution for washing components other than nucleic acids from eluate, and an elution solution for eluting nucleic acids from a solid phase carrier. In the kit of the present invention, the configuration other than the solid phase carrier, the binding solution, the washing solution and the eluate is not particularly limited. For example, a known configuration used in the boom method may be included.
以下に実施例を示して本発明を具体的に説明するが、本発明は実施例に限定されるものではない。なお、明細書中の評価法は以下の通りである。
［各種評価法］EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to the examples. The evaluation methods in the specification are as follows.
[Various evaluation methods]
＜１．多孔性無機粒子の含有率＞
下記式（１）にて算出した。
含有率（％）＝（多孔性無機粒子の重量／固相担体の重量）×１００ （１）<1. Content of porous inorganic particles>
It calculated with the following formula (1).
Content (%) = (weight of porous inorganic particles / weight of solid phase carrier) × 100 (1)
＜２．多孔性無機粒子の形状＞
各粒子を走査型電子顕微鏡（ＳＥＭ）で観察し、［１］球また楕円体である球状 ［２］立方体または直方体であるシート状 ［３］その他のランダムな形状である破砕状 に分類した。<2. Shape of porous inorganic particles>
Each particle was observed with a scanning electron microscope (SEM) and classified into [1] a sphere or an ellipsoidal sphere [2] a cube or a rectangular parallelepiped sheet [3] other random shapes such as a crushed shape.
＜３．多孔性無機粒子の平均粒子径＞
各粒子を走査型電子顕微鏡（ＳＥＭ）で観察し、１００個の粒子の直径を測定し、平均直径（平均粒子径）を算出した。<3. Average particle size of porous inorganic particles>
Each particle was observed with a scanning electron microscope (SEM), the diameter of 100 particles was measured, and the average diameter (average particle diameter) was calculated.
＜４．固相担体の目付け＞
２００ｍｍ×２００ｍｍの固相担体を、乾燥機で８０℃×３０分加熱し、デシケータ（乾燥剤：シリカゲル）で室温×３０分静置した。その後、重量を測定し、１ｍ２当りの重量（目付け）に換算した。<4. Weight of solid support>
A 200 mm × 200 mm solid phase carrier was heated with a dryer at 80 ° C. for 30 minutes, and allowed to stand at room temperature for 30 minutes with a desiccator (desiccant: silica gel). Thereafter, the weight was measured and converted to a weight per 1 m 2 (weight per unit area).
＜５．固相担体の厚み＞
２００ｍｍ×２００ｍｍの固相担体の１０箇所で、荷重６８６Ｐａの圧力を加えた時の厚みを測定し、平均厚みを算出した。<5. Thickness of solid support>
The thickness when a load of 686 Pa was applied was measured at 10 locations on a 200 mm × 200 mm solid phase carrier, and the average thickness was calculated.
＜６．核酸の濃度：１０ｎｇ／μＬ以上＞
測定対象の核酸溶液について、核酸濃度が１０ｎｇ／μＬ以上の場合は、下記条件で核酸濃度を測定した。
（１）ｔｏｔａｌ ＲＮＡの場合
装置名 ： Ｔｈｅｒｍｏ Ｓｃｉｅｎｔｉｆｉｃ社製
ｎａｎｏ ｄｒｏｐ（登録商標）２０００
測定モード ： 核酸−ＲＮＡ
試料液量 ： ２μＬ
Ｂｌａｎｋ ： 水
（２）Ｇｅｎｏｍｉｃ ＤＮＡの場合
装置名 ： Ｔｈｅｒｍｏ Ｓｃｉｅｎｔｉｆｉｃ社製
ｎａｎｏ ｄｒｏｐ（登録商標）２０００
測定モード ： 核酸−ＤＮＡ
試料液量 ： ２μＬ
Ｂｌａｎｋ ： 水<6. Nucleic acid concentration: 10 ng / μL or more>
For the nucleic acid solution to be measured, when the nucleic acid concentration was 10 ng / μL or more, the nucleic acid concentration was measured under the following conditions.
(1) In the case of total RNA, apparatus name: nano drop (registered trademark) 2000 manufactured by Thermo Scientific
Measurement mode: Nucleic acid-RNA
Sample volume: 2 μL
Blank: Water (2) In the case of Genomic DNA Device name: nano drop (registered trademark) 2000 manufactured by Thermo Scientific
Measurement mode: Nucleic acid-DNA
Sample volume: 2 μL
Blank: Water
＜７．核酸の濃度：１０ｎｇ／μＬ以下＞
測定対象の核酸溶液について、核酸濃度が１０ｎｇ／μＬ以下の場合は、下記条件で核酸濃度を測定した。
（１）ｔｏｔａｌ ＲＮＡの場合
装置名 ： Ｉｎｖｉｔｒｏｇｅｎ社製 Ｑｕｂｉｔ（登録商標）２．０
測定キット ： Ｉｎｖｉｔｒｏｇｅｎ社製 Ｑｕｂｉｔ（登録商標）
ＲＮＡ Ａｓｓａｙ Ｋｉｔ
容器 ： Ｉｎｖｉｔｒｏｇｅｎ社製 Ｑｕｂｉｔ（登録商標）
ａｓｓａｙ ｔｕｂｅｓ
測定モード ： ＲＮＡ−ＲＮＡ
試料液量 ： １０μＬ
Ｂｌａｎｋ ： 水
（２）Ｇｅｎｏｍｉｃ ＤＮＡの場合
装置名 ： Ｉｎｖｉｔｒｏｇｅｎ社製 Ｑｕｂｉｔ（登録商標）２．０
測定キット ： Ｉｎｖｉｔｒｏｇｅｎ社製 Ｑｕｂｉｔ（登録商標）
ｄｓＤＮＡ ＨＳ Ａｓｓａｙ Ｋｉｔ
容器 ： Ｉｎｖｉｔｒｏｇｅｎ社製 Ｑｕｂｉｔ（登録商標）
ａｓｓａｙ ｔｕｂｅｓ
測定モード ： ＤＮＡ−ｄｓＤＮＡ Ｈｉｇｈ Ｓｅｎｓｉｔｉｖｉｔｙ
試料液量 ： １０μＬ
Ｂｌａｎｋ ： 水<7. Nucleic acid concentration: 10 ng / μL or less>
For the nucleic acid solution to be measured, when the nucleic acid concentration was 10 ng / μL or less, the nucleic acid concentration was measured under the following conditions.
(1) In the case of total RNA Device name: Qubit (registered trademark) 2.0 manufactured by Invitrogen
Measurement kit: Qubit (registered trademark) manufactured by Invitrogen
RNA Assay Kit
Container: Qubit (registered trademark) manufactured by Invitrogen
assay tubes
Measurement mode: RNA-RNA
Sample liquid volume: 10 μL
Blank: Water (2) In the case of Genomic DNA Device name: Qubit (registered trademark) 2.0 manufactured by Invitrogen
Measurement kit: Qubit (registered trademark) manufactured by Invitrogen
dsDNA HS Assay Kit
Container: Qubit (registered trademark) manufactured by Invitrogen
assay tubes
Measurement mode: DNA-dsDNA High Sensitivity
Sample liquid volume: 10 μL
Blank: Water
＜８．ｔｏｔａｌ ＲＮＡ分解度（ＲＩＮ値）＞
測定対象のｔｏｔａｌ ＲＮＡ溶液について、下記条件でｔｏｔａｌ ＲＮＡ分解度（ＲＩＮ値）を測定した。
装置名 ： アジレント社製 Ａｇｉｌｅｎｔ ２２００ ＴａｐｅＳｔａｔｉｏｎ
測定キット ： アジレント社製 ＨＳ ＲＮＡ Ｓｃｒｅｅｎ Ｔａｐｅ、
Ｓａｍｐｌｅ Ｂｕｆｆｅｒ、Ｌａｄｄｅｒ
試料液量 ： ２μＬ<8. Total RNA degradation (RIN value)>
For the total RNA solution to be measured, the total RNA degradation degree (RIN value) was measured under the following conditions.
Device name: Agilent 2200 TapeStation manufactured by Agilent
Measurement kit: HS RNA Screen Tape manufactured by Agilent,
Sample Buffer, Ladder
Sample volume: 2 μL
［実施例１］
多孔性シリカ粒子を５重量％と、ガラス繊維を６６．５重量％と、ポリエステルバインダーを２８．５重量％の比率で、湿式抄紙装置（東洋紡エンジニアリング社製）を使い固相担体１を作成した。得られた固相担体１の詳細を表１に示す。
次いで、固相担体１をベルトポンチＴＰＯ−７０（トラスコ社製）にて７ｍｍφの大きさに打ち抜いた。その後、エコノスピン（登録商標）ＩＩａ（ジーンデザイン社製）のシリカメンブレン、およびＯ−リングをシリカメンブレンフィルターハウジングから取り除き、シリカメンブレンの代わりに固相担体１を２枚シリカメンブレンフィルターハウジング内に配し、Ｏ−リングで固定し、スピンカラム１（図１参照）を作成した。
次いで、固相担体１をベルトポンチＴＰＯ−４０（トラスコ社製）にて４ｍｍφの大きさに打ち抜いた。その後、テルモシリンジ ＳＳ−０２ＳＺ（テルモ社製）の先端に、固相担体１を２枚介してメスルアーフィッティング ＶＰＲＦ２０６（アイシス社製）を接続し、シリンジ１（図２参照）を作成した。[Example 1]
A solid support 1 was prepared using a wet papermaking machine (manufactured by Toyobo Engineering Co., Ltd.) at a ratio of 5% by weight of porous silica particles, 66.5% by weight of glass fiber, and 28.5% by weight of polyester binder. . Details of the obtained solid phase carrier 1 are shown in Table 1.
Next, the solid phase carrier 1 was punched into a size of 7 mmφ with a belt punch TPO-70 (manufactured by Trusco). Thereafter, the silica membrane and O-ring of Econospin (registered trademark) IIa (manufactured by Gene Design) are removed from the silica membrane filter housing, and two solid phase carriers 1 are placed in the silica membrane filter housing instead of the silica membrane. Then, the spin column 1 (see FIG. 1) was prepared by fixing with an O-ring.
Next, the solid phase carrier 1 was punched into a size of 4 mmφ with a belt punch TPO-40 (manufactured by Trusco). Thereafter, a female luer fitting VPRF206 (manufactured by Isis) was connected to the tip of a Terumo syringe SS-02SZ (manufactured by Terumo) via two solid phase carriers 1 to prepare a syringe 1 (see FIG. 2).
［実施例２〜５］
多孔性シリカ粒子、ガラス繊維、ポリエステルバインダーの配合比率が異なる以外は、実施例１と同様にして、固相担体２〜５を作成した。得られた固相担体２〜５の詳細を表１に示す。
また、実施例１と同様にして、スピンカラム２〜５、シリンジ２〜５を作成した。[Examples 2 to 5]
Solid phase carriers 2 to 5 were prepared in the same manner as in Example 1 except that the mixing ratios of porous silica particles, glass fibers, and polyester binder were different. Details of the obtained solid phase carriers 2 to 5 are shown in Table 1.
Moreover, it carried out similarly to Example 1, and created the spin columns 2-5 and the syringes 2-5.
［実施例６、７］
多孔性シリカ粒子の平均粒子径が異なる以外は、実施例１と同様にして、固相担体６、７を作成した。得られた固相担体６、７の詳細を表１に示す。
また、実施例１と同様にして、スピンカラム６、７、シリンジ６、７を作成した。[Examples 6 and 7]
Solid phase carriers 6 and 7 were prepared in the same manner as in Example 1 except that the average particle size of the porous silica particles was different. The details of the obtained solid phase carriers 6 and 7 are shown in Table 1.
In the same manner as in Example 1, spin columns 6 and 7 and syringes 6 and 7 were prepared.
［実施例８、９］
多孔性シリカ粒子の形状が異なる以外は、実施例１と同様にして、固相担体８、９を作成した。得られた固相担体８、９の詳細を表２に示す。
また、実施例１と同様にして、スピンカラム８、９、シリンジ８、９を作成した。[Examples 8 and 9]
Solid phase carriers 8 and 9 were prepared in the same manner as in Example 1 except that the shape of the porous silica particles was different. Details of the obtained solid phase carriers 8 and 9 are shown in Table 2.
Also, spin columns 8 and 9 and syringes 8 and 9 were prepared in the same manner as in Example 1.
［実施例１０］
ガラス繊維の代わりにセルロース繊維を用いる以外は、実施例１と同様にして、固相担体１０を作成した。得られた固相担体１０の詳細を表２に示す。
また、実施例１と同様にして、スピンカラム１０、シリンジ１０を作成した。[Example 10]
A solid support 10 was prepared in the same manner as in Example 1 except that cellulose fibers were used instead of glass fibers. Details of the obtained solid phase carrier 10 are shown in Table 2.
Moreover, the spin column 10 and the syringe 10 were created in the same manner as in Example 1.
［実施例１１、１２］
ポリエステルバインダーの代わりにポリエチレンバインダー、ポリプロピレンバインダーを用いる以外は、実施例１と同様にして、固相担体１１、１２を作成した。得られた固相担体１１、１２の詳細を表２に示す。
また、実施例１と同様にして、スピンカラム１１、１２、シリンジ１１、１２を作成した。[Examples 11 and 12]
Solid phase carriers 11 and 12 were prepared in the same manner as in Example 1 except that a polyethylene binder and a polypropylene binder were used instead of the polyester binder. Details of the obtained solid phase carriers 11 and 12 are shown in Table 2.
Further, spin columns 11 and 12 and syringes 11 and 12 were produced in the same manner as in Example 1.
［実施例１３、１４］
固相担持の目付け、厚みが異なる以外は、実施例１と同様にして、固相担体１３、１４を作成した。得られた固相担体１３、１４の詳細を表２に示す。
また、実施例１と同様にして、スピンカラム１３、１４、シリンジ１３、１４を作成した。[Examples 13 and 14]
Solid phase carriers 13 and 14 were prepared in the same manner as in Example 1 except that the basis weight and thickness of the solid phase support were different. The details of the obtained solid phase carriers 13 and 14 are shown in Table 2.
Also, spin columns 13 and 14 and syringes 13 and 14 were produced in the same manner as in Example 1.
［比較例１、２］
多孔性シリカ粒子、ガラス繊維、ポリエステルバインダーの配合比率が異なる以外は、実施例１と同様にして、固相担体１５、１６を作成した。得られた固相担体１５、１６の詳細を表３に示す。固相担体１６は、品位が悪く、また非常に脆いため、後の評価を行なうことができなかった。
また、実施例１と同様にして、スピンカラム１５、シリンジ１５を作成した。[Comparative Examples 1 and 2]
Solid phase carriers 15 and 16 were prepared in the same manner as in Example 1 except that the mixing ratios of porous silica particles, glass fibers, and polyester binder were different. The details of the obtained solid phase carriers 15 and 16 are shown in Table 3. Since the solid phase carrier 16 was poor in quality and very brittle, later evaluation could not be performed.
Further, a spin column 15 and a syringe 15 were prepared in the same manner as in Example 1.
［比較例３］
多孔性シリカ粒子の平均粒子径が異なる以外は、実施例１と同様にして、固相担体１７を作成した。得られた固相担体１７の詳細を表３に示す。
また、実施例１と同様にして、スピンカラム１７、シリンジ１７を作成した。[Comparative Example 3]
A solid support 17 was prepared in the same manner as in Example 1 except that the average particle diameter of the porous silica particles was different. The details of the obtained solid phase carrier 17 are shown in Table 3.
Further, in the same manner as in Example 1, a spin column 17 and a syringe 17 were prepared.
［比較例４］
ポリエステルバインダーの代わりにＰＶＡバインダーを用いる以外は、実施例１と同様にして、固相担体１８を作成した。得られた固相担体１８の詳細を表３に示す。
また、実施例１と同様にして、スピンカラム１８、シリンジ１８を作成した。[Comparative Example 4]
A solid support 18 was prepared in the same manner as in Example 1 except that a PVA binder was used instead of the polyester binder. Details of the obtained solid phase carrier 18 are shown in Table 3.
Further, in the same manner as in Example 1, a spin column 18 and a syringe 18 were prepared.
［比較例５〜７］
市販品である、エコノスピン（登録商標）ＩＩａ（ジーンデザイン社製）、ＲＮｅａｓｙ（登録商標）（キアゲン社製）、ＰｕｒｅＬｉｎｋ（登録商標）（ライフテクノロジーズ社製）のカラムをそのまま用いた。[Comparative Examples 5 to 7]
Commercially available columns of Econospin (registered trademark) IIa (manufactured by Gene Design), RNeasy (registered trademark) (manufactured by Qiagen), and PureLink (registered trademark) (manufactured by Life Technologies) were used as they were.
＜固相担体の通液性＞
前記スピンカラム１〜１８を用いて、固相担体の通液性を評価した。
Ｂｏｖｉｎｅ Ｂｌｏｏｄ（フナコシ社製）６００μＬをスピンカラム１〜１８にアプライし、微量高速冷却遠心機ＭＸ−３０７（トミー精工社製）にて１，０００〜２０，０００Ｇでそれぞれ１分間遠心し、Ｂｏｖｉｎｅ Ｂｌｏｏｄ（フナコシ社製）がスピンカラム中の固相担体を通液するために必要な最小の遠心力を評価した。得られた最小通液遠心力を表４に示す。<Liquid permeability of solid support>
Using the spin columns 1 to 18, the liquid permeability of the solid phase carrier was evaluated.
Bovine Blood (manufactured by Funakoshi Co., Ltd.) (600 μL) was applied to spin columns 1 to 18 and centrifuged at 1,000 to 20,000 G for 1 minute each in a micro high-speed cooling centrifuge MX-307 (Tommy Seiko Co., Ltd.), and Bovine Blood (Funakoshi Co., Ltd.) evaluated the minimum centrifugal force required for passing the solid phase carrier in the spin column. Table 4 shows the obtained minimum flow centrifugal force.
＜ｔｏｔａｌ ＲＮＡの回収率、ｔｏｔａｌ ＲＮＡ分解度：スピンカラム法＞
前記スピンカラム１〜１８を用いてｔｏｔａｌ ＲＮＡ水溶液からのｔｏｔａｌ ＲＮＡ回収率、ｔｏｔａｌ ＲＮＡ分解度の評価を行なった。試料溶液として、ＨｅＬａ Ｓ３細胞よりセパゾール（登録商標）ＲＮＡＩ ＳｕｐｅｒＧ（ナカライテスク社製）を用いてプロトコール通りに分離精製した１００ｎｇ／μＬのｔｏｔａｌ ＲＮＡ水溶液（ＲＩＮ値＝９．９）１００μＬと、結合液としてＲＮｅａｓｙ（登録商標）（キアゲン社製）のＢｕｆｆｅｒ ＲＬＴ ３５０μＬとを、ボルテックスミキサーにて混合した。次いで、エタノールＳＰ ２５０μＬ（ナカライテスク社製）を混合した後、スピンカラム１〜１８にアプライし、小型微量遠心機ＰＭＣ−０６０（トミー精工社製）にて１９３０Ｇで１分間遠心し、ろ液を廃棄した。次いで、洗浄液としてＲＮｅａｓｙ（登録商標）（キアゲン社製）のＢｕｆｆｅｒ ＲＰＥ ５００μＬをスピンカラム１〜１８にアプライし、小型微量遠心機ＰＭＣ−０６０（トミー精工社製）にて１９３０Ｇで１分間遠心し、ろ液を廃棄する操作を２回繰り返した。最後に、溶出液としてＮｕｃｌｅａｓｅ−Ｆｒｅｅ Ｗａｔｅｒ（ライフテクノロジーズ社製）３０μＬをスピンカラム１〜１８にアプライし、小型微量遠心機ＰＭＣ−０６０（トミー精工社製）にて１９３０Ｇで１分間遠心し、ｔｏｔａｌ ＲＮＡ水溶液を得た。得られたｔｏｔａｌ ＲＮＡ回収率、ｔｏｔａｌ ＲＮＡ分解度を表５〜７に示す。なお、ｔｏｔａｌ ＲＮＡ回収率は、下記式（２）より算出した。
ｔｏｔａｌ ＲＮＡ回収率（％）＝｛精製ｔｏｔａｌ ＲＮＡ濃度（ｎｇ／μＬ）×溶出液量３０（μＬ）｝÷｛投入ｔｏｔａｌ ＲＮＡ濃度１００（ｎｇ／μＬ）×投入ｔｏｔａｌ ＲＮＡ液量１００（μＬ）｝×１００ （２）<Recovery rate of total RNA, total RNA degradation degree: spin column method>
Using the spin columns 1 to 18, the total RNA recovery rate from the total RNA aqueous solution and the total RNA degradation degree were evaluated. As a sample solution, 100 μL of 100 ng / μL total RNA aqueous solution (RIN value = 9.9) 100 μL separated and purified from HeLa S3 cells using Sepasol (registered trademark) RNAI SuperG (manufactured by Nacalai Tesque) according to the protocol, and binding solution RNeasy (registered trademark) (manufactured by Qiagen) as a buffer RLT (350 μL) was mixed with a vortex mixer. Next, 250 μL of ethanol SP (manufactured by Nacalai Tesque) was mixed, and then applied to spin columns 1 to 18, and centrifuged at 1930 G for 1 minute in a small microcentrifuge PMC-060 (manufactured by Tommy Seiko Co., Ltd.). Discarded. Next, 500 μL of Buffer RPE of RNeasy (registered trademark) (manufactured by Qiagen) was applied to the spin columns 1 to 18 as a washing solution, and centrifuged at 1930 G for 1 minute in a small microcentrifuge PMC-060 (manufactured by Tommy Seiko). The operation of discarding the filtrate was repeated twice. Finally, 30 μL of Nuclease-Free Water (Life Technologies) is applied to the spin columns 1 to 18 as an eluate, centrifuged at 1930 G for 1 minute in a small microcentrifuge PMC-060 (Tomy Seiko), and total. An aqueous RNA solution was obtained. The obtained total RNA recovery rate and total RNA degradation degree are shown in Tables 5-7. The total RNA recovery rate was calculated from the following formula (2).
Total RNA recovery rate (%) = {purified total RNA concentration (ng / μL) × eluate volume 30 (μL)} ÷ {input total RNA concentration 100 (ng / μL) × input total RNA solution volume 100 (μL)} × 100 (2)
次いで、前記スピンカラム１〜１８を用いて健常者血液からのｔｏｔａｌ ＲＮＡ回収率、ｔｏｔａｌ ＲＮＡ分解度の評価を行なった。試料溶液として、健常者血液（全血）２００μＬと、結合液としてＰｕｒｅＬｉｎｋ（登録商標）（ライフテクノロジーズ社製）のＬｙｓｉｓ Ｂｕｆｆｅｒ ２００μＬとを混合し、ボルテックスミキサーにて混合した。次いで、エタノールＳＰ ２００μＬ（ナカライテスク社製）を混合した後、スピンカラム１〜１８にアプライし、小型微量遠心機ＰＭＣ−０６０（トミー精工社製）にて１９３０Ｇで１分間遠心し、ろ液を廃棄した。次いで、ＰｕｒｅＬｉｎｋ（登録商標）（ライフテクノロジーズ社製）のＷａｓｈ ＢｕｆｆｅｒＩ ７００μＬをスピンカラム１〜１８にアプライし、小型微量遠心機ＰＭＣ−０６０（トミー精工社製）にて１９３０Ｇで１分間遠心し、ろ液を廃棄した。次いで、ＰｕｒｅＬｉｎｋ（登録商標）（ライフテクノロジーズ社製）のＷａｓｈ ＢｕｆｆｅｒＩＩ ５００μＬをスピンカラム１〜１８にアプライし、小型微量遠心機ＰＭＣ−０６０（トミー精工社製）にて１９３０Ｇで１分間遠心し、ろ液を廃棄する操作を２回繰り返した。最後に、溶出液としてＮｕｃｌｅａｓｅ−Ｆｒｅｅ Ｗａｔｅｒ（ライフテクノロジーズ社製）３０μＬをスピンカラム１〜１８にアプライし、小型微量遠心機 ＰＭＣ−０６０（トミー精工社製）にて１９３０Ｇで１分間遠心し、ｔｏｔａｌ ＲＮＡ水溶液を得た。得られたｔｏｔａｌ ＲＮＡ回収率、ｔｏｔａｌ ＲＮＡ分解度を表５〜７に示す。なお、ｔｏｔａｌ ＲＮＡ回収率は下記式（３）より算出した。
ｔｏｔａｌ ＲＮＡ回収率（％）＝｛精製ｔｏｔａｌ ＲＮＡ濃度（ｎｇ／μＬ）×溶出液量３０（μＬ）｝÷｛単位血液量当りのｔｏｔａｌ ＲＮＡ量１．５（ｎｇ／μＬ）×投入血液量２００（μＬ）｝×１００ （３）Subsequently, the total RNA recovery rate and the total RNA degradation degree from the blood of healthy subjects were evaluated using the spin columns 1-18. As a sample solution, 200 μL of healthy human blood (whole blood) and 200 μL of PureLink (registered trademark) (manufactured by Life Technologies) as a binding solution were mixed and mixed with a vortex mixer. Subsequently, 200 μL of ethanol SP (manufactured by Nacalai Tesque) was mixed, and then applied to spin columns 1 to 18 and centrifuged at 1930 G for 1 minute in a small microcentrifuge PMC-060 (manufactured by Tommy Seiko). Discarded. Next, 700 μL of PureLink (registered trademark) (manufactured by Life Technologies) was applied to spin columns 1 to 18 and centrifuged at 1930 G for 1 minute in a small microcentrifuge PMC-060 (manufactured by Tommy Seiko). The liquid was discarded. Next, 500 μL of Pure Buffer (registered trademark) (manufactured by Life Technologies) was applied to spin columns 1 to 18 and centrifuged at 1930 G for 1 minute in a small microcentrifuge PMC-060 (manufactured by Tommy Seiko). The operation of discarding the liquid was repeated twice. Finally, 30 μL of Nuclease-Free Water (Life Technologies) is applied to the spin columns 1 to 18 as an eluent, centrifuged at 1930 G for 1 minute in a small microcentrifuge PMC-060 (Tomy Seiko), and total. An aqueous RNA solution was obtained. The obtained total RNA recovery rate and total RNA degradation degree are shown in Tables 5-7. The total RNA recovery rate was calculated from the following formula (3).
Total RNA recovery rate (%) = {purified total RNA concentration (ng / μL) × eluate volume 30 (μL)} ÷ {total RNA amount per unit blood volume 1.5 (ng / μL) × input blood volume 200 (ΜL)} × 100 (3)
＜ｔｏｔａｌ ＲＮＡの回収率、ＲＮＡ分解度：シリンジ法＞
前記シリンジ１〜１８を用いてｔｏｔａｌ ＲＮＡ水溶液からのｔｏｔａｌ ＲＮＡ回収率、ｔｏｔａｌ ＲＮＡ分解度の評価を行なった。試料溶液として、ＨｅＬａ Ｓ３細胞よりセパゾール（登録商標）ＲＮＡＩ ＳｕｐｅｒＧ（ナカライテスク社製）を用いてプロトコール通りに分離精製した１００ｎｇ／μＬのｔｏｔａｌ ＲＮＡ水溶液（ＲＩＮ値＝９．９）５０μＬと、結合液としてＲＮｅａｓｙ（登録商標）（キアゲン社製）のＢｕｆｆｅｒ ＲＬＴ １７５μＬとを、ボルテックスミキサーにて混合した。次いで、エタノールＳＰ １２５μＬ（ナカライテスク社製）を混合した後、シリンジ１〜１８にて吸引吐出を１０回繰り返した後、ろ液を廃棄した。次いで、洗浄液としてＲＮｅａｓｙ（登録商標）（キアゲン社製）のＢｕｆｆｅｒ ＲＰＥ ５００μＬをシリンジ１〜１８にて吸引吐出を１０回繰り返した後、ろ液を廃棄する操作を２回繰り返した。最後に、溶出液としてＮｕｃｌｅａｓｅ−Ｆｒｅｅ Ｗａｔｅｒ（ライフテクノロジーズ社製）５０μＬをシリンジ１〜１８にて吸引吐出を１０回繰り返し、ｔｏｔａｌ ＲＮＡ水溶液を得た。得られたｔｏｔａｌ ＲＮＡ回収率、ｔｏｔａｌ ＲＮＡ分解度を表５〜７に示す。なお、ｔｏｔａｌ ＲＮＡ回収率は下記式（４）より算出した。
ｔｏｔａｌ ＲＮＡ回収率（％）＝｛精製ｔｏｔａｌ ＲＮＡ濃度（ｎｇ／μＬ）×溶出液量 ５０（μＬ）｝÷｛投入ｔｏｔａｌ ＲＮＡ濃度 １００（ｎｇ／μＬ）×投入ｔｏｔａｌ ＲＮＡ液量 ５０（μＬ）｝×１００ （４）<Total RNA recovery rate, RNA degradation degree: syringe method>
Using the syringes 1 to 18, the total RNA recovery rate from the total RNA aqueous solution and the total RNA degradation degree were evaluated. As a sample solution, 50 μL of 100 ng / μL total RNA aqueous solution (RIN value = 9.9) separated and purified according to the protocol using Sepasol (registered trademark) RNAI SuperG (manufactured by Nacalai Tesque) from HeLa S3 cells, and binding solution RNeasy (registered trademark) (manufactured by Qiagen) as Buffer RLT 175 μL was mixed with a vortex mixer. Subsequently, 125 μL of ethanol SP (manufactured by Nacalai Tesque) was mixed, and then suction and discharge were repeated 10 times with syringes 1 to 18, and then the filtrate was discarded. Next, 500 μL of Buffer RPE of RNeasy (registered trademark) (manufactured by Qiagen) as a cleaning solution was repeatedly aspirated and discharged 10 times with syringes 1 to 18, and then the operation of discarding the filtrate was repeated twice. Finally, 50 μL of Nuclease-Free Water (manufactured by Life Technologies) as an eluent was repeatedly aspirated and discharged 10 times with syringes 1 to 18 to obtain a total RNA aqueous solution. The obtained total RNA recovery rate and total RNA degradation degree are shown in Tables 5-7. The total RNA recovery rate was calculated from the following formula (4).
Total RNA recovery rate (%) = {purified total RNA concentration (ng / μL) × eluate amount 50 (μL)} ÷ {input total RNA concentration 100 (ng / μL) × input total RNA solution amount 50 (μL)} × 100 (4)
次いで、前記シリンジ１〜１８を用いて健常者血液からのｔｏｔａｌ ＲＮＡ回収率、ｔｏｔａｌ ＲＮＡ分解度の評価を行なった。試料溶液として、健常者血液（全血）３０μＬと、Ｎｕｃｌｅａｓｅ−Ｆｒｅｅ Ｗａｔｅｒ（ライフテクノロジーズ社製）７０μＬと、結合液としてＰｕｒｅＬｉｎｋ（登録商標）（ライフテクノロジーズ社製）のＬｙｓｉｓ Ｂｕｆｆｅｒ １００μＬとを混合し、ボルテックスミキサーにて混合した。次いで、エタノールＳＰ １００μＬ（ナカライテスク社製）を混合した後、シリンジ１〜１８にて吸引吐出を１０回繰り返した後、ろ液を廃棄した。次いで、ＰｕｒｅＬｉｎｋ（登録商標）（ライフテクノロジーズ社製）のＷａｓｈ ＢｕｆｆｅｒＩ ７００μＬをシリンジ１〜１８にて吸引吐出を１０回繰り返した後、ろ液を廃棄した。次いで、ＰｕｒｅＬｉｎｋ（登録商標）（ライフテクノロジーズ社製）のＷａｓｈ ＢｕｆｆｅｒＩＩ ５００μＬをシリンジ１〜１８にて吸引吐出を１０回繰り返した後、ろ液を廃棄する操作を２回繰り返した。最後に、溶出液としてＮｕｃｌｅａｓｅ−Ｆｒｅｅ Ｗａｔｅｒ（ライフテクノロジーズ社製）５０μＬをシリンジ１〜１８にて吸引吐出を１０回繰り返し、ｔｏｔａｌ ＲＮＡ水溶液を得た。得られたｔｏｔａｌ ＲＮＡ回収率、ｔｏｔａｌ ＲＮＡ分解度を表５〜７に示す。なお、ｔｏｔａｌ ＲＮＡ回収率は下記式（５）より算出した。
ｔｏｔａｌ ＲＮＡ回収率（％）＝｛精製ｔｏｔａｌ ＲＮＡ濃度（ｎｇ／μＬ）×溶出液量５０（μＬ）｝÷｛単位血液量当りのｔｏｔａｌ ＲＮＡ量１．５（ｎｇ／μＬ）×投入血液量１００（μＬ）｝×１００ （５）Subsequently, using the syringes 1 to 18, the total RNA recovery rate from the blood of healthy subjects and the total RNA degradation degree were evaluated. As a sample solution, 30 μL of healthy human blood (whole blood), 70 μL of Nuclease-Free Water (Life Technologies), and 100 μL of PureLink (registered trademark) (Life Technologies) Lysis Buffer as a binding solution, Mix with a vortex mixer. Subsequently, 100 μL of ethanol SP (manufactured by Nacalai Tesque) was mixed, and then suction and discharge were repeated 10 times with syringes 1 to 18, and then the filtrate was discarded. Subsequently, 700 μL of Pure Buffer (registered trademark) (manufactured by Life Technologies) was repeatedly sucked and discharged 10 times with syringes 1 to 18 and then the filtrate was discarded. Subsequently, after suction and discharge of 500 μL of PureLink (registered trademark) (manufactured by Life Technologies) with a syringe 1 to 18 was repeated 10 times, an operation of discarding the filtrate was repeated twice. Finally, 50 μL of Nuclease-Free Water (manufactured by Life Technologies) as an eluent was repeatedly aspirated and discharged 10 times with syringes 1 to 18 to obtain a total RNA aqueous solution. The obtained total RNA recovery rate and total RNA degradation degree are shown in Tables 5-7. The total RNA recovery rate was calculated from the following formula (5).
Total RNA recovery rate (%) = {purified total RNA concentration (ng / μL) × eluate volume 50 (μL)} ÷ {total RNA volume per unit blood volume 1.5 (ng / μL) × input blood volume 100 (ΜL)} × 100 (5)
＜Ｇｅｎｏｍｉｃ ＤＮＡの回収率：スピンカラム法＞
前記スピンカラム１〜１８を用いてＧｅｎｏｍｉｃ ＤＮＡ水溶液からのＧｅｎｏｍｉｃ ＤＮＡ回収率の評価を行なった。試料溶液として、１００ｎｇ／μＬのＨｕｍａｎ Ｇｅｎｏｍｉｃ ＤＮＡ（ロシュ社製）１００μＬと、結合液としてジーンキューブ専用前処理セット（東洋紡社製）の溶解吸着液３５０μＬとを混合し、ボルテックスミキサーにて混合した。次いで、エタノールＳＰ ２５０μＬ（ナカライテスク社製）を混合した後、スピンカラム１〜１８にアプライし、小型微量遠心機ＰＭＣ−０６０（トミー精工社製）にて１９３０Ｇで１分間遠心し、ろ液を廃棄した。次いで、洗浄液としてジーンキューブ専用前処理セット（東洋紡社製）の洗浄液５００μＬをスピンカラム１〜１８にアプライし、小型微量遠心機ＰＭＣ−０６０（トミー精工社製）にて１９３０Ｇで１分間遠心し、ろ液を廃棄する操作を２回繰り返した。最後に、溶出液としてＮｕｃｌｅａｓｅ−Ｆｒｅｅ Ｗａｔｅｒ（ライフテクノロジーズ社製）３０μＬをスピンカラム１〜１８にアプライし、小型微量遠心機ＰＭＣ−０６０（トミー精工社製）にて１９３０Ｇで１分間遠心し、Ｇｅｎｏｍｉｃ ＤＮＡ水溶液を得た。得られたＧｅｎｏｍｉｃ ＤＮＡ回収率を表５〜７に示す。なお、Ｇｅｎｏｍｉｃ ＤＮＡ回収率は下記式（６）より算出した。
Ｇｅｎｏｍｉｃ ＤＮＡ回収率（％）＝｛精製Ｇｅｎｏｍｉｃ ＤＮＡ濃度（ｎｇ／μＬ）×溶出液量３０（μＬ）｝÷｛投入Ｇｅｎｏｍｉｃ ＤＮＡ濃度１００（ｎｇ／μＬ）×投入Ｇｅｎｏｍｉｃ ＤＮＡ液量１００（μＬ）｝×１００ （６）<Recovery rate of Genomic DNA: Spin column method>
Using the spin columns 1 to 18, the genomic DNA recovery rate from the aqueous genomic DNA solution was evaluated. As a sample solution, 100 μL of 100 ng / μL Human Genomic DNA (manufactured by Roche) and 350 μL of a dissolved adsorption solution of Gene Cube pretreatment set (manufactured by Toyobo Co., Ltd.) as a binding solution were mixed and mixed with a vortex mixer. Next, 250 μL of ethanol SP (manufactured by Nacalai Tesque) was mixed, and then applied to spin columns 1 to 18, and centrifuged at 1930 G for 1 minute in a small microcentrifuge PMC-060 (manufactured by Tommy Seiko Co., Ltd.). Discarded. Next, 500 μL of Gene Cube exclusive pretreatment set (manufactured by Toyobo Co., Ltd.) was applied to the spin columns 1 to 18 as a cleaning liquid, and centrifuged at 1930 G for 1 minute in a small microcentrifuge PMC-060 (Tomy Seiko Co., Ltd.) The operation of discarding the filtrate was repeated twice. Finally, 30 μL of Nuclease-Free Water (Life Technologies) is applied to the spin columns 1 to 18 as an eluent, centrifuged at 1930 G for 1 minute in a small microcentrifuge PMC-060 (Tomy Seiko), and Genomic An aqueous DNA solution was obtained. The obtained genomic DNA recovery rates are shown in Tables 5-7. The genomic DNA recovery rate was calculated from the following formula (6).
Genomic DNA recovery (%) = {purified Genomic DNA concentration (ng / μL) × eluate volume 30 (μL)} ÷ {input Genomic DNA concentration 100 (ng / μL) × input Genomic DNA volume 100 (μL)} × 100 (6)
＜Ｇｅｎｏｍｉｃ ＤＮＡの回収率：シリンジ法＞
前記シリンジ１〜１８を用いてＧｅｎｏｍｉｃ ＤＮＡ水溶液からのＧｅｎｏｍｉｃ ＤＮＡ回収率の評価を行なった。試料溶液として、１００ｎｇ／μＬのＨｕｍａｎ Ｇｅｎｏｍｉｃ ＤＮＡ（ロシュ社製）５０μＬと、結合液としてジーンキューブ専用前処理セット（東洋紡社製）の溶解吸着液１７５μＬとを混合し、ボルテックスミキサーにて混合した。次いで、エタノールＳＰ １２５μＬ（ナカライテスク社製）を混合した後、シリンジ１〜１８にて吸引吐出を１０回繰り返した後、ろ液を廃棄した。次いで、洗浄液としてジーンキューブ専用前処理セット（東洋紡社製）の洗浄液５００μＬをシリンジ１〜１８にて吸引吐出を１０回繰り返した後、ろ液を廃棄する操作を２回繰り返した。最後に、溶出液としてＮｕｃｌｅａｓｅ−Ｆｒｅｅ Ｗａｔｅｒ（ライフテクノロジーズ社製）５０μＬをシリンジ１〜１８にて吸引吐出を１０回繰り返し、Ｇｅｎｏｍｉｃ ＤＮＡ水溶液を得た。得られたＧｅｎｏｍｉｃ ＤＮＡ回収率を表５〜７に示す。なお、Ｇｅｎｏｍｉｃ ＤＮＡ回収率は下記式（７）より算出した。
Ｇｅｎｏｍｉｃ ＤＮＡ回収率（％）＝｛精製Ｇｅｎｏｍｉｃ ＤＮＡ濃度（ｎｇ／μＬ）×溶出液量５０（μＬ）｝÷｛投入Ｇｅｎｏｍｉｃ ＤＮＡ濃度１００（ｎｇ／μＬ）×投入Ｇｅｎｏｍｉｃ ＤＮＡ液量５０（μＬ）｝×１００ （７）<Recovery rate of Genomic DNA: Syringe method>
Using the syringes 1 to 18, the recovery rate of Genomic DNA from the Genomic DNA aqueous solution was evaluated. As a sample solution, 50 μL of 100 ng / μL Human Genomic DNA (manufactured by Roche) and 175 μL of a dissolved adsorption solution of Gene Cube pretreatment set (manufactured by Toyobo Co., Ltd.) as a binding solution were mixed and mixed with a vortex mixer. Subsequently, 125 μL of ethanol SP (manufactured by Nacalai Tesque) was mixed, and then suction and discharge were repeated 10 times with syringes 1 to 18, and then the filtrate was discarded. Next, 500 μL of the Gene Cube pretreatment set (manufactured by Toyobo Co., Ltd.) as the cleaning liquid was repeatedly sucked and discharged 10 times with the syringes 1 to 18, and then the operation of discarding the filtrate was repeated twice. Finally, 50 μL of Nuclease-Free Water (manufactured by Life Technologies) was repeatedly aspirated and discharged 10 times with syringes 1 to 18 as an eluent to obtain an aqueous Genomic DNA solution. The obtained genomic DNA recovery rates are shown in Tables 5-7. The Genomic DNA recovery rate was calculated from the following formula (7).
Genomic DNA recovery rate (%) = {purified Genomic DNA concentration (ng / μL) × eluate volume 50 (μL)} ÷ {input Genomic DNA concentration 100 (ng / μL) × input Genomic DNA volume 50 (μL)} × 100 (7)
本発明により、高い核酸の回収率と高い固相担体の通液性を両立することができ、より迅速・簡便な核酸抽出可能となり、また核酸の分離精製工程の自動化も非常に容易であることからも、産業界に大きく寄与することが期待される。
According to the present invention, it is possible to achieve both a high recovery rate of nucleic acid and a high liquid permeability of a solid phase carrier, to enable more rapid and simple nucleic acid extraction, and it is very easy to automate the nucleic acid separation and purification process. Therefore, it is expected to greatly contribute to the industry.
Claims (14)
（Ａ）核酸を含む試料溶液と結合液とを混合して混合液を得る工程
（Ｂ）前記混合液と固相担体とを接触させ、固相担体に核酸を吸着させる工程
（Ｃ）固相担体から核酸以外の成分を洗浄する工程
（Ｄ）固相担体から核酸を脱着させる工程At least the following steps (A) to (D) are carried out using a solid phase carrier comprising a composition comprising at least (1) porous inorganic particles, (2) organic or inorganic fibers, and (3) a hydrophobic organic binder. A method for separating and purifying nucleic acid, which is performed in order.
(A) A step of mixing a sample solution containing nucleic acid and a binding solution to obtain a mixed solution (B) A step of bringing the mixed solution into contact with a solid phase carrier and adsorbing the nucleic acid to the solid phase carrier (C) A solid phase Step of washing components other than nucleic acid from carrier (D) Step of desorbing nucleic acid from solid phase carrier
A nucleic acid separation and purification kit for performing a method for separating and purifying nucleic acid, comprising: a solid phase carrier according to claim 12; a device according to claim 13; a binding solution for adsorbing nucleic acid on the solid phase carrier; A nucleic acid separation and purification kit comprising a washing solution for washing components other than nucleic acids from a phase carrier and an elution solution for eluting nucleic acids from a solid phase carrier.
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