JP2009065849A - Nucleic acid extraction method - Google Patents
Nucleic acid extraction method Download PDFInfo
- Publication number
- JP2009065849A JP2009065849A JP2007234914A JP2007234914A JP2009065849A JP 2009065849 A JP2009065849 A JP 2009065849A JP 2007234914 A JP2007234914 A JP 2007234914A JP 2007234914 A JP2007234914 A JP 2007234914A JP 2009065849 A JP2009065849 A JP 2009065849A
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- JP
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- Prior art keywords
- nucleic acid
- acid extraction
- extraction method
- solid phase
- phase carrier
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 182
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 182
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 182
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- 239000007790 solid phase Substances 0.000 claims abstract description 50
- 238000000034 method Methods 0.000 claims abstract description 43
- FZHAPNGMFPVSLP-UHFFFAOYSA-N silanamine Chemical compound [SiH3]N FZHAPNGMFPVSLP-UHFFFAOYSA-N 0.000 claims abstract description 34
- 125000003277 amino group Chemical group 0.000 claims abstract description 30
- -1 phosphate compound Chemical class 0.000 claims abstract description 30
- 229910019142 PO4 Inorganic materials 0.000 claims abstract description 24
- 239000010452 phosphate Substances 0.000 claims abstract description 24
- 238000006911 enzymatic reaction Methods 0.000 claims abstract description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 42
- 239000003960 organic solvent Substances 0.000 claims description 30
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 24
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 24
- 239000007864 aqueous solution Substances 0.000 claims description 21
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Abstract
【課題】安全な溶液中で固相担体に多量の核酸を効率良く吸着させるとともに、吸着された該核酸を高効率で脱離させ、簡単・迅速に高純度核酸抽出・精製を可能にする核酸抽出方法を提供する。また、抽出した核酸を断片化や増幅するための酵素反応に利用することを特徴とする遺伝子操作方法を提供すること。
【解決手段】表面にアミノシランを有する固相担体に前記アミノシランのアミノ基を介して静電的な結合により核酸を吸着させた後、該核酸をリン酸化合物溶液と反応させることにより、該核酸を溶媒中に再溶解させ、核酸抽出を行う。
【選択図】図３A nucleic acid capable of efficiently adsorbing a large amount of nucleic acid on a solid phase carrier in a safe solution and desorbing the adsorbed nucleic acid with high efficiency, enabling simple and rapid extraction and purification of high-purity nucleic acid. An extraction method is provided. The present invention also provides a genetic manipulation method characterized by utilizing the extracted nucleic acid in an enzymatic reaction for fragmentation or amplification.
The nucleic acid is adsorbed to a solid phase carrier having aminosilane on the surface through an electrostatic bond via an amino group of the aminosilane, and then reacted with a phosphate compound solution to thereby remove the nucleic acid. Re-dissolve in solvent and perform nucleic acid extraction.
[Selection] Figure 3
Description
本発明は、固相担体を用いて核酸の吸着・脱離を行う核酸抽出方法に関するものであり、特に核酸抽出用溶液中にリン酸化合物を添加した高効率な核酸抽出方法に関するものである。 The present invention relates to a nucleic acid extraction method for adsorbing and desorbing nucleic acids using a solid phase carrier, and more particularly to a highly efficient nucleic acid extraction method in which a phosphate compound is added to a nucleic acid extraction solution.
ヒトゲノム解読が完了し、近年、個人の遺伝情報を利用した研究開発が活発に行われている。遺伝子を対象とした解析研究や検査などの分野においては、血液などの生体サンプル中に含まれている核酸を簡便且つ高効率に抽出することが望まれる。
従来、分子生物学やバイオテクノロジー分野で用いられてきた核酸抽出方法としては、フェノール抽出などの有毒な有機溶剤を使用する古典的な方法に代わり、シリカビーズやシリカメンブレンフィルタなどのシリカ担体を用いて、これらシリカ担体の表面に核酸を高濃度のカオトロピック塩（塩酸グアニジン、グアニジンチオシアネートなど）を含む溶液中で選択的に吸着させる方法が用いられている（非特許文献１参照）。この方法によれば、危険な試薬を用いることなく、シリカ担体から核酸を容易に単離させることができ、効率的に核酸を回収することが可能である。
かかる方法として代表的なものは、1990年Boomらによって提唱された核酸抽出法（所謂、Boom法）が知られている。この核酸抽出法は、シリカコーティングした磁気ビーズを用い、カオトロピック反応により核酸の吸着、脱離を行う方法である(非特許文献２参照）。
また、これと同様の原理に基づく技術で、ポリエチレングリコール（PEG）存在下において、カルボキシル基で修飾された磁性ビーズに核酸が選択的に結合する現象を利用した核酸精製法（所謂、solid-phase reversible immobilization(SPRI) 法）が開発された（非特許文献３参照）。この核酸精製法によれば、遠心、ろ過、沈殿などの操作を必要とせず、簡単・迅速に高純度核酸抽出・精製が可能である。
これらの方法は、核酸抽出から遺伝子増幅・DNAシークエンスに至るまで同一プロトコルで処理することができ、自動化システムへの応用が可能であることから、現在最も汎用的な方法として用いられている。
しかしながら、Boom法は、核酸吸着工程において、刺激性、毒性を有するカオトロピック塩を高濃度条件において使用することが必須であるため、高濃度の塩が洗浄工程を経た後も残存し、核酸精製工程に続いて行われる遺伝子増幅やDNAの酵素切断などの際に悪影響を及ぼす可能性がある。また、Boom法は、種子サンプルなどの多糖類を多く含むサンプルを用いる場合には、カオトロピック塩の存在下で溶液が糊化するという欠点を有している。更に、核酸に結合した磁気ビーズを洗浄する操作では、70％エタノールが使用されるが、このエタノールが核酸精製工程後のプロセスに影響を及ぼすことが指摘されている。特に、マイクロチップデバイスのように、非常に微量な反応容量で核酸をハンドリングする必要がある場合には、その混入の危険性が高い。
SPRI法でも同様に、核酸吸着工程で用いられる高濃度塩（NaCl）の残存や洗浄工程における70％エタノールの混入による悪影響が問題となっている。
これらの問題点に対し、核酸を固定化させる固相表面と核酸との電荷相互作用を利用した核酸の単離方法が公表されている（特許文献１，２及び非特許文献４参照）。また、この単離方法とほぼ同様の原理（Charge-Switch technology）に基づくDNA抽出キットが市販されている。
これらの方法は、生体サンプル中の核酸をあるｐＨ条件下で活性化固相と接触せしめ、負電荷を持つ核酸を、固相表面に導入されたキトサンなどの正に帯電した極性基と静電的に結合させた後、溶液のｐＨを変化させて固相表面の電荷を正から負に切り替えることにより、固相表面から核酸を容易に脱離させる方法である。これらの方法は、カオトロピック塩や高濃度塩、70％エタノールを使用しないため、安全性の点で優れている。
Human genome decoding has been completed, and research and development using individual genetic information has been actively carried out in recent years. In the fields of analytical research and testing for genes, it is desirable to easily and efficiently extract nucleic acids contained in biological samples such as blood.
Conventionally, nucleic acid extraction methods used in the fields of molecular biology and biotechnology use silica carriers such as silica beads and silica membrane filters instead of the classical methods using toxic organic solvents such as phenol extraction. Thus, a method of selectively adsorbing nucleic acids on the surface of these silica supports in a solution containing a high concentration chaotropic salt (guanidine hydrochloride, guanidine thiocyanate, etc.) is used (see Non-Patent Document 1). According to this method, the nucleic acid can be easily isolated from the silica carrier without using a dangerous reagent, and the nucleic acid can be efficiently recovered.
A typical example of such a method is a nucleic acid extraction method (so-called Boom method) proposed by Boom et al. In 1990. This nucleic acid extraction method is a method of adsorbing and desorbing nucleic acid by chaotropic reaction using silica-coated magnetic beads (see Non-Patent Document 2).
In addition, a technology based on the same principle, a nucleic acid purification method (so-called solid-phase) using a phenomenon in which nucleic acid selectively binds to a magnetic bead modified with a carboxyl group in the presence of polyethylene glycol (PEG). reversible immobilization (SPRI) method) was developed (see Non-Patent Document 3). According to this nucleic acid purification method, high-purity nucleic acid extraction and purification can be performed easily and quickly without requiring operations such as centrifugation, filtration, and precipitation.
These methods can be processed with the same protocol from nucleic acid extraction to gene amplification / DNA sequencing, and can be applied to automated systems, so they are currently used as the most versatile methods.
However, in the Boom method, in the nucleic acid adsorption process, it is essential to use a chaotropic salt having irritation and toxicity under high concentration conditions. Therefore, the high concentration salt remains after the washing step, and the nucleic acid purification step. This may have an adverse effect on subsequent gene amplification or DNA enzymatic cleavage. In addition, the Boom method has a drawback that the solution is gelatinized in the presence of a chaotropic salt when a sample containing a large amount of polysaccharide such as a seed sample is used. Furthermore, 70% ethanol is used in the operation of washing the magnetic beads bound to the nucleic acid, and it has been pointed out that this ethanol affects the process after the nucleic acid purification step. In particular, when it is necessary to handle nucleic acids with a very small reaction volume as in a microchip device, the risk of contamination is high.
Similarly, in the SPRI method, there are problems such as the remaining high-concentration salt (NaCl) used in the nucleic acid adsorption process and the adverse effect of 70% ethanol in the washing process.
In order to solve these problems, methods for isolating nucleic acids using charge interaction between a solid phase surface on which nucleic acids are immobilized and nucleic acids have been published (see Patent Documents 1 and 2 and Non-Patent Document 4). A DNA extraction kit based on the principle (Charge-Switch technology) almost the same as this isolation method is commercially available.
In these methods, a nucleic acid in a biological sample is brought into contact with an activated solid phase under certain pH conditions, and a negatively charged nucleic acid is electrostatically charged with a positively charged polar group such as chitosan introduced on the surface of the solid phase. This is a method of easily desorbing nucleic acid from the solid phase surface by changing the pH of the solution and switching the charge on the solid phase surface from positive to negative. These methods are superior in terms of safety because they do not use chaotropic salts, high-concentration salts, or 70% ethanol.
しかしながら、上述の単離方法では、核酸の吸着反応・脱離反応がｐＨ依存的に制御されるため、核酸精製工程後のプロセスにおける溶液のｐＨ条件を管理する必要がある。従って、この単離方法では、ｐＨ6付近でDNAを吸着させ、ｐＨ8付近でDNAを脱離させるというプロトコールを改変することはできない。
そこで、本発明者は、核酸を結合させる担体材料として、表面がアミノシラン処理によりアミノ基で修飾された粒子を用いることにより、核酸を効率よく回収する方法を開発した（特開2006-280277号公報参照）。
従来より、アミノシランは、DNAマイクロアレイの核酸吸着担体材料として知られているが、核酸との結合力が強固であるがゆえ、この特性が核酸の単離工程で支障になることが容易に想像され、上述の単離方法でも利用されていない。
これに対し、本発明者は、多価アニオンによりアミノシランから核酸を高効率で脱離できることを見出し、この現象を利用した、新たな核酸単離方法を提案した。
以上のように、生体サンプル中から効率的に核酸を回収するには、核酸の担体への吸着と脱離を的確に行うための条件検討が重要である。特に、遺伝子解析技術の高度化に伴い、微量サンプルからの高感度解析・診断が進むにしたがって、核酸抽出効率の向上が一層望まれている。
However, in the isolation method described above, since the nucleic acid adsorption / desorption reaction is controlled in a pH-dependent manner, it is necessary to manage the pH conditions of the solution in the process after the nucleic acid purification step. Therefore, this isolation method cannot modify the protocol for adsorbing DNA near pH 6 and desorbing DNA near pH 8.
Therefore, the present inventor has developed a method for efficiently recovering nucleic acids by using particles whose surfaces are modified with amino groups by aminosilane treatment as a carrier material for binding nucleic acids (Japanese Patent Laid-Open No. 2006-280277). reference).
Conventionally, aminosilane has been known as a nucleic acid adsorption carrier material for DNA microarrays, but it is easily imagined that this property may interfere with the nucleic acid isolation process because of its strong binding force with nucleic acids. It is not used in the above-described isolation method.
In contrast, the present inventor has found that nucleic acids can be desorbed from aminosilane with high efficiency by a polyvalent anion, and has proposed a new nucleic acid isolation method utilizing this phenomenon.
As described above, in order to efficiently recover a nucleic acid from a biological sample, it is important to examine conditions for accurately performing adsorption and desorption of the nucleic acid on a carrier. In particular, with the advancement of gene analysis technology, the improvement of nucleic acid extraction efficiency is further desired as highly sensitive analysis / diagnosis from a small amount of sample proceeds.
本発明者等は、上述の課題を解決すべく鋭意研究した結果、固相担体表面のアミノシランと結合した核酸を脱離させる反応において、図１に示すように、溶出液にリン酸化合物を加えることにより、該リン酸化合物と前記核酸とが置き換わり、容易に核酸を脱離できること、また低濃度の有機溶剤の添加により脱離効率が飛躍的に向上することを見出し、本発明を完成するに至った。
即ち、本発明は以下の事項に関する。
（１）生体試料を表面にアミノシランを有する固相担体と混合し、前記アミノシランのアミノ基と前記生体試料中の核酸のリン酸基を静電的に結合させた後、該核酸と該核酸より前記アミノ基に対する官能性が高いリン酸基を有するリン酸化合物を含む水溶液とを接触させ、リン酸基置換反応により該核酸を前記水溶液中に溶出させることを特徴とする核酸抽出方法。
（２）前記リン酸化合物がリン酸ジエステル結合を有することを特徴とする（１）に記載の核酸抽出方法
（３）前記リン酸化合物はヌクレオチドであることを特徴とする（２）に記載の核酸抽出方法。
（４）前記リン酸化合物を含む水溶液中に有機溶媒が添加されることを特徴とする（１）乃至（３）のいずれかに記載の核酸抽出方法。
（５）前記有機溶媒は、メタノール、エタノール、プロパノール及びその異性体、ブタノール及びその異性体から選ばれる少なくとも１つであることを特徴とする（４）に記載の核酸抽出方法。
（６）前記有機溶媒の濃度が０．５〜２０％であることを特徴とする（４）又は（５）に記載の核酸抽出方法。
（７）前記固相担体は、シリカ、ガラス、金属、ポリスチレン、ポリサッカライド、セラミック、人工磁性体、バクテリア由来の磁性体から選ばれる少なくとも１つであることを特徴とする（１）乃至（６）のいずれか１項に記載の核酸抽出方法。
（８）（１）乃至（７）のいずれか１項に記載の核酸抽出方法により、抽出した核酸を断片化や増幅するための酵素反応に利用することを特徴とする遺伝子操作方法。
（９）（１）乃至（７）のいずれか1項に記載の核酸抽出方法を行うための核酸抽出用試薬キット。
As a result of diligent research to solve the above-mentioned problems, the present inventors have added a phosphate compound to the eluate in a reaction for desorbing nucleic acid bound to aminosilane on the surface of the solid phase carrier as shown in FIG. Thus, the phosphoric acid compound and the nucleic acid are replaced and the nucleic acid can be easily desorbed, and the desorption efficiency is dramatically improved by the addition of a low concentration organic solvent, and the present invention is completed. It came.
That is, the present invention relates to the following matters.
(1) A biological sample is mixed with a solid phase carrier having aminosilane on the surface, and the amino group of the aminosilane and the phosphate group of the nucleic acid in the biological sample are electrostatically bound, and then the nucleic acid and the nucleic acid A nucleic acid extraction method comprising contacting an aqueous solution containing a phosphate compound having a phosphate group having high functionality with respect to the amino group, and eluting the nucleic acid into the aqueous solution by a phosphate group substitution reaction.
(2) The nucleic acid extraction method according to (1), wherein the phosphate compound has a phosphodiester bond (3), wherein the phosphate compound is a nucleotide. Nucleic acid extraction method.
(4) The nucleic acid extraction method according to any one of (1) to (3), wherein an organic solvent is added to the aqueous solution containing the phosphate compound.
(5) The nucleic acid extraction method according to (4), wherein the organic solvent is at least one selected from methanol, ethanol, propanol and its isomer, butanol and its isomer.
(6) The nucleic acid extraction method according to (4) or (5), wherein the concentration of the organic solvent is 0.5 to 20%.
(7) The solid phase carrier is at least one selected from silica, glass, metal, polystyrene, polysaccharide, ceramic, artificial magnetic substance, and bacterial-derived magnetic substance (1) to (6) The nucleic acid extraction method according to any one of (1).
(8) A gene manipulation method characterized by being used in an enzymatic reaction for fragmenting or amplifying the nucleic acid extracted by the nucleic acid extraction method according to any one of (1) to (7).
(9) A nucleic acid extraction reagent kit for performing the nucleic acid extraction method according to any one of (1) to (7).
本発明では、固相担体に核酸を吸着させるに際し、核酸と前記固相担体表面に形成されたアミノシランを結合させることにより、固相担体表面に多量の核酸を吸着させることができる。
また、核酸を脱離させる際に固相担体表面に吸着させた核酸を脱離させる際に、リン酸化合物を使用することにより、前記核酸が該化合物と置き換わり、容易に核酸を水溶液中に再溶解させることができる。
従って、本発明によれば、高濃度のフェノールやクロロホルムなどの有機溶媒を用いることなく、また洗浄・精製後に行われる遺伝子増幅やDNAの酵素切断などの際に、酵素阻害等の影響を受けずに、核酸を効率よく回収することができる。
更に、本発明では、低濃度の有機溶剤を添加することにより、脱離効率を飛躍的に向上させることができる。このため、特にごく少量のサンプル中から対象とする核酸を効率よく単離する必要がある場合や、サンプル中に含まれる核酸の量が微量である場合でも、高純度・高回収率で核酸抽出を行うことが可能となる。
かかる核酸の吸着・脱離工程は、核酸精製後に続く遺伝子増幅・DNAシークエンスに至るまでの工程と同一プロトコルで処理することができる。従って、本発明によれば、自動化システムへの応用が可能であり、ＰＣＲ法及びＳＮＰ自動検出の統合化が可能な核酸の抽出方法を提供することが可能になる。
In the present invention, when adsorbing a nucleic acid on a solid phase carrier, a large amount of nucleic acid can be adsorbed on the surface of the solid phase carrier by binding the nucleic acid and aminosilane formed on the surface of the solid phase carrier.
Further, when the nucleic acid adsorbed on the surface of the solid phase carrier is desorbed when the nucleic acid is desorbed, by using a phosphate compound, the nucleic acid is replaced with the compound, and the nucleic acid is easily re-introduced into the aqueous solution. Can be dissolved.
Therefore, according to the present invention, it is not affected by enzyme inhibition or the like in gene amplification or DNA enzymatic cleavage performed after washing and purification without using a high concentration organic solvent such as phenol or chloroform. In addition, the nucleic acid can be efficiently recovered.
Furthermore, in the present invention, the desorption efficiency can be dramatically improved by adding a low concentration organic solvent. For this reason, even when it is necessary to efficiently isolate the target nucleic acid from a very small amount of sample or when the amount of nucleic acid contained in the sample is very small, nucleic acid extraction with high purity and high recovery rate is possible. Can be performed.
The nucleic acid adsorption / desorption step can be processed by the same protocol as the steps up to gene amplification / DNA sequencing following the nucleic acid purification. Therefore, according to the present invention, it is possible to provide a nucleic acid extraction method that can be applied to an automation system and can integrate the PCR method and SNP automatic detection.
以下、本発明の実施の形態を詳細に説明する。
本発明の核酸の抽出方法は、生体試料を表面にアミノシランを有する固相担体と混合し、前記アミノシランのアミノ基を介して前記生体試料中の核酸を固相担体に静電的に結合させた後、該核酸と該核酸より前記アミノ基に対する官能性が高いリン酸基を有するリン酸化合物を含む水溶液とを接触させ、リン酸基置換反応により該核酸を前記水溶液中に溶出させることを特徴としている。
Hereinafter, embodiments of the present invention will be described in detail.
In the nucleic acid extraction method of the present invention, a biological sample is mixed with a solid phase carrier having aminosilane on the surface, and the nucleic acid in the biological sample is electrostatically bound to the solid phase carrier via the amino group of the aminosilane. Thereafter, the nucleic acid is brought into contact with an aqueous solution containing a phosphate compound having a phosphate group having higher functionality for the amino group than the nucleic acid, and the nucleic acid is eluted into the aqueous solution by a phosphate group substitution reaction. It is said.
＜アミノシランを有する固相担体＞
[固相担体について]
本発明の核酸の抽出方法において使用される固相担体は、その表面に核酸を多量に吸着させるためのアミノシランが導入されてなる。表面がアミノシランにより修飾された固相担体では、化学的に安定なアミノ基による正電荷が付与される。このため、生体試料中の負電荷を有する成分を強い接着力により非特異的に吸着できる。中でも、核酸はアミノシランに対する親和性が高いため、前記アミノ基を介して該固相担体に強固に結合される。これにより、効率的に核酸を分離することができる。
アミノシランを表面に有する担体の核酸吸着量は、核酸の濃度との間に次式（１）のラングミュア型吸着等温式で示される関係が成り立つ。
<Solid phase carrier having aminosilane>
[About solid support]
The solid phase carrier used in the nucleic acid extraction method of the present invention has an aminosilane introduced on its surface for adsorbing a large amount of nucleic acid. In the solid phase carrier whose surface is modified with aminosilane, a positive charge is imparted by a chemically stable amino group. For this reason, the component which has a negative charge in a biological sample can be adsorb | sucked nonspecifically with strong adhesive force. Among them, since nucleic acid has high affinity for aminosilane, it is firmly bound to the solid phase carrier via the amino group. Thereby, nucleic acids can be separated efficiently.
The nucleic acid adsorption amount of the carrier having aminosilane on the surface has a relationship represented by the Langmuir type adsorption isotherm of the following formula (1) with the nucleic acid concentration.
Ｎ＝ＮｍａｘＫＣ／（１＋ＫＣ）・・・（１） N = N max KC / (1 + KC) (1)
但し、上式（１）中、Ｎは吸着量（ｐｇ／ｃｍ2）、Ｎｍａｘは最大吸着量（ｐｇ／ｃｍ2）、Ｋは吸着定数（ｍｌ／ｍｏｌ）、Ｃは溶質濃度（ｐｇ/μｌ）をそれぞれ示す。
例えば、（３−アミノプロピル）モノエトキシジメチルシラン[（3-aminopropyl）monoethoxydimethoxydimethylsilane]を固定化したシリカ担体へのオリゴヌクレオチド（溶質）の吸着においては、オリゴヌクレオチドの濃度に依存してシリカ担体に吸着する（図２参照）。
この場合、吸着定数は4.67×1012 ml/molであり、吸着速度は極めて速く、脱離速度は極めて遅い。このような現象は、アミノシランの高い吸着力によるものであり、前記シリカ担体への牛血清アルブミンの吸着・脱離における吸着定数Ｋはオリゴヌクレオチドの場合と同様に大きく、0.5×1010cm3/molである（J. Colloid Interface Sci. Vol.194, p408-418参照）。この事実から明らかなように、かかる現象は核酸特異的に起こるものではない。
In the above formula (1), N is the adsorption amount (pg / cm 2 ), N max is the maximum adsorption amount (pg / cm 2 ), K is the adsorption constant (ml / mol), and C is the solute concentration (pg / cm 2 ). μl) is shown respectively.
For example, in the adsorption of oligonucleotides (solutes) to a silica support on which (3-aminopropyl) monoethoxydimethylsilane [(3-aminopropyl) monoethoxydimethoxydimethylsilane] is immobilized, the adsorption to the silica support depends on the concentration of the oligonucleotide. (See FIG. 2).
In this case, the adsorption constant is 4.67 × 10 12 ml / mol, the adsorption rate is extremely fast, and the desorption rate is very slow. Such a phenomenon is due to the high adsorption force of aminosilane, and the adsorption constant K in the adsorption / desorption of bovine serum albumin on the silica carrier is as large as in the case of oligonucleotide, 0.5 × 10 10 cm 3 / mol (see J. Colloid Interface Sci. Vol.194, p408-418). As is apparent from this fact, this phenomenon does not occur in a nucleic acid specific manner.
本発明では、かかるアミノシランの特性に着目し、核酸の固相担体への吸着工程において、この原理を利用する。これにより、極めて強力に核酸の吸着を行うことができる。
この固相担体としては、例えばシリカ、ガラス、金属、ポリスチレン、ポリサッカライド、セラミック、人工磁性体、バクテリア由来の磁性体等が挙げられ、磁性及び大きさ等の観点から、特にバクテリア由来の磁性体（図３参照）が好適である。
前記バクテリア由来の磁性体は、酸化鉄で構成された単磁区構造を有し、その大きさは５０〜１００ｎｍ程度である。
In the present invention, paying attention to the characteristics of such aminosilane, this principle is used in the adsorption process of nucleic acid to a solid phase carrier. Thereby, nucleic acid can be adsorbed extremely strongly.
Examples of the solid support include silica, glass, metal, polystyrene, polysaccharides, ceramics, artificial magnetic materials, and bacterial-derived magnetic materials. From the viewpoint of magnetism and size, the bacterial-derived magnetic materials are particularly preferable. (See FIG. 3) is preferable.
The bacterium-derived magnetic substance has a single magnetic domain structure composed of iron oxide, and its size is about 50 to 100 nm.
この固相担体は、その表面構造が単層とされることは勿論、さらに表面処理を施すことによって二層以上に分岐を繰り返すデンドリマー状とされてもよく、抽出したい核酸の性質に応じて適宜設定することができる。 This solid phase carrier may be of a dendrimer shape that repeats branching into two or more layers by subjecting it to a surface treatment, as well as having a single layer surface structure, and is appropriately selected according to the nature of the nucleic acid to be extracted. Can be set.
[アミノシランについて]
前記固相担体表面にアミノシランを保持せしめる方法としては、固相担体表面をアミノシラン処理する方法などが挙げられる。
前記アミノシラン処理は、特に限定されるものではないが、例えばアミノシリル化剤やアミノシランカップリング剤などの薬剤による処理などが挙げられる。
前記アミノシランカップリング剤を用いて処理する際には、固相担体に存在するヒドロキシル基を表面に露出させることが好ましい。例えば、固相担体としてバクテリア由来の磁性体等の磁性体を採択した場合、該固相担体表面に存在する脂質二重膜を除去することによって表面のヒドロキシル基を活性化させ、アミノシリル化反応、アミノシランカップリング反応を促進することができる。該固相担体上の脂質二重膜を除去する方法は、特に限定されるものではないが、例えば該固相担体を有機溶媒、界面活性剤、強アルカリ等で処理する方法が使用可能である。
該アミノシランカップリング剤は、前記固相担体表面のヒドロキシル基をアミノシランカップリングすることができれば、特に制限されるものでないが、入手し易さの観点から、例えば３-[２-(２-アミノエチル)-エチルアミン]-プロピルトリメトキシシラン（ＡＥＥＡ）や３-アミノプロピルトリエトキシシラン（ＡＰＴＥＳ）等が好ましい。また前記シランカップリング剤を溶解させる溶媒としては、該アミノカップリング剤を溶解することができれば特に制限されるものではなく、アルコール、炭化水素、芳香族炭化水素等の従来公知の溶媒がいずれも使用可能である。好ましくはアミノ基を多量に導入することができることから芳香族炭化水素とされ、より好ましくはトルエンとされる。
このように、界面活性剤等により前記固相担体上に存在する脂質二重膜を除去した後、アミノシランカップリング剤を所定時間反応させることによりアミノシラン処理を施せば、固相担体表面にアミノ基を導入することができる。
ここで、該アミノ基の導入数は、スクシイミジル基を有し、第１級アミンと１対１で反応するサルフォサクシイミド６-[３'-(２-ピリジルジチオール)-プロピオンアミド]ヘキサノエイト（sulfo-LC-SPDP）と反応させ、ＤＴＴ等のチオール性還元剤によってジスルフィド結合を切断し、遊離する化合物の吸光度を測定することにより定量することができる。
[About aminosilane]
Examples of the method for retaining aminosilane on the surface of the solid phase carrier include a method of treating the surface of the solid phase carrier with aminosilane.
The aminosilane treatment is not particularly limited, and examples thereof include treatment with an agent such as an aminosilylating agent and an aminosilane coupling agent.
When the treatment is performed using the aminosilane coupling agent, it is preferable to expose the hydroxyl group present on the solid support to the surface. For example, when a magnetic substance such as a magnetic substance derived from bacteria is adopted as the solid phase carrier, the hydroxyl group on the surface is activated by removing the lipid bilayer present on the surface of the solid phase carrier, an aminosilylation reaction, The aminosilane coupling reaction can be promoted. The method for removing the lipid bilayer membrane on the solid phase carrier is not particularly limited. For example, a method of treating the solid phase carrier with an organic solvent, a surfactant, a strong alkali, or the like can be used. .
The aminosilane coupling agent is not particularly limited as long as the hydroxyl group on the surface of the solid phase carrier can be aminosilane-coupled. However, from the viewpoint of availability, for example, 3- [2- (2-amino Ethyl) -ethylamine] -propyltrimethoxysilane (AEEA), 3-aminopropyltriethoxysilane (APTES) and the like are preferable. The solvent for dissolving the silane coupling agent is not particularly limited as long as the amino coupling agent can be dissolved, and any conventionally known solvents such as alcohols, hydrocarbons, and aromatic hydrocarbons can be used. It can be used. Preferably, it is an aromatic hydrocarbon because it can introduce a large amount of amino groups, and more preferably toluene.
Thus, after removing the lipid bilayer present on the solid phase carrier with a surfactant or the like, if aminosilane treatment is performed by reacting the aminosilane coupling agent for a predetermined time, an amino group is formed on the surface of the solid phase carrier. Can be introduced.
Here, the number of amino groups introduced is sulphosuccinimide 6- [3 ′-(2-pyridyldithiol) -propionamide] hexanoate having a succinimidyl group and reacting one-to-one with a primary amine ( It can be quantified by reacting with sulfo-LC-SPDP), cleaving the disulfide bond with a thiol reducing agent such as DTT, and measuring the absorbance of the released compound.
なお、アミノ基が表面に導入された固相担体は、炭化水素、アルコール、超純水等に安定に保存することができる。 The solid phase carrier having amino groups introduced on the surface can be stably stored in hydrocarbon, alcohol, ultrapure water, or the like.
＜核酸の吸着＞
本発明の核酸の抽出方法においては、核酸が溶解した溶液中で前記固相担体と該核酸を接触させ、該核酸を前記固相担体表面に形成されたアミノシランのアミノ基に吸着させる。これにより、ターゲットとなる核酸を前記固相担体表面に多量に効率よく吸着させることができる。
<Nucleic acid adsorption>
In the nucleic acid extraction method of the present invention, the solid phase carrier is brought into contact with the nucleic acid in a solution in which the nucleic acid is dissolved, and the nucleic acid is adsorbed to the amino group of aminosilane formed on the surface of the solid phase carrier. Thereby, a large amount of target nucleic acid can be efficiently adsorbed on the surface of the solid phase carrier.
例えば、前記固相担体としてバクテリア由来の磁性体を用いた場合、図３に示すように、該磁性体表面に形成されたアミノシランのアミノ基と核酸のリン酸基が静電相互作用により結合し、該磁性体への核酸の吸着が起こる。
前記生体試料としては、培養細胞、動物由来の細胞又は組織（血液、血清、バフィーコート、体液、リンパ球等）、植物由来の細胞又は組織、あるいは細菌、真菌、ウィルス等、種々の生物を対象とした材料がいずれも使用可能である。
かかる生体試料は、該生体試料に含まれる核酸成分が溶液中に溶解された状態であることが好ましい。前記溶液としては、用いられる生体試料の種類に応じて、適宜選定されれば良く、例えば2-(Ｎ-ｍorpholino)ethanesulfonic acid（ＭＥＳ）緩衝液、トリス塩酸緩衝液(Ｔｒｉｓ−ＨＣｌバッファー)などの一般に使用される溶液がいずれも使用可能である。
For example, when a bacterium-derived magnetic substance is used as the solid phase carrier, as shown in FIG. 3, the amino group of aminosilane formed on the surface of the magnetic substance and the phosphate group of nucleic acid are bound by electrostatic interaction. Adsorption of nucleic acid to the magnetic material occurs.
Examples of the biological sample include cultured cells, animal-derived cells or tissues (blood, serum, buffy coat, body fluid, lymphocytes, etc.), plant-derived cells or tissues, or various organisms such as bacteria, fungi, and viruses. Any of these materials can be used.
Such a biological sample is preferably in a state in which a nucleic acid component contained in the biological sample is dissolved in a solution. The solution may be appropriately selected according to the type of biological sample to be used. For example, 2- (N-morpholino) ethanesulfonic acid (MES) buffer, Tris-HCl buffer (Tris-HCl buffer), etc. Any of the commonly used solutions can be used.
かかる溶液のｐＨは５〜９の範囲内、より好ましくは５〜８の範囲内に調節される。ｐＨ条件をかかる範囲内とすることにより、安定した核酸吸着量を実現することができる。
また、該溶液には、低濃度の有機溶媒が添加されることが好ましい。該溶液中の有機溶媒は核酸の固相担体への吸着を促進することができる。
この低濃度有機溶媒としては、例えばアセトン、アセトニトリル、アルコール類、ジメチルホルムアミド等がいずれも使用可能であり、これらは単独又は組み合わせて使用されれば良い。
尚、固相担体に対する核酸の吸着量は、前記溶液中に添加した核酸量と固相担体表面のアミノ基と反応させ洗浄後に上清液から回収された核酸量との差をとることによって算出することができる。核酸の定量は、核酸定量試薬により行うことができる。
The pH of such a solution is adjusted within the range of 5-9, more preferably within the range of 5-8. By setting the pH condition within this range, a stable nucleic acid adsorption amount can be realized.
Moreover, it is preferable that a low concentration organic solvent is added to the solution. The organic solvent in the solution can promote the adsorption of the nucleic acid to the solid phase carrier.
As the low-concentration organic solvent, for example, acetone, acetonitrile, alcohols, dimethylformamide and the like can be used, and these may be used alone or in combination.
The amount of nucleic acid adsorbed to the solid phase carrier is calculated by taking the difference between the amount of nucleic acid added to the solution and the amount of nucleic acid recovered from the supernatant after washing with the amino group on the surface of the solid phase carrier. can do. Nucleic acid quantification can be performed with a nucleic acid quantification reagent.
＜核酸の脱離＞
[リン酸化合物を含む水溶液について]
本発明においては、前記固相担体表面に形成されたアミノ基と結合した多量の核酸を効率よく脱離するために、リン酸化合物を使用する。図３に示すように、核酸抽出用の水溶液中に電離した状態で存在する前記リン酸化合物が前記核酸と置き換わって前記アミノ基と結合し、前記核酸は前記水溶液中に再溶解するため、前記固相担体からの前記核酸の脱離が容易に起こる。
<Desorption of nucleic acid>
[Aqueous solution containing phosphate compound]
In the present invention, a phosphate compound is used in order to efficiently desorb a large amount of nucleic acid bound to the amino group formed on the surface of the solid phase carrier. As shown in FIG. 3, the phosphate compound existing in an ionized state in an aqueous solution for nucleic acid extraction is replaced with the nucleic acid and bonded to the amino group, and the nucleic acid is redissolved in the aqueous solution. Desorption of the nucleic acid from the solid support easily occurs.
該リン酸化合物としては、アミノ基との静電相互作用が核酸よりも大きいものであれば良く、例えばリン酸ジエステル結合を有する化合物等が好ましい。具体的に例示すれば、ADP、GDP、TDP、CDP、UDPのリボヌクレオシド二リン酸、ATP、GTP、TTP、CTP、UTPのリボヌクレオシド三リン酸、dADP、dTDP、dGDP、dCDPのデオキシリボヌクレオシド二リン酸、dATP、dTTP、dGTP、dCTP又はこれら４種の混合物（dNTP）、dUTPのデオキシリボヌクレオシド三リン酸、ポリリン酸等である。特に、デオキシリボヌクレオシド三リン酸は通常PCRの試薬として使用されるため、上述の脱離工程に次いで行われる遺伝子増幅の際に影響を及ぼすことがない点で優れている。
これらリン酸化合物の濃度は、１．０ｍＭ〜５００ｍＭであることが必要である。濃度が１．０ｍＭ未満であると、核酸の脱離が不十分となり、一方、濃度が５００ｍＭを超えると、ＰＣＲ法による核酸増幅に障害が生じる。
The phosphate compound may be any compound having an electrostatic interaction with an amino group larger than that of the nucleic acid, and for example, a compound having a phosphate diester bond is preferable. Specifically, ADP, GDP, TDP, CDP, UDP ribonucleoside diphosphate, ATP, GTP, TTP, CTP, UTP ribonucleoside triphosphate, dADP, dTDP, dGDP, dCDP deoxyribonucleoside diphosphate Phosphoric acid, dATP, dTTP, dGTP, dCTP, or a mixture of these four (dNTP), dUTP deoxyribonucleoside triphosphate, polyphosphoric acid, and the like. In particular, since deoxyribonucleoside triphosphate is usually used as a PCR reagent, it is excellent in that it does not affect the gene amplification performed after the above-described desorption step.
The concentration of these phosphate compounds needs to be 1.0 mM to 500 mM. When the concentration is less than 1.0 mM, nucleic acid is insufficiently desorbed. On the other hand, when the concentration exceeds 500 mM, nucleic acid amplification by the PCR method is impaired.
また、該リン酸化合物を含む水溶液には、低濃度の有機溶媒が添加されることが好ましい。これにより、核酸の脱離効率を飛躍的に向上させることができる。
該有機溶媒としては、前記水溶液に可溶なものであれば良く、例えばアセトン、アセトニトリル、アルコール類、ジメチルホルムアミド等から適宜選択し又は組み合わせて使用可能である。中でもメタノール、エタノール、プロパノール及びその異性体、ブタノール及びその異性体等のアルコール類が好適であるが、環境負荷の点や安全性の面から、特にエタノールが好適である。
Moreover, it is preferable that a low concentration organic solvent is added to the aqueous solution containing the phosphoric acid compound. Thereby, the nucleic acid desorption efficiency can be dramatically improved.
The organic solvent is not particularly limited as long as it is soluble in the aqueous solution, and can be appropriately selected or used in combination from acetone, acetonitrile, alcohols, dimethylformamide, and the like. Among them, alcohols such as methanol, ethanol, propanol and isomers thereof, butanol and isomers thereof are preferable, but ethanol is particularly preferable from the viewpoint of environmental burden and safety.
かかる有機溶媒の濃度は、０．５〜２０％であることが好ましい。有機溶媒の濃度が０．５％を下回る範囲では、十分な効果を期待することができず、逆に２０％を超える範囲では、核酸の脱離工程に続く洗浄工程後での残存により、次いで行われる遺伝子増幅やDNAの酵素切断などの際に、酵素阻害等の影響を受ける虞が生じる。
更に、前記水溶液に少量の塩化ナトリウム等の電解質を添加することにより、該水溶液のイオン強度を適宜調整しても良い。
The concentration of the organic solvent is preferably 0.5 to 20%. If the concentration of the organic solvent is less than 0.5%, a sufficient effect cannot be expected. Conversely, if the concentration exceeds 20%, the remaining after the washing step following the nucleic acid desorption step, When gene amplification or DNA enzymatic cleavage is performed, there is a risk of being affected by enzyme inhibition or the like.
Furthermore, the ionic strength of the aqueous solution may be appropriately adjusted by adding a small amount of an electrolyte such as sodium chloride to the aqueous solution.
以上の核酸脱離工程においては、多量の核酸が吸着した固相担体上に前記リン酸化合物を含む水溶液を加え、所定の温度にてインキュベートした後、核酸を脱離する。インキュベートする温度としては、１０℃〜９０℃、好ましくは２０℃〜８０℃である。インキュベートする温度が１０℃未満であると核酸の脱離が起こりにくく、９０℃以上であると脱離した核酸に障害を与えることとなり好ましくない。なお、前記リン酸化合物の濃度とインキュベートする温度の最適化、ＰＣＲ法の適用を考えると、核酸の脱離する割合が同程度ならば、核酸抽出用の前記水溶液は低塩下であることが好ましいので、リン酸化合物の濃度を低く設定するとよい。 In the nucleic acid desorption step described above, an aqueous solution containing the phosphate compound is added to a solid phase carrier on which a large amount of nucleic acid has been adsorbed, and incubated at a predetermined temperature, and then the nucleic acid is desorbed. The incubation temperature is 10 ° C to 90 ° C, preferably 20 ° C to 80 ° C. If the incubation temperature is less than 10 ° C., the nucleic acid is hardly detached, and if it is 90 ° C. or higher, the detached nucleic acid is undesirably damaged. In consideration of optimization of the phosphate compound concentration and incubation temperature, and application of the PCR method, the nucleic acid extraction solution may be under low salt if the rate of nucleic acid desorption is comparable. It is preferable to set the concentration of the phosphate compound low.
本発明において、前記固相担体表面に吸着した核酸の、前記リン酸化合物を含む水溶液によって脱離した量を定量するには、該核酸が吸着した固相担体に前記水溶液を加えた後の上澄み液中に遊離している核酸量を測定することにより行う。 In the present invention, in order to quantify the amount of nucleic acid adsorbed on the surface of the solid phase carrier by the aqueous solution containing the phosphate compound, the supernatant after adding the aqueous solution to the solid phase carrier adsorbed with the nucleic acid This is done by measuring the amount of nucleic acid released in the solution.
＜ＰＣＲ法への適用＞
本発明においては、前記核酸の抽出方法を従来のＰＣＲ法に統合することができる。すなわち、本発明の核酸の抽出方法により吸着、脱離し、抽出した核酸をテンプレートし、核酸増幅物とすることができる。特に、本発明の場合、水溶液で核酸抽出を行うことができるので酵素阻害等の悪影響を防止することができる。ここで、ＰＣＲは、核酸抽出物を所定の温度で、所定時間反応、変性させた後、伸長させる。ＰＣＲ法を適用して得られた核酸増幅物は電気泳動後、染色により生じるバンドの有無により、確認することができる。
<Application to PCR method>
In the present invention, the nucleic acid extraction method can be integrated into a conventional PCR method. That is, it can be adsorbed and desorbed by the nucleic acid extraction method of the present invention, and the extracted nucleic acid can be used as a template to obtain a nucleic acid amplification product. In particular, in the case of the present invention, since nucleic acid extraction can be performed with an aqueous solution, adverse effects such as enzyme inhibition can be prevented. Here, in the PCR, the nucleic acid extract is reacted and denatured at a predetermined temperature for a predetermined time and then extended. The nucleic acid amplification product obtained by applying the PCR method can be confirmed by the presence or absence of a band generated by staining after electrophoresis.
さらに本発明においては、核酸抽出方法によって抽出され、ＰＣＲ法により増幅された核酸増幅産物を用いて、更にＳＮＰ自動検出を行うことができる。ＳＮＰ検出は、特に限定されるものでなく、従来の方法にしたがって行うことができる。例えば、核酸増幅産物を緩衝溶液中で固定化した後、アルカリ処理により核酸を１本鎖化とし、標識した検出プローブとハイブリタイズを行い、標識した検出プローブを遊離させることによって行うことができる。 Furthermore, in the present invention, automatic SNP detection can be further performed using a nucleic acid amplification product extracted by the nucleic acid extraction method and amplified by the PCR method. SNP detection is not particularly limited, and can be performed according to a conventional method. For example, after the nucleic acid amplification product is immobilized in a buffer solution, the nucleic acid is made into a single strand by alkali treatment, hybridized with a labeled detection probe, and the labeled detection probe is released.
＜核酸抽出用試薬キットとしての応用＞
本発明の核酸抽出用試薬キットは、少なくとも上述した固相担体と、核酸が溶解した溶液と、核酸抽出用の水溶液とから構成されてなる。これらは別個の容器に収容され、上述の各工程において、順次添加混合されて使用される。該核酸抽出用試薬キットにより、生体試料中から所望の核酸を回収するに際し、必要な試薬を用意・調製する手間が省略でき、迅速に操作を行うことができる。
<Application as a reagent kit for nucleic acid extraction>
The reagent kit for nucleic acid extraction of the present invention comprises at least the above-mentioned solid phase carrier, a solution in which nucleic acid is dissolved, and an aqueous solution for nucleic acid extraction. These are accommodated in separate containers, and are used by being sequentially added and mixed in each step described above. With this nucleic acid extraction reagent kit, when recovering a desired nucleic acid from a biological sample, it is possible to dispense with the trouble of preparing and preparing the necessary reagents, and to perform operations quickly.
以下、本発明につき、実施例を用いて説明するが、本発明は、これになんら限定されるものではない。 Hereinafter, although this invention is demonstrated using an Example, this invention is not limited to this at all.
＜粒子の調製及び磁性細菌粒子の脂質二重膜の除去＞
本実施例では、固相担体として磁性細菌粒子（平均粒径８０ｎｍ）を使用した。磁性細菌粒子は磁性細菌MagnetospirilluｍＭagneticum ＡＭＢ−１を従来公知の手順に従って分離・調製した後、粒子表面の脂質二重膜の除去を行うことにより得た（Biotechnology and Bioengineering; Volume 94, Issue 5 , Pages 862 868 (2006)参照）。
即ち、ＭＳＧＭ培地１００ｌとし、室温にて７日間、微好気条件下で培養した菌体から分離した。さらに、１００００×ｇ、４℃にて連続遠心集菌し、２０ｍＭトリス塩酸緩衝液（Ｔｒｉｓ−ＨＣｌバッファー、ｐＨ７．０)で３回洗浄をした。遠心分離で得られた磁性細菌をトリス塩酸緩衝液（ｐＨ７．０)に懸濁し、フレンチプレス（有限会社大岳製作所製、商品名５５０１Ｍ）を使用して１５００ｋｇ／cｍ2で破砕した。その後、ネオジムーボロン（Ｎｄ−Ｂ）磁石を用いて菌体破砕液から磁性細菌粒子を磁気分離した。回収した磁性粒子は、１０ｍＭＰＢＳ緩衝液中に保存した。
<Preparation of particles and removal of lipid bilayer from magnetic bacterial particles>
In this example, magnetic bacterial particles (average particle size of 80 nm) were used as the solid support. Magnetic bacteria particles were obtained by separating and preparing the magnetic bacteria Magnetospirillum mMagneticum AMB-1 according to a conventionally known procedure, and then removing the lipid bilayer on the particle surface (Biotechnology and Bioengineering; Volume 94, Issue 5, Pages 862). 868 (2006)).
That is, it was set to 100 liters of MSGM medium and separated from the cells cultured under microaerobic conditions for 7 days at room temperature. Further, the cells were collected by continuous centrifugation at 10,000 × g and 4 ° C., and washed three times with 20 mM Tris-HCl buffer (Tris-HCl buffer, pH 7.0). The magnetic bacterium obtained by centrifugation was suspended in Tris-HCl buffer (pH 7.0) and crushed at 1500 kg / cm 2 using a French press (trade name 5501M, manufactured by Otake Seisakusho Co., Ltd.). Thereafter, magnetic bacterial particles were magnetically separated from the bacterial cell disruption solution using a neodymium-boron (Nd-B) magnet. The collected magnetic particles were stored in 10 mM PBS buffer.
次に、ＰＢＳ緩衝液に保存した前記磁性細菌粒子を形成するマグネタイトの結晶表面に存在するヒドロキシル基を露出させるために、ドジデル硫酸ナトリウム（ＳＤＳ：界面活性剤）により脂質二重膜を除去した。先ず、磁性細菌粒子１０ｍｇを１％界面活性剤（ＳＤＳ）溶液１ｍｌに懸濁し、１時間煮沸させた。煮沸段階においては、５分ごとに磁性細菌粒子を超音波分散させた。その後、超純水及びメタノールで洗浄した。 Next, in order to expose the hydroxyl group present on the surface of the magnetite crystals forming the magnetic bacterial particles stored in the PBS buffer, the lipid bilayer was removed with sodium dodider sulfate (SDS: surfactant). First, 10 mg of magnetic bacterial particles were suspended in 1 ml of a 1% surfactant (SDS) solution and boiled for 1 hour. In the boiling stage, magnetic bacterial particles were ultrasonically dispersed every 5 minutes. Thereafter, it was washed with ultrapure water and methanol.
＜アミノ基修飾磁性細菌粒子の作製＞
この磁性細菌粒子１０ｍｇを超純水で洗浄し、粒子表面のヒドロキシル基を活性化させるために、室温で１０分間ＡＰＭ溶液（アンモニア過水、水：水酸化アンモニウム：過酸化水素＝５：１：１の割合で混合した溶液、Fluka Chemical社製）に懸濁させ、表面処理を行った。
<Preparation of amino group-modified magnetic bacterial particles>
To wash 10 mg of the magnetic bacterial particles with ultrapure water and activate the hydroxyl groups on the surface of the particles, an APM solution (ammonia peroxide, water: ammonium hydroxide: hydrogen peroxide = 5: 1: 10 minutes at room temperature) Surface treatment was carried out by suspending in a solution mixed at a ratio of 1 (Fluka Chemical Co., Ltd.).
次に、該磁性細菌粒子をエタノール洗浄した後、アミノシランカップリング剤として、３-[２-(２-アミノエチル)-エチルアミン]-プロピルトリメトキシシラン（ＡＥＥＡ）を使用し、２％ＡＥＥＡエタノール溶液２０ｍｌ中で室温にて１０分間反応させた。反応後、該磁性細菌粒子をジメチルホルムアミド（ＤＭＦ、和光純薬工業社製）で洗浄した。 Next, after washing the magnetic bacterial particles with ethanol, 3- [2- (2-aminoethyl) -ethylamine] -propyltrimethoxysilane (AEEA) was used as an aminosilane coupling agent, and a 2% AEEA ethanol solution was used. The reaction was carried out in 20 ml at room temperature for 10 minutes. After the reaction, the magnetic bacterial particles were washed with dimethylformamide (DMF, manufactured by Wako Pure Chemical Industries, Ltd.).
続いて、該磁性細菌粒子を１２０℃のＤＭＦ２０ｍｌ中で３０分間加熱し、１０分毎に超音波で分散させた。かかる反応条件に設定した場合、後述する実施例１のように、磁性細菌粒子表面に単位粒子当り約２．７×１０４個と、従来法における条件下（６０℃１０分間で、常時超音波分散を行う）よりも安定で多量のアミノ基を導入することができた。
そして、カップリング反応後の磁性細菌粒子を磁気回収し、メタノール洗浄を３回繰り返した後、４℃の条件下にてメタノール中に保存した。
Subsequently, the magnetic bacterial particles were heated in 20 ml of DMF at 120 ° C. for 30 minutes and dispersed with ultrasonic waves every 10 minutes. When such reaction conditions are set, as in Example 1 to be described later, about 2.7 × 10 4 particles per unit particle on the surface of the magnetic bacteria particles, the conditions under the conventional method (continuous ultrasonic waves at 60 ° C. for 10 minutes). A larger amount of amino groups can be introduced, which is more stable than that of dispersion.
Then, the magnetic bacterial particles after the coupling reaction were magnetically collected, and the methanol washing was repeated three times, and then stored in methanol at 4 ° C.
このように作製したアミノ基修飾磁性細菌粒子の表面に存在するアミノ基の数をサルフォサクシイミド６-[3'-(2-ピリジルジチオール)-プロピオンアミド]ヘキサノエイト（Sulfo-LC-SPDP、Pierce Chemical社製）を使用し測定した。
先ず、前記アミノ基修飾磁性細菌粒子を２５０μgとり、ＰＢＳで置換した後、１０ｍＭ Sulfo-LC-SPDPＰＢＳ緩衝液中で懸濁して、室温にて３０分インキュベートした。
The number of amino groups present on the surface of the amino group-modified magnetic bacterial particles prepared in this way was determined by sulfosuccinimide 6- [3 '-(2-pyridyldithiol) -propionamide] hexanoate (Sulfo-LC-SPDP, Pierce (Manufactured by Chemical).
First, 250 μg of the amino group-modified magnetic bacterial particles were taken and replaced with PBS, then suspended in 10 mM Sulfo-LC-SPDP PBS buffer and incubated at room temperature for 30 minutes.
次に、ＰＢＳで洗浄することにより非特異的に吸着したSulfo-LC-SPDPを除去した。この工程を３回繰り返した。
そして、ＰＢＳ緩衝液中の２０ｍＭＤＴＴ２００μｌに粒子を懸濁し、Sulfo-LC-SPDPのジスルフィド結合の開裂により２−ピリジルチオールを遊離させるためにインキュベートした。遊離した２−ピリジルチオールの３４３ｎｍの吸光度を測定することにより磁性細菌粒子表面上のアミノ基を測定した。なお、２−ピリジルチオールの検出には紫外線可視分光度計（島津製作所 製ＵＶ−１６００）を使用した。また、磁性細菌粒子表面のアミノ基と反応するSulfo-LC-SPDPの濃度は、２０ｍＭ２−ピリジルチオール溶液中のSulfo-LC-SPDPの異なる濃度について標準曲線を作成して求めた。
Next, non-specifically adsorbed Sulfo-LC-SPDP was removed by washing with PBS. This process was repeated three times.
The particles were then suspended in 200 μl of 20 mM DTT in PBS buffer and incubated to release 2-pyridylthiol by cleavage of the Sulfo-LC-SPDP disulfide bond. The amino group on the surface of the magnetic bacterium particle was measured by measuring the absorbance of the released 2-pyridylthiol at 343 nm. In addition, the ultraviolet visible spectrophotometer (Shimadzu Corporation UV-1600) was used for the detection of 2-pyridylthiol. The concentration of Sulfo-LC-SPDP that reacts with amino groups on the surface of magnetic bacteria particles was determined by preparing standard curves for different concentrations of Sulfo-LC-SPDP in a 20 mM 2-pyridylthiol solution.
また、磁性細菌粒子表面のアミノ基の数は、単位面積当り概ね１．１個／ｎｍ２であるので（粒子形状を５０×５０×１００ｎｍ３と仮定すると、その表面積は２．５×１０４ｎｍ２と見積もられるため）、約２．７×１０４個／粒子であることがわかった。 In addition, since the number of amino groups on the surface of the magnetic bacterial particle is approximately 1.1 / nm 2 per unit area (assuming the particle shape is 50 × 50 × 100 nm 3 , the surface area is 2.5 × 10 4. It was found to be about 2.7 × 10 4 particles / particle (because it is estimated to be nm 2 ).
＜アミノ基修飾磁性細菌粒子によるλDNAの吸着＞
以上のようにして得られたアミノ基修飾磁性細菌粒子を使用し、核酸としてλDNA（48,502bp、タカラバイオ社製）を用いて、前記粒子への該λDNAの吸着を行った。
即ち、アミノ基修飾磁性細菌粒子５０μｇに所定の濃度のλDNAを含む溶液４０μｌを加えた後、室温でインキュベートした。そして、磁性細菌粒子に結合したλDNAを磁気的に回収した。さらに磁性細菌粒子の洗浄を３回繰り返した。
<Adsorption of λDNA by amino group-modified magnetic bacterial particles>
The amino group-modified magnetic bacterial particles obtained as described above were used, and λDNA (48,502 bp, manufactured by Takara Bio Inc.) was used as a nucleic acid to adsorb the λDNA to the particles.
That is, 40 μl of a solution containing a predetermined concentration of λDNA was added to 50 μg of amino group-modified magnetic bacterial particles, followed by incubation at room temperature. Then, λDNA bound to the magnetic bacterial particles was magnetically recovered. Furthermore, washing of the magnetic bacteria particles was repeated 3 times.
そして、加えたλDNAの量から洗浄後に上澄み溶液に存在するλDNAの量の合計を差し引きして、磁性細菌粒子によるλDNAの吸着量を求めた。λDNAの吸着量は、２本鎖DNA定量試薬である PicoGreen（モレキュラープローブ社製）を使用し、fluorescence microplate reader (商品名：FLUO Star Galaxy,BMG Labtechnologies社製)により502ｎｍと523ｎｍにおける蛍光強度を測定し、異なる濃度におけるλDNAの吸着量から標準曲線を作成して求めた。 The total amount of λDNA present in the supernatant solution after washing was subtracted from the amount of λDNA added to determine the amount of λDNA adsorbed by the magnetic bacterial particles. The amount of λDNA adsorbed is measured using a double-stranded DNA quantitative reagent, PicoGreen (manufactured by Molecular Probes), and fluorescence intensity at 502 nm and 523 nm using a fluorescence microplate reader (trade name: FLUO Star Galaxy, manufactured by BMG Labtechnologies) A standard curve was prepared from the amount of λDNA adsorbed at different concentrations.
図４に、磁性細菌粒子５０μｇに対して、λDNAの添加量を０〜１７００ｎｇの範囲で変化させたときのλDNAの吸着量を示す。尚、該磁性細菌粒子はトリス塩酸緩衝液（ｐＨ８.０)に懸濁させた。
この結果から、最大で約１２０ｎｇのλDNAを吸着できることが明らかとなった。
FIG. 4 shows the amount of λDNA adsorbed when the amount of λDNA added is changed in the range of 0 to 1700 ng with respect to 50 μg of magnetic bacterial particles. The magnetic bacterial particles were suspended in Tris-HCl buffer (pH 8.0).
From this result, it was revealed that about 120 ng of λDNA could be adsorbed at maximum.
次に、該磁性細菌粒子を含む前記トリス塩酸緩衝液中にλDNAを添加してからの経過時間とλDNA吸着量の関係について調べた。測定は、５、１０、２０、４０、８０分経過後にそれぞれ行った。
この結果を図５に示す。図５より、２０分間経過した時点で、吸着量はほぼ最大となり、更に４０分間経過後は殆ど一定値に収束することが確認できた。
Next, the relationship between the elapsed time after addition of λDNA and the amount of λDNA adsorbed in the Tris-HCl buffer containing the magnetic bacterial particles was examined. Measurement was performed after 5, 10, 20, 40, and 80 minutes.
The result is shown in FIG. From FIG. 5, it was confirmed that the adsorption amount was almost the maximum when 20 minutes passed and converged to a substantially constant value after a further 40 minutes.
また、前記磁性細菌粒子を懸濁させる溶液（核酸吸着用の溶液）の種類を次の通り変化させ、ｐＨによる吸着量への影響を検討した。
即ち、前記磁性細菌粒子とλDNAを混合させる溶液として、２５ｍＭ2-(Ｎｍorpholino)ethanesulfonic acid（ＭＥＳ）緩衝液（ｐＨ５、ｐＨ６及びｐＨ７の場合）、２５ｍＭトリス塩酸緩衝液（ｐＨ８、ｐＨ９の場合)及び２５ｍＭカーボネート緩衝液（ｐＨ１０の場合）を用い、核酸吸着用の溶液のｐＨを５〜１０の範囲で変化させたときの前記磁性細菌粒子に対するλDNAの吸着量をそれぞれ調べた。
In addition, the type of the solution for suspending the magnetic bacterial particles (solution for adsorbing nucleic acid) was changed as follows, and the influence of pH on the amount of adsorption was examined.
That is, as a solution for mixing the magnetic bacterial particles and λDNA, 25 mM 2- (Nmorpholino) ethanesulfonic acid (MES) buffer (for pH 5, pH 6 and pH 7), 25 mM Tris-HCl buffer (for pH 8 and pH 9) and 25 mM. Using a carbonate buffer solution (in the case of pH 10), the amount of λDNA adsorbed to the magnetic bacterial particles when the pH of the solution for nucleic acid adsorption was changed in the range of 5 to 10 was examined.
この結果、図６に示すように、前記核酸吸着用溶液のｐＨが９以下の範囲内で吸着が起こり、特にｐＨが５〜８の範囲に設定されることにより、優れた吸着効率が得られるのに対して、ｐＨが１０の条件下では、ほぼ１０％程度しか吸着が起こらなかった。ここで、ＡＥＥＡの酸解離定数ｐＫaは９．７であるので、ｐＨが９を超えるとアミノ基は脱プロトン化され、逆にｐＨが８以下になると、アミノ基のプロトン化が十分に起こると予想される。つまり、λDNAの吸着反応は前記磁性細菌粒子表面の電荷に関係していると言える。 As a result, as shown in FIG. 6, the adsorption of the nucleic acid adsorption solution occurs within a range of 9 or less, and in particular, when the pH is set in the range of 5 to 8, excellent adsorption efficiency can be obtained. On the other hand, only about 10% of adsorption occurred under the condition of pH 10. Here, since the acid dissociation constant pKa of AEEA is 9.7, when the pH exceeds 9, the amino group is deprotonated. On the other hand, when the pH is 8 or less, the amino group is sufficiently protonated. is expected. That is, it can be said that the adsorption reaction of λDNA is related to the charge on the surface of the magnetic bacterial particle.
更に、核酸吸着用の溶液に種々の有機溶媒を添加した場合の吸着効率について調べた。
即ち、該磁性細菌粒子を含む前記トリス塩酸緩衝液（ｐＨ８．０）中に、アセトニトリル、ＤＭＦ、メタノール又はエタノールを添加した場合と、有機溶媒を添加しない場合について、前記磁性細菌粒子に対するλDNAの吸着量をそれぞれ測定した。尚、上述の各種有機溶媒はいずれも５％となるように調製した。この結果を表１に示す。
Furthermore, the adsorption efficiency when various organic solvents were added to the nucleic acid adsorption solution was examined.
That is, adsorption of λDNA to the magnetic bacterial particles in the case where acetonitrile, DMF, methanol or ethanol is added to the Tris-HCl buffer (pH 8.0) containing the magnetic bacterial particles and no organic solvent is added. Each amount was measured. In addition, all the above-mentioned various organic solvents were prepared so that it might become 5%. The results are shown in Table 1.
表１に示すように、有機溶媒を添加しない場合に比べ、低濃度の各種有機溶媒を添加することにより、良好な吸着効果が得られることがわかった。また、メタノールやエタノールなどのプロトン性溶媒を使用する場合に比べ、アセトニトリルやＤＭＦなどの非プロトン性溶媒を使用する場合の方が高い吸着効果を得ることができた。これは、有機溶媒によってλDNAの周囲に存在する水分子構造の規則性が失われ、その結果λDNAとアミノ基間の静電的な結合がより速く進んだためと考えられる。また、有機溶媒非存在下ではλDNA中のリン酸が水和しており、アミノ基との相互作用が起こりにくく、結果として核酸が十分に吸着しない可能性があることが示唆される。 As shown in Table 1, it was found that a good adsorption effect can be obtained by adding low concentrations of various organic solvents compared to the case where no organic solvent is added. In addition, a higher adsorption effect was obtained when an aprotic solvent such as acetonitrile or DMF was used than when a protic solvent such as methanol or ethanol was used. This is presumably because the regularity of the water molecule structure existing around λDNA was lost by the organic solvent, and as a result, electrostatic bonding between λDNA and the amino group proceeded faster. In addition, in the absence of an organic solvent, phosphoric acid in λDNA is hydrated, and it is unlikely that interaction with an amino group occurs, and as a result, nucleic acid may not be sufficiently adsorbed.
＜アミノ基修飾磁性細菌粒子に吸着したλDNAの脱離＞
（実施例１〜２及び比較例１〜５）
前記アミノ基修飾磁性細菌粒子５０μｇに１Ｍリン酸ナトリウムが添加されたトリス塩酸緩衝液（ＰＨ８．０）を加え、８０℃で４０分間インキュベートした。インキュベートと同時に５分ごとに超音波で磁性細菌粒子を攪拌した。磁性細菌粒子を磁気回収して、上澄み液中に存在する脱離したλDNAを定量した（実施例１）。尚、磁性細菌粒子はλDNAを最大吸着量の１２０ｎｇを吸着させたものを用いた。
<Desorption of λDNA adsorbed on amino group-modified magnetic bacterial particles>
(Examples 1-2 and Comparative Examples 1-5)
Tris-HCl buffer (PH 8.0) supplemented with 1M sodium phosphate was added to 50 μg of the amino group-modified magnetic bacterial particles and incubated at 80 ° C. for 40 minutes. Simultaneously with the incubation, the magnetic bacterial particles were stirred with an ultrasonic wave every 5 minutes. Magnetic bacteria particles were magnetically recovered to quantify the desorbed λDNA present in the supernatant (Example 1). The magnetic bacterial particles used were λDNA adsorbed with a maximum adsorption amount of 120 ng.
また、比較例として、実施例１で使用した１Ｍリン酸ナトリウムを、１Ｍクエン酸ナトリウム（比較例１）、１Ｍ酒石酸ナトリウム（比較例２）、塩化ナトリウム（比較例３）、１Ｍ硝酸アンモニウム（比較例４）、１Ｍ硫酸ナトリウム（比較例５）にそれぞれ変え、その他の条件は実施例１と同様にした場合についても併せて調べた。この結果を図７に示す。 As a comparative example, 1M sodium phosphate used in Example 1 was replaced with 1M sodium citrate (Comparative Example 1), 1M sodium tartrate (Comparative Example 2), sodium chloride (Comparative Example 3), 1M ammonium nitrate (Comparative Example). 4) The case where the conditions were changed to 1M sodium sulfate (Comparative Example 5) and the other conditions were the same as in Example 1 was also investigated. The result is shown in FIG.
図７に示すように、核酸抽出用の溶液中にリン酸ナトリウムを用いることにより、λDNAを効率的に脱離、抽出することができることがわかった。
これに対して、比較例１〜５の場合では十分な効果が得られなかったことから、核酸の脱離に対する効果は、添加するアニオンのホフマイスター系列に依存するのではなく、実施例１のようにリン酸基が存在することにより顕著となることがわかった。
As shown in FIG. 7, it was found that λDNA can be efficiently desorbed and extracted by using sodium phosphate in the solution for nucleic acid extraction.
On the other hand, in the case of Comparative Examples 1-5, since sufficient effect was not acquired, the effect with respect to detachment | desorption of a nucleic acid does not depend on the Hofmeister series of the anion to add, Example 1 It was found that the presence of a phosphate group becomes remarkable.
次に、核酸抽出用の溶液中に有機溶剤を添加させたときの脱離効果について検討した。ここで、有機溶剤としては、メタノールを用いた。
即ち、実施例１及び比較例１〜５において使用したトリス塩酸緩衝液中に、メタノールを５、１０、１５％の濃度範囲で段階的に変化させて添加した以外は、上述の実施例１や比較例１〜５と同様にしてλDNAの脱離量をそれぞれ調べた。この結果、図７に併せて示す。
Next, the desorption effect when an organic solvent was added to the solution for nucleic acid extraction was examined. Here, methanol was used as the organic solvent.
That is, in the Tris-HCl buffer used in Example 1 and Comparative Examples 1 to 5, except that methanol was added stepwise in the concentration range of 5, 10, and 15%, the above-described Example 1 and The amount of λDNA desorption was examined in the same manner as in Comparative Examples 1-5. The result is also shown in FIG.
図７より、リン酸化合物とメタノールの共存下では、飛躍的に脱離効果を向上させることができ、特にメタノールの濃度を１５％とした場合では、約９０％近くλDNAを脱離、抽出することが明らかとなった。これは、メタノールの添加により、正電荷を持つアミノ基と相互作用することができるリン酸イオンの周りに存在する水分子が奪われることに起因すると考えられる。 From FIG. 7, it is possible to dramatically improve the desorption effect under the coexistence of the phosphate compound and methanol. In particular, when the concentration of methanol is 15%, λDNA is desorbed and extracted by about 90%. It became clear. This is thought to be due to the depletion of water molecules present around phosphate ions that can interact with positively charged amino groups by the addition of methanol.
また、実施例１で使用した１Ｍリン酸ナトリウムをdNTPに変え、その他は実施例１と同様にしたときの核酸の脱離効率を調べた（実施例２とする）。
図８に、核酸抽出用の溶液中に１Ｍリン酸ナトリウムを添加した場合のλDNAの脱離量を１００％とし、添加するリン酸化合物としてdNTPを用いたときの脱離効率に対するdNTPの濃度依存性を示す。
この結果から、リン酸ナトリウムよりdNTPを用いることで、より低モル濃度の媒質で核酸の脱離が可能となることが明らかとなった。
In addition, the 1M sodium phosphate used in Example 1 was replaced with dNTPs, and the nucleic acid desorption efficiency was examined in the same manner as in Example 1 (referred to as Example 2).
FIG. 8 shows the concentration dependence of dNTP on the desorption efficiency when λDNA is added to a solution for nucleic acid extraction with 100% λDNA and dNTP is used as the added phosphate compound. Showing gender.
From this result, it was clarified that nucleic acid can be eliminated in a medium having a lower molar concentration by using dNTP from sodium phosphate.
＜抽出した大腸菌ゲノムDNAのＰＣＲ法による増幅＞
（実施例３）
上述のアミノ基修飾磁性細菌粒子（５０μｇ/４０μl）に対し、大腸菌精製ゲノム１０００ｎｇを添加し、核酸の吸着を行った。
即ち、前記磁性細菌粒子を上述と同様にして洗浄した後、２．５ ｍＭ のdNTPを含む溶液を使用して磁性細菌粒子からλDNAを脱離した。
この核酸抽出用の溶液を１μｌ採取し、全量２５μｌで、ＰＣＲを行った。ＰＣＲのターゲットとしては、大腸菌のgyrB遺伝子に特異な配列を使用した。その配列は、フォワードプライマー:5'-ＴＧＣＧＴＧＡＧＴＴＧＴＣＧＴＴＣＣＴＣ-３'およびリバースプライマー: 5'-ＡＣＧＣＣＡＡＴＡＣＣＧＴＣＴＴＴＴＴＣＡ-３'である。
<Amplification of extracted Escherichia coli genomic DNA by PCR>
(Example 3)
To the amino group-modified magnetic bacterial particles (50 μg / 40 μl), 1000 ng of E. coli purified genome was added to adsorb nucleic acid.
That is, after washing the magnetic bacterial particles in the same manner as described above, λDNA was detached from the magnetic bacterial particles using a solution containing 2.5 mM dNTP.
1 μl of this nucleic acid extraction solution was collected, and PCR was performed with a total volume of 25 μl. As a PCR target, a sequence specific to the gyrB gene of E. coli was used. The sequences are forward primer: 5′-TGCGTGGAGTTGTCGTTCTC-3 ′ and reverse primer: 5′-ACGCCAATACCGTCTTTTTCA-3 ′.
ＰＣＲ反応は０．７５Ｕ ＴａｑDNAポリメラーゼ 、０．２５ｍＭ dNTPs、０．４μＭ プライマー、１．５ｍＭの塩化マグネシウムを含むＰＣＲバッファー中で行った。ＰＣＲ反応の条件は、９４℃で２０秒、５５℃で３０秒、 ７２℃で４０秒を３５サイクルで行い、その後７２℃で３分間伸長させた。ＰＣＲ反応後のＰＣＲ増幅産物は２．０％アガロースを用いて電気泳動を行い、電気泳動後、エチルブロマイドで染色して確認をした。結果を図９に示す。 The PCR reaction was performed in a PCR buffer containing 0.75 U Taq DNA polymerase, 0.25 mM dNTPs, 0.4 μM primer, 1.5 mM magnesium chloride. The PCR reaction conditions were 94 ° C for 20 seconds, 55 ° C for 30 seconds, and 72 ° C for 40 seconds in 35 cycles, followed by extension at 72 ° C for 3 minutes. The PCR amplification product after the PCR reaction was subjected to electrophoresis using 2.0% agarose, and after electrophoresis, it was confirmed by staining with ethyl bromide. The results are shown in FIG.
図９より、１Ｍリン酸ナトリウム緩衝溶液で溶出したλDNAをサンプルとした場合には、添加するメタノール濃度に関わらず遺伝子増幅がみられないのに対し（図９中、レーン１〜４）、２．５ ｍＭ dNTPを加えた場合にはコントロール（図９中、レーンＣ）と同等の遺伝子増幅が認められた。尚、コントロールには、大腸菌の精製ゲノム１ｎｇをサンプルとして用いた。
このことから、dNTPを溶出溶液とした場合にはＰＣＲ阻害性が全く見られないことが明らかとなった。
From FIG. 9, when λDNA eluted with 1M sodium phosphate buffer was used as a sample, no gene amplification was observed regardless of the concentration of methanol added (lanes 1 to 4 in FIG. 9), 2 When 0.5 mM dNTP was added, gene amplification equivalent to that of the control (lane C in FIG. 9) was observed. For control, 1 ng of purified E. coli genome was used as a sample.
This revealed that no PCR inhibition was observed when dNTP was used as the elution solution.
本発明の核酸の抽出方法は、従来法において、核酸抽出後の遺伝子増幅や酵素処理などの反応における阻害効果とされていた高塩濃度や高い有機溶媒含有の試薬を必要としないため、核酸抽出の自動化技術に大いに貢献することができる。このため、本発明は、遺伝子診断やオーダーメイド医療等の発展が著しい医療分野において、有効な核酸抽出技術を提供することができる。
また、上述のように高塩濃度や高い有機溶媒を用いないことから、本発明は、例えば多糖を多く含む生体試料を対象とする解析・検査の際にも適用でき、遺伝子解析技術の向上に大きく寄与することが可能である。
The nucleic acid extraction method of the present invention does not require a reagent containing a high salt concentration or a high organic solvent, which has been regarded as an inhibitory effect in reactions such as gene amplification and enzyme treatment after nucleic acid extraction in the conventional method. Can greatly contribute to the automation technology. For this reason, the present invention can provide an effective nucleic acid extraction technique in the medical field where developments such as genetic diagnosis and custom-made medicine are remarkable.
In addition, since a high salt concentration or a high organic solvent is not used as described above, the present invention can be applied to, for example, analysis / testing on biological samples containing a large amount of polysaccharides, thereby improving gene analysis technology. It is possible to contribute greatly.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2011055751A (en) * | 2009-09-09 | 2011-03-24 | Tokyo Univ Of Agriculture & Technology | Method for preparing dendrimer-modified magnetic fine particle |
| JP2011057813A (en) * | 2009-09-09 | 2011-03-24 | Tokyo Univ Of Agriculture & Technology | Dendrimer coated magnetic fine particle, manufacturing method thereof, and application |
| KR101834820B1 (en) | 2017-06-02 | 2018-03-07 | 한국과학기술원 | Gene extraction device using magnetic force |
| CN111514871A (en) * | 2020-05-27 | 2020-08-11 | 合肥中科易康达生物医学有限公司 | Preparation method and application of solid phase substrate for nucleic acid extraction |
| CN112921028A (en) * | 2019-12-06 | 2021-06-08 | 深圳华大基因科技服务有限公司 | DNA purification method, genomic DNA extraction method, sequencing method and kit |
| CN116510994A (en) * | 2023-07-03 | 2023-08-01 | 中国农业大学 | PCR tube for integrally extracting genome |
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2007
- 2007-09-11 JP JP2007234914A patent/JP2009065849A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2011055751A (en) * | 2009-09-09 | 2011-03-24 | Tokyo Univ Of Agriculture & Technology | Method for preparing dendrimer-modified magnetic fine particle |
| JP2011057813A (en) * | 2009-09-09 | 2011-03-24 | Tokyo Univ Of Agriculture & Technology | Dendrimer coated magnetic fine particle, manufacturing method thereof, and application |
| KR101834820B1 (en) | 2017-06-02 | 2018-03-07 | 한국과학기술원 | Gene extraction device using magnetic force |
| CN112921028A (en) * | 2019-12-06 | 2021-06-08 | 深圳华大基因科技服务有限公司 | DNA purification method, genomic DNA extraction method, sequencing method and kit |
| CN111514871A (en) * | 2020-05-27 | 2020-08-11 | 合肥中科易康达生物医学有限公司 | Preparation method and application of solid phase substrate for nucleic acid extraction |
| CN116510994A (en) * | 2023-07-03 | 2023-08-01 | 中国农业大学 | PCR tube for integrally extracting genome |
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