JP2004298005A - Method for separating polynucleotide and apparatus for separation - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for separation with which a polynucleotide having a target base sequence can readily be separated from a mixed solution of the polynucleotides and to provide an apparatus for separation. <P>SOLUTION: The method for separating the polynucleotides is characterized as follows. The mixed solution containing the polynucleotide (A) having the target base sequence and the polynucleotide (B) without the target base sequence is arranged in a cell in which a probe having a base sequence complementary to the target base sequence is immobilized in the inner surface of the cell or the solid phase in the cell. The mixed solution is provided with a temperature distribution in which a first region regulated to a first temperature higher than a dissociation temperature of a hybrid chain of the polynucleotide (B) with the probe is mutually brought into contact with a second region regulated to a second temperature lower than the first temperature and below a temperature for dissociating the total amount of the hybrid chain of the polynucleotide (A) with the probe. The temperature distribution is continuously moved in one direction to thereby hybridize the polynucleotide (A) with the probe. As a result, the polynucleotide (A) is separated from the mixed solution. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

【００３０】
【表２】

（ただし、ｘ、ｙはそれぞれ０以上１以下で、それぞれのポリヌクレオチドの解離曲線に依存する値である。）
【００３１】
［分離方法］
本発明の分離方法は、標的塩基配列を有するポリヌクレオチド（Ａ）と、標的塩基配列を有さないポリヌクレオチド（Ｂ）とを含有する混合溶液から、ポリヌクレオチド（Ａ）を分離するポリヌクレオチドの分離方法であって、標的塩基配列に対して相補的な塩基配列を有するプローブが、セルの内表面またはセル内の固相の少なくともいずれか一方に固定されたセルに混合溶液を配し、セル内に配された混合溶液に、ポリヌクレオチド（Ｂ）とプローブとが形成するハイブリッド鎖の解離温度より高い第一温度に調節された第一領域、および、前記第一温度より低く、かつポリヌクレオチド（Ａ）とプローブとが形成するハイブリッド鎖の全量が解離する温度未満である第二温度に調節された第二領域が互いに接する温度分布を持たせ、該温度分布を一方向に連続的に移動させることにより、ポリヌクレオチド（Ａ）をプローブとハイブリダイズさせて混合溶液から分離することを特徴とする。
【００３２】
第一温度は、ポリヌクレオチド（Ｂ）とプローブとが形成するハイブリッド鎖の解離温度（Ｔ_ＬＢ）より高い温度である。第一温度をＴ_ＬＢ未満の温度範囲（α）内とすると、第一温度より低い第二温度も同領域（α）に入り、溶液中のポリヌクレオチド（Ａ）もポリヌクレオチド（Ｂ）もハイブリダイゼーションにより固相に吸着されていて、セル中のいずれの領域においても溶液中に解離したこれらポリヌクレオチドが無く、分離は行われない。
【００３３】
第二温度は、第一温度より低く、かつポリヌクレオチド（Ａ）とプローブとが形成するハイブリッド鎖の全量が解離する温度（Ｔ_ＨＡ）未満である。第二温度を温度範囲ε、即ち、Ｔ_ＨＡ以上にすると、第二温度より高い第一温度も温度範囲εに入り、セル内の全領域に於いてポリヌクレオチド（Ａ）もポリヌクレオチド（Ｂ）もハイブリダイゼーションせず、全量が溶液に溶解していることになる。このため、この条件では本発明の分離は行われない。
【００３４】
従って、本発明では、第一温度をＴ_ＬＢを越える温度とし、かつ第二温度を第一温度より低く、Ｔ_ＨＡ未満の温度とする。この条件では、ポリヌクレオチド（Ａ）とポリヌクレオチド（Ｂ）とに関する二つの解離曲線が非重畳的である場合にも重畳的である場合にも、第一領域に於ける両ポリヌクレオチドの全吸着量は、第二領域に於けるそれと同等以下であり、かつ、第一領域の溶液中に於けるポリヌクレオチド（Ｂ）が溶解している割合（溶解している量／全量）は、ポリヌクレオチド（Ａ）が溶解している割合（溶解している量／全量）と同等以上である。
【００３５】
本発明の分離機構はまだ明確ではないが、次のように推定される。
セル内の任意の部分が第一温度に成されたときに、ポリヌクレオチド（Ｂ）の少なくとも一部が解離（脱ハイブリダイゼーション）した状態となる。この際、第一温度がＴ_ＨＡ未満であると、ポリヌクレオチド（Ａ）の少なくとも一部がプローブとのハイブリッド鎖を形成して固相に吸着されるため溶液中のポリヌクレオチド（Ｂ）の組成比が上昇する。従って、該第一領域から、拡散や送液によって温度分布の移動方向や下流方向に流出する割合は、ポリヌクレオチド（Ａ）よりポリヌクレオチド（Ｂ）の方が高くなる。その後、同部分が連続的な温度変化により第二温度となることで、プローブとポリヌクレオチド（Ａ）とが優先的にハイブリダイゼーションすることにより、溶液中のポリヌクレオチド（Ｂ）の組成比がさらに上昇する。従って、この領域に於いても、拡散や送液によって該領域から温度分布の移動方向や下流方向に流出する割合は、ポリヌクレオチド）Ａ）よりポリヌクレオチド（Ｂ）ぼ方が高くなる。またこの時、第一温度がＴ_ＨＡ以上であっても、第一温度から第二温度への連続的な温度変化の際に、固定されたプローブがポリヌクレオチド（Ａ）を吸着する速度がポリヌクレオチド（Ｂ）を吸着する速度より速いため、温度の変化する領域において、溶液中のポリヌクレオチド（Ｂ）の組成比が上昇することで分離されるが、第一温度がＴ_ＨＡ未満であるとより効率よくポリヌクレオチド（Ａ）とポリヌクレオチド（Ｂ）とを分離できるため好ましい。
【００３６】
さらに、第一温度はポリヌクレオチド（Ｂ）の全量が解離する温度（Ｔ_ＨＢ）以上であり、第二温度はポリヌクレオチド（Ｂ）の融解温度（Ｔ_ＭＢ）以下であることが好ましい。第一温度がＴ_ＨＢ以上であると、セル内の任意の部分が第一領域に入ったとき、ポリヌクレオチド（Ｂ）がほぼ全量が解離されるため溶液中のポリヌクレオチド（Ｂ）の組成比を上昇させることができ、セル内での移動量が大きくなって、分離効率、即ち第一温度から第二温度への一回の変化による分離の程度が向上する。また、第二温度がＴ_ＭＢ以下であるとポリヌクレオチド（Ｂ）を充分高い割合でプローブにハイブリダイゼーションさせることができるため、第一領域と第二領域との溶液中のポリヌクレオチドの濃度差が大きくなって、分離効率が向上する。この時、第二温度は、ポリヌクレオチド（Ｂ）の解離温度Ｔ_ＬＢ以下（温度範囲α）であることが、同じ理由でさらに好ましい。
また、第二温度が温度範囲αにあり、第一温度がＴ_ＭＢ以上であることが、第一領域と第二領域との溶液中のポリヌクレオチドの濃度差が大きくなって、分離効率が向上するため好ましく、第一温度が温度範囲β、γ、β’、又はγ’にあることが、さらに好ましい。
【００３７】
これとは逆に、セル内の任意の部分が第二温度から第一温度に変化する場合には、上記した場合とは逆に、セル内の任意の部分が第二温度に成されたときに、ポリヌクレオチド（Ａ）の少なくとも一部がハイブリダイゼーションによって固相に吸着される。この際、第二温度がＴ_ＬＢより高いと、ポリヌクレオチド（Ｂ）の少なくとも一部がプローブから解離して溶液中に溶出するため、第二領域において溶液中のポリヌクレオチド（Ｂ）の組成比が上昇する。従って、該第二領域から、拡散や送液によって温度分布の移動方向や下流方向に流出する割合は、ポリヌクレオチド（Ａ）よりポリヌクレオチド（Ｂ）の方が高くなる。その後、同部分が連続的な温度変化により第一温度となることで、プローブとポリヌクレオチド（Ｂ）のハイブリッド鎖が優先的に解離することにより、溶液中のポリヌクレオチド（Ｂ）の組成比がさらに上昇する。従って、この領域に於いても、拡散や送液によって該領域から温度分布の移動方向や下流方向に流出する割合は、ポリヌクレオチド（Ａ）よりポリヌクレオチド（Ｂ）の方が高くなる。また、第一温度がＴ_ＬＢ以下であっても、第二温度から第一温度への連続的な温度変化の際に、プローブとポリヌクレオチド（Ｂ）とのハイブリダイゼーションが優先的に解離することで、該温度の変化する領域において、溶液中のポリヌクレオチド（Ｂ）の組成比が上昇することで分離されるが、第二温度がＴ_ＬＢ未満であるとより効率よくポリヌクレオチド（Ａ）とポリヌクレオチド（Ｂ）とを分離できるため好ましい。
【００３８】
さらにまた、第一温度がＴ_ＨＡ以上であり、かつ、第二温度がＴ_ＬＢ未満であっても、第一温度から第二温度への連続的な温度変化の際に、固定されたプローブにポリヌクレオチド（Ａ）が吸着される速度は、ポリヌクレオチド（Ｂ）が吸着される速度より速く、また、第二温度から第一温度への連続的な温度変化の際に、固定されたプローブに吸着されたポリヌクレオチド（Ｂ）が解離する速度は、ポリヌクレオチド（Ａ）が解離する速度より速いことで、温度の変化する領域において、溶液中のポリヌクレオチド（Ｂ）の組成比が上昇し、分離される。
【００３９】
勿論、上記の説明は概念的な原理であり、実際の系ではもっと複雑な現象が生じていると推定される。例えば、第一温度から第二温度への変化やその逆の変化は中間温度を経由すること、ハイブリッド形成も脱ハイブリダイゼーションも、所定温度に達してから時間的な遅れがあること、等の速度的な効果である。よって、第一領域と第二領域の移動方向の長さ、温度分布の移動速度、プローブの長さ、ポリヌクレオチド（Ａ）やポリヌクレオチド（Ｂ）の長さ、溶液の流量などによって、上記の速度的な関係が変化して、複雑な様相を呈しうる。
【００４０】
従来技術（特許文献１）においては、固相に固定されたオリゴヌクレオチド（本発明で言うプローブ）に溶液中の野生型ポリヌクレオチドを選択的にハイブリッド形成させるが、一定温度での処理では、野生型のみを充分な選択性でハイブリッド形成させるには、長時間、例えば１〜１０時間を要した。これに対し、本発明に於いては、各第一温度及び第二温度の保持時間は１分未満でよく、このような短時間でも充分な選択性が発揮されて分離される。その理由は、第一に、ハイブリッド形成時より脱ハイブリダイゼーション時の方が選択性が高いこと、第二に、各段階での分離度は低くても、それを繰り返すことで、分離度が累乗的に増加することによると考えられる。
【００４１】
上記したように、温度分布の移動は、第一および第二領域のどちらを先にしてもよい。従来の結晶精製に於けるゾーンメルティング法においては、結晶析出の際に不純物が融液中に押し出されるので、高温部を先にしないと精製されないのに対し、本発明では、ポリヌクレオチド（Ａ）やポリヌクレオチド（Ｂ）がプローブとハイブリダイゼーションする際にも、解離する際にも精製されうるためである。しかしながら本発明においては、従来のゾーンメルティング法とは逆に、第二領域を先にして移動させるものであることが、分離効率が向上するため好ましい。第二領域を先にすることにより、セル内の任意の位置が、第二温度になされたのち、次いで第一温度になされることになる。従って、ポリヌクレオチド（Ｂ）やポリヌクレオチド（Ａ）が解離する際に分離されることになる。このような条件が好ましい理由は明確ではないが、ポリヌクレオチドはハイブリッド形成速度より解離速度の方が速く、又、解離時の方が選択性も高いことによるものと推定される。
本発明の分離方法を室温雰囲気で実施する場合には、ポリヌクレオチド（Ｂ）が極端に短い場合以外は、セルは最初第二温度にあることになり、自ずと第二領域を先にして温度分布を移動させることになる（第二領域の先端はセルの端より先にあると考える）。
【００４２】
さらに、本発明に於いては、第一領域と第二領域とを、交互に２回以上繰り返して移動させることが好ましく、その際には、第一領域と第二領域の組を一組として、それを繰り返すと考える。第一領域と第二領域の寸法が同じである場合には、セルの任意の部分を第一領域と第二領域が交互に通過するため、両者の違いは僅少と成るが、第一領域と第二領域の寸法を違える方法や、第一領域から第二領域へ移行する部分と第二領域から第一領域へ移行する部分の温度勾配に差を設けることで、順序の効果を増すことが出来る。
【００４３】
本発明の分離方法の分離メカニズムは未だ明確ではないが、ポリヌクレオチド（Ａ）および（Ｂ）が溶液の全領域で解離した状態でも、全領域で吸着された状態でも分離が行われないこと、及び、第一領域を先にした場合でも第二温度を先にした場合でも分離が行われることから、解離量の差、解離速度の差、ハイブリッド形成速度の差の少なくともいずれかが理由であると思われる。例えば、第一領域でポリヌクレオチド（Ｂ）の溶液中組成比が高い状態では、拡散、送液、電気浸透流、電気泳動などによりポリヌクレオチド（Ｂ）が第一領域から出てゆく割合が高くなることに寄るのであろう。
【００４４】
また、本発明においては、準静的な条件より速い温度変化で行うことも好ましいことから、ポリヌクレオチド（Ａ）とポリヌクレオチド（Ｂ）の解離率が同じ条件であるように見える条件であっても、速度的な差や、温度の移行部分でこれらが異なる条件があり、分離される。例えば、セル各部の溶液が第一領域に入ったとき、溶液が昇温してゆく時間の遅延と、解離に要する時間の遅延がある。一般に、解離に要する時間はハイブリッド形成に要する時間より短く、又、ポリヌクレオチド（Ａ）とポリヌクレオチド（Ｂ）の選択性も解離時の方が高い。従って、短時間だけ第一温度とし、その他の時間を第二温度とすることによって、ポリヌクレオチド（Ｂ）は、拡散によって或いは他の移動手段によって、選択的に第一領域から流出する。従って、第二温度が温度範囲αにあり、第一温度が温度範囲εにあっても、分離は可能である。
【００４５】
本発明に於いては、第一領域および第二領域が互いに接する温度分布を、セルに添って一方向に連続的に移動させる。移動の速度は、任意であり、セルの寸法、セル壁の厚さ、プローブの長さ、ポリヌクレオチド（Ａ）やポリヌクレオチド（Ｂ）の長さ、ポリヌクレオチド（Ａ）とポリヌクレオチド（Ｂ）の配列の異なり具合、セル内の溶液を移動させるか否かなどにより、適宜最適速度を選択できる。最適速度は用いる系における簡単な実験により見いだすことが出来る。
【００４６】
例えば、セル中の溶液を流動させないときであって、セルの幅又は深さ方向の直径又は短径が１０〜１００μｍであり、充填剤がない場合には、移動速度は、０．１〜１０ｍｍ／分が好ましい。この好ましい範囲の下限以上とすることで、分離時間を短くでき、この好ましい範囲の上限以下とすることで、分離能率の低下を防ぐことが出来る。セルの幅又は深さ方向の直径又は短径がこれより大である場合には、それに応じて速度を下げることが好ましい。セルの幅又は深さ方向の直径又は短径が上記より大であっても、多孔質体や充填剤が充填されていて、細孔の寸法が上記寸法より小である場合には、温度調節方法にもよるが、充分に速い昇降温速度で温度調節可能であれば、速くすることができる。また、セル内の溶液を前記温度分布の移動方向に流動させる場合などには、速くすることが好ましい。
【００４７】
セル中の溶液を第一温度及び第二温度に温度調節する方法は任意である。例えば、接触又は近接された温調ブロックとの相対的位置の移動、温度調節された気流との接触とその位置の移動、温度調節された液体との接触とその位置の移動、赤外線や電磁波の照射と照射位置の移動等を挙げることができる。
【００４８】
第一領域および第二領域が互いに接する温度分布は、勿論、その間に温度勾配を有する。第一領域と第二領域の移行部分の温度勾配は、特に強制的な（即ち、拡散以外の）ポリヌクレオチドの移送を行わないときには１〜１０００℃／ｍｍが好ましく、２〜１００℃／ｍｍがさらに好ましい。強制的なポリヌクレオチドの移送を行うときに０．１〜１００℃／ｍｍが好ましく、０．５〜１０℃／ｍｍがさらに好ましい。この範囲とすることによって、分離効率が高く、分離装置を小型に出来、また、温度調節も容易である。
【００４９】
このような大きな温度勾配を形成するために、セルを有する容器は、温度分布の移動方向における熱伝導率が１０ｗ・ｍ^−１・Ｋ^−１以下であることが好ましく、１ｗ・ｍ^−１・Ｋ^−１以下であることがさらに好ましく、０．３ｗ・ｍ^−１・Ｋ^−１以下であることが最も好ましい。上記方向の熱伝導率の下限は、自ずと限界はあろうが低いことそれ自体による不都合はないため下限を限定する必要はないが、例えば０．０１ｗ・ｍ^−１・Ｋ^−１であり得る。温度分布の移動方向における熱伝導率をこのような値にするには、該当する熱伝導率を有する素材で形成する方法、該方向に空隙や発泡体を形成することにより該方向の熱伝導率を低くする方法などにより実施できる。温度分布に直角な方向における熱伝導率は任意であるが、容器の温度分布の移動方向における熱伝導率以上であることが好ましい。そのようにすることによって、セルの第一領域及び第二領域の温度を均一にすることが容易になる。
【００５０】
本発明に於いては、セル中でポリヌクレオチドを強制的に（即ち、拡散によって自発的に移動すること以外の方法によることをいう。但し、説明の簡略化のため、以下、「強制的に」は省略する）移送しつつ行うことも好ましい。ポリヌクレオチドを移送することにより、分離の効率と速度を向上させることが出来る。ポリヌクレオチドの移送方法は任意であるが、例えば、前記溶液の送液（即ち、ポリヌクレオチドと溶媒を同時に移動させる方法）、溶液中のポリヌクレオチドの移送（即ち、溶媒は移動させない方法）、及びこれらが混合した移送（即ち、ポリヌクレオチドと溶媒の移動速度が異なる移送方法）であり得る。これらのうち、溶液の送液、すなわち、ポリヌクレオチドの混合溶液をセル内で一方向に流動させつつ温度分布の移動を行うことが、簡易な装置により行うことができるため好ましい。溶液の送液方法は任意であり、押し出し又は吸引による定量的送液、加圧気体や減圧気体による圧力差による送液、吸収体による吸収による送液、ダイヤフラムポンプなどによる間欠的な送液、電磁波による移送、振動や超音波による移送を例示できる。ポリヌクレオチドの移送は、電気泳動を例示できる。それらが混同した移送は、電気浸透流を例示できる。勿論上記の複数の方法を併用してもよい。
【００５１】
以下説明の簡略化のために、特に断らない限り、ポリヌクレオチドの移送を溶液の移送で代表させて説明する。溶液を移送する方向は温度分布の移動方向の同じでも反対方向でもよいが、同じ方向であることが、より効果的であり好ましい。温度分布の移動方向と同じ方向に溶液を移送する場合には、温度分布の移動速度の０．０１倍〜１．５倍が好ましく、０．１倍〜１倍がさらに好ましい。この範囲の速度で移送することにより、分離の効率と速度を増すことが出来る。電気泳動や電気浸透流の場合には、ポリヌクレオチドの移動速度がこの値になるように、印加電圧を調節する。
【００５２】
また、温度分布の移動は、２回以上繰り返し行われることが好ましく、５回以上、特に１０回以上繰り返して行われることがさらに好ましい。移動を２回以上繰り返すことによって、分離効率をより向上させることができる。
さらに、上記を実施するに当たって、第一領域と第二領域から成る一組の温度分布をセルの一端から他端まで移動させる操作を繰り返してもよいが、温度分布は、第一領域と第二領域とを温度分布の移動方向に交互に２回以上繰り返して設けられたものであることが好ましい。さらに５回以上、特に１０回以上繰り返して設けられることが、一回の操作で、セル中の任意の位置の溶液に多数回の温度変化を与えられるため好ましい。なお、この時、第一領域の温度は全て同一である必要はなく、第二領域の温度も全て同一である必要はない。
【００５３】
本発明の分離方法は、溶液を流動させない場合には、処理後に、セルの温度分布の移動末端にある溶液を採取し、ポリヌクレオチド（Ｂ）の組成比を向上させた溶液を得ることができる。また、溶液を流動させる場合には、セルから流出する溶液を採取することによって得られる。さらに、温度分布の移動方向と溶液の流動方向を同一にして、溶液の流動方向および温度分布の移動方向の最終端から流出する溶液を採取して、ポリヌクレオチド（Ｂ）の組成比を向上させた溶液を得ることが好ましい。
【００５４】
或いは、溶液を流動させる場合も流動させない場合も、処理後に、セル内のポリヌクレオチド溶液を洗浄液に置換し、セル内、好ましくはセル内の温度分布の移動の前端若しくは上流部分にある固相に吸着されたポリヌクレオチドを溶出させて採取し、分離されたポリヌクレオチド（Ａ）を得ることも出来る。
【００５５】
［分離装置］
本発明のポリヌクレオチドの分離装置は、標的塩基配列を有するポリヌクレオチド（Ａ）と、前記標的塩基配列を有さないポリヌクレオチド（Ｂ）とを含有する混合溶液から、ポリヌクレオチド（Ａ）分離するポリヌクレオチドの分離装置であって、容器内のセルの内表面および／または該セル内の固相に、前記標的塩基配列に対して相補的な塩基配列を有するプローブが固定された容器と、該容器内の前記セル内に収納された前記混合溶液に、ポリヌクレオチド（Ｂ）とプローブとが形成するハイブリッド鎖の解離温度より高い第一温度に調節された第一領域、および、前記第一温度より低く、かつポリヌクレオチド（Ａ）とプローブとが形成するハイブリッド鎖の全量が解離する温度未満である第二温度に調節された第二領域が互いに接する温度分布を持たせるための熱源と、該熱源を移動させる移動手段とを備えることを特徴とする。
【００５６】
セル内の混合溶液に第一領域および第二領域が互いに接する温度分布を持たせるための熱源としては、温調ブロック、温度調節された気流、温度調節された液体、赤外線や電磁波の照射等が挙げられる。これらのうち、装置の簡易さの観点から、温調ブロックを用いることが好ましい。これらの熱源は、セル内の溶液に所望の温度分布を持たせることができれば設置位置は任意であり、容器に直接接していてもよいし、近接されているだけでもよい。
また、熱源を移動させる移動手段としては、パルスモーター、直流モーター、交流モーターなどの電磁モーターを使用した移動機構等を挙げることができる。この熱源の移動は、熱源自体を移動させるものであってもよいし、容器の方を移動させることにより、容器と熱源との位置を相対的に移動させるものであってもよい。
【００５７】
本発明の装置に用いられる容器、およびセルとしては、それぞれ上述したものを好適に用いることができる。特にセルは、熱源の移動方向に対して垂直な断面の直径若しくは一辺が１μｍ〜１ｍｍの毛細管状であることが好ましい。この直径範囲内の毛細管とすることによって、熱伝導度に優れ所望の温度分布を達成することが容易であり、かつ製造容易な装置とすることができる。
【００５８】
【実施例】
以下本発明の実施例を説明するが、本発明の範囲はこれらの実施例に限定されるものではない。なお、以下の実施例において、「部」及び「％」は、特に断りがない限り、各々「質量％」、「質量部」を表す。
【００５９】
［組成物（Ｘ１）］
容器Ｄ１の製造例で使用する紫外線硬化性の組成物（Ｘ１）の調製方法を示す。
重合性化合物として、平均分子量約２０００の大日本インキ化学工業株式会社製３官能ウレタンアクリレートオリゴマー「ユニディックＶ−４２６３」６０部、第一工業製薬株式会社製ヘキサンジオールジアクリレート「ニューフロンティアＨＤＤＡ」３０部、和光純薬工業株式会社製メタクリル酸グリシジル１０部、光重合開始剤としてチバスペシャルテッィケミカルズ社製１−ヒドロキシシクロヘキシルフェニルケトン「イルガキュアー１８４」５部、及び重合遅延剤として関東化学株式会社製２，４−ジフェニル−４−メチル−１−ペンテン０．５部を混合して、組成物（Ｘ１）を調製する。
【００６０】
［組成物（Ｘ２）］
容器Ｄ２の製造例で使用する紫外線硬化性の組成物（Ｘ２）の調製方法を示す。
重合性化合物として、上記「ユニディックＶ−４２６３」６０部、上記「ニューフロンティアＨＤＤＡ」２０部、上記メタクリル酸グリシジル２０部、光重合開始剤として上記「イルガキュアー１８４」」５部、及び重合遅延剤として上記２，４−ジフェニル−４−メチル−１−ペンテン０．５部を混合して、組成物（Ｘ２）を調製する。
【００６１】
［製膜液（Ｙ）］
容器Ｄ１の製造例で使用する紫外線硬化性の組成物（Ｙ）の調製方法を示す。
重合性の化合物として、上記「ユニディックＶ−４２６３」７２部、日本化薬株式会社製ジシクロペンタニルジアクリレート「Ｒ−６８４」１８部、上記メタクリル酸グリシジル１０部、貧溶剤（Ｒ）として和光純薬工業株式会社製デカン酸メチル１２０部、揮発性の良溶剤としてアセトン１０部、紫外線重合開始剤として上記「イルガキュアー１８４」１３部、を均一に混合して製膜液（Ｙ）を調製する。
【００６２】
［紫外線の照射］
製造例に於ける紫外線照射は、例えば、下記の装置と方法で実施できる。
２００Ｗメタルハライドランプを光源とするウシオ電機株式会社製のマルチライト２００型露光装置用光源ユニットを用い、３６５ｎｍにおける紫外線強度が１００ｍＷ／ｃｍ^２の紫外線を、特に指定が無い限り室温、窒素雰囲気中で照射する。
【００６３】
［容器Ｄ１］
図３は、本発明の分離方法に好適に用いられる分離装置の一実施例のうち、セルを有する容器を示したもので、図３（ａ）は平面模式図、図３（ｂ）は断面模式図である。
図３に示すように、本実施例の容器１００は、平面状の支持体１上に、多孔質層２、第一樹脂層３、及び第二樹脂層４が順に積層され、これらが一体化されて概略構成されており、全体の外形は長さ７５ｍｍ、幅５０ｍｍ、厚さ約１．１８ｍｍの板状となっている。なお、外形は分離するポリヌクレオチド混合溶液の量等によって決定すればよく任意である。
【００６４】
支持体１としては、アクリル板、ポリスチレン板、ポリカーボネート板、硝子板等を用いることができるが、容器Ｄ１においてはアクリル板が使用されている。
多孔質層２は、支持体１上に固着されて形成されており、上述の製膜液（Ｙ）の塗膜を硬化させ、貧溶剤を除去したものである。その厚さは任意であるがＤ１においては約５μｍである。
第一樹脂層３は、上述の組成物（Ｘ１）を硬化させた層である。この第一樹脂層３には、層の表面から裏面に貫通するコの字型の細線状欠損部５が形成され、これが容器Ｄ１におけるセル５となっている。このセル５は、いずれの部分も幅が５００μｍ、高さが８５μｍであって、長さが３０ｍｍである直線状の欠損部５ｄと、その両端から４５度の角度で伸びたそれぞれ長さ３０ｍｍの直線状の欠損部５ｃおよび５ｅからなっている。
このセル５の内壁の各面には、長さ２０塩基の１本鎖ＤＮＡであるプローブが結合されている。
【００６５】
第二樹脂層４は、組成物（Ｘ１）を硬化させたものであり、その厚さは約９０μｍである。
第二樹脂層４には、層の表面から裏面に貫通する孔である流入口６および流出口７が形成されている。流入口６は、第一樹脂層３のセル５の上流端５ａに連通している。流出口７は、第一樹脂層３のセル５の下流端５ｂに連通している。
また、容器Ｄ１中心部のセル５ｄ付近にはセル５の温度測定用の熱電対８が封入されており、そのリード線９が容器外に引き出されている。
【００６６】
以下、この容器Ｄ１の製造方法について説明する。
〔工程１：多孔質層の形成〕
厚さ１ｍｍのアクリル板製支持体１の上に、スピンコーターを用いて、製膜液（Ｙ）を塗工し、紫外線を６０秒照射して硬化させ、相分離した貧溶剤（Ｒ）をｎ−ヘキサンで洗浄除去して多孔質層２を形成する。
【００６７】
〔工程２：欠損部（セル）の形成〕
上記多孔質層２の上に、バーコーターを用いて組成物（Ｘ１）を塗工して未硬化塗膜を形成し、該未硬化塗膜のセル５と成すべき部分、熱電対８装着部およびリード線９引き出し部以外の部分にフォトマスクを通して紫外線を１０秒間照射して未硬化塗膜を半硬化させて第一樹脂層３と成した。次いで、非照射部分の未硬化の組成物（Ｘ１）をエタノールで除去して、第一樹脂層３の欠損部として、多孔質層２が底面に形成されたセル５及び熱電対装着部８ａおよびリード線引き出し部９ａとなる欠損部を形成する。
【００６８】
〔工程３：蓋の固着〕
それぞれ太さ８５μｍのアルメル線、トクロメル線を電気溶接して作製し、熱電対８およびリード線９を、上記で作製した熱電対装着部８ａ及びリード線引き出し部９ａに装着する。
二村化学株式会社製二軸延伸ポリプロピレンフィルム「ＯＰＰフィルム」上に組成物（Ｘ１）を、スピンコーターを用いて塗工し、全面に紫外線を３秒間照射して半硬化させて第二樹脂層４と成し、上記工程２で作製した部材の第一樹脂層２の上に貼り合わせ、紫外線を４０秒照射して、第一樹脂層２、第二樹脂層４共に完全に硬化させたのち、ＯＰＰフィルムを剥離して、容器Ｄ１前駆体を製造した。
【００６９】
〔流入口、流出口の形成〕
容器Ｄ１前駆体のセル５の両端部５ａ，５ｂの位置に於いて、ドリルを用いて第二樹脂層４に直径１ｍｍの孔６、７を穿ち、セル５に連結した流入口６と流出口７を形成して、容器Ｄ１を製造した。
【００７０】
〔ＤＮＡの固定〕
流入部６からセル内に、５％ポリアリルアミン（日東紡株式会社）水溶液を注入し、５０℃で２時間静置した後、セルに流水を１５分間通して洗浄し余剰のポリアリルアミンを除去し、アミノ基を流路内側の固相（多孔質層及びその他のセル内壁）に導入する。
次いで、セル５に５％グルタルアルデヒド水溶液を注入し、５０℃で２時間静置した後、流水を通して１０分間洗浄して余剰のグルタルアルデヒドを除去し、乾燥窒素を通して多孔質層とセル内壁を乾燥させて、ホルミル基導入多孔質膜を得る。
次いで、セル５内にエスペックオリゴサービス株式会社製の５’末端にアミノ基を修飾した長さ２０塩基のＤＮＡの緩衝液溶液（ＤＮＡ濃度５０μＭ）を注入し、６０℃で１５時間静置した後、緩衝液を通して洗浄し、セル５内の固相にプローブとなるＤＮＡを共有結合で固定する。
【００７１】
〔接続部品Ｆの作製〕
図４に、本発明にかかる容器Ｄ１等を接続するために好適に用いられる接続部品Ｆの斜視図を示す。接続部品Ｆは、図４に示すように、５ｍｍ×５ｍｍ×厚さ２ｍｍのポリスチレン板１１の平面の中心部に、直径１．６ｍｍの貫通孔１２を穿ち、該貫通孔１２にポリエチレン製のチューブ１３を接着して、接続部材（Ｆ）を作製する。本接続部材（Ｆ）は、オーリング１４を挟んで容器Ｄ１の流入口６や流出口７にクランプ（図示せず）で密着させることによって、マイクロシリンジ（図示せず）やポンプ（図示せず）などに接続することができる。
【００７２】
［容器Ｄ２の製造例］
次に、本発明の分離装置に好適に用いられる容器の別の例として、チューブ状の容器Ｄ２を示す。
外径６ｍｍ、内径４ｍｍの硬質塩化ビニルパイプを加熱延伸して、外径１．０ｍｍ、内径０．５ｍｍの硬質塩化ビニル製のチューブ（図示せず）を作製する。
長さ１０ｃｍの前記チューブに、紫外線硬化性の組成物（Ｘ２）を注入し、加圧窒素で押し出して、チューブ内面に塗工し、紫外線を６０秒間照射して、該チューブ内面をエポキシ基導入硬化塗膜でコートする。
次いで、エスペックオリゴサービス株式会社製の５‘末端にアミノ基を修飾した長さ２０塩基のＤＮＡの緩衝液溶液（ＤＮＡ濃度５０μＭ）を前記チューブに注入し、該チューブの両端部を加熱してポリスチレンを軟化させつクランプで封じて、６０℃、で１５時間静置した後、緩衝液を前記チューブ内に流して余剰のＤＮＡを除去し、前記チューブ内面にプローブとなるＤＮＡを共有結合で固定する。
以上のようにして、硬質塩化ビニル製の長さ１０ｃｍ、外径１．０ｍｍ、内径０．５ｍｍのチューブの内面に、２０塩基の１本鎖ＤＮＡであるプローブが結合した容器Ｄ２を作製する。
【００７３】
［温調ブロック（Ｈ）］
図５に、本発明の分離装置に好適に用いられる温調ブロックＨのブロック斜視図を示す。
図５に示したように、この温調ブロックＨは、２２．５ｍｍ×２０ｍｍ×厚さ３ｍｍのアルミニウム製板状部２１ａに、１０ｍｍ×２０ｍｍ×厚さ１．５ｍｍの８枚のフィン２１ｂが１．５ｍｍの等間隔で形成されたアルミニウム製の部材２１に、板状の電気ヒーター２２とシース型の熱電対２３とが装着されてなっている。
【００７４】
［ポリヌクレオチドの分離］
例えば、マイクロシリンジ（図示せず）を用いて流入口７から混合ＤＮＡ溶液をセルに注入した容器Ｄ１又はＤ２を、室温の空気中で、例えば８５℃に調節された２つの上記温調ブロックＨ及び温調ブロックＨ’（以下、第二の温調ブロックＨ’の各部の名称には「’」を付ける）のフィン２１ｂとフィン２１ｂ’を突き合わせた間に挟んで移動させると、容器Ｄ１又はＤ２の両側からフィン２１ｂとフィン２１ｂ’で挟まれた領域が前記第一領域となり、フィン２１ｂ間の空隙２４とフィン２１ｂ’間の空隙２４’とで挟まれた領域が第二領域となる。
必要に応じて、流入口７又は流出口８にポンプを接続して、ＤＮＡ混合溶液を移動させる。
これにより、標的塩基配列を有するＤＮＡはプローブとハイブリダイズされて分離され、流出口８から、標的塩基配列を有さないＤＮＡの組成比が上昇したＤＮＡ溶液を得ることができる。
【００７５】
【発明の効果】
以上説明したように、本発明の分離方法によると、ポリヌクレオチドの混合溶液から、標的塩基配列を有するポリヌクレオチドを容易に分離することができ、特に全ポリヌクレオチドに対する組成比の低いポリヌクレオチドの溶液中組成比を上昇させることができる。また、本発明によると、上記分離方法に好適に用いることができ、かつμ−ＴＡＳ（マイクロ・トータル・アナリティカル・システム）の一部に組み込むことが容易な分離装置を提供することができる。
【図面の簡単な説明】
【図１】非重畳的な場合の、ポリヌクレオチド（Ａ）とポリヌクレオチド（Ｂ）の解離曲線を示した模式図である。
【図２】重畳的な場合の、ポリヌクレオチド（Ａ）とポリヌクレオチド（Ｂ）の解離曲線を示した模式図である。
【図３】本発明の製造装置に好適に用いられる容器Ｄ１の平面模式図および断面模式図である。
【図４】本発明の製造装置に好適に用いられる接続部品Ｆの斜視図である。
【図５】本発明の製造装置に好適に用いられる温調ブロックＨの斜視図である。
【符号の説明】
Ｄ１・・容器、Ｆ・・接続部品、Ｈ・・温調ブロック
１・・支持体、２・・多孔質層、３・・第一樹脂層、４・・第二樹脂層、５・・セル（欠損部）、６・・流入口、７・・流出口、８・・熱電対、９・・リード線、１１・・ポリスチレン板、１２・・貫通孔、１３・・チューブ、１４・・オーリング、２１・・アルミニウム製部材、２１ａ・・板状部、２１ｂ・・フィン、２２・・電気ヒーター、２３・・熱電対、２４・・空隙部[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a polynucleotide separation method and apparatus for separating a polynucleotide having a specific base sequence from a polynucleotide mixed solution.
[0002]
[Prior art]
As a method for separating and purifying the mutant DNA from the mixed solution of the wild-type DNA and the mutant DNA, a probe complementary to the wild-type DNA is immobilized on the powder packed in the column, and the mixed solution is purified. A method is disclosed in which a wild-type DNA is selectively adsorbed to a powder by hybridization and removed from a solution by hybridization, and a mutant DNA is concentrated in an outflowing solution (for example, see Patent Document 1). .).
However, when the base sequence of the target polynucleotide to be removed and the non-target polynucleotide differ by only one base (for example, single nucleotide polymorphism; SNP), it is necessary to exhibit sufficiently high selectivity in hybridization. It took several hours or more for separation and purification. In addition, even if it takes a long time, the selectivity of hybridization is limited, so that the separation rate is limited. In particular, when the composition ratio of the target polynucleotide for analysis or the like to the total polynucleotide is low, it has been difficult to separate the target polynucleotide from the target and obtain a target polynucleotide with a sufficiently high composition ratio.
On the other hand, affinity chromatography and affinity electrophoresis can be expected to have high resolving power and can be easily separated at a high level, but have a problem that the throughput is small for the size of the apparatus. In addition, when a purified polynucleotide is used in a reaction or the like, a large and complicated apparatus was required for automation.
[0003]
[Patent Document 1]
JP 2001-514504 A
[0004]
[Problems to be solved by the invention]
The problem to be solved by the present invention is to provide a method for easily separating a polynucleotide having a target base sequence from a mixed solution of polynucleotides, and in particular, has a low composition ratio to the total polynucleotides in the solution. It is an object of the present invention to provide a method capable of increasing the composition ratio of a polynucleotide, and to provide a separation device which can be easily incorporated into a part of μ-TAS (micro total analytical system).
[0005]
[Means for Solving the Problems]
The present inventors have conducted intensive studies on a method for solving the above-mentioned problem, and as a result, between the polynucleotide in the solution and the probe immobilized on the solid phase, while repeating hybridization and dehybridization, only one of the components was used. Or by moving one of the components faster to allow for a high degree of separation, and such an approach is possible by moving regions of partially different temperatures. This led to the completion of the present invention.
[0006]
That is, the present invention relates to a polynucleotide for separating a polynucleotide (A) from a mixed solution containing a polynucleotide (A) having a target base sequence and a polynucleotide (B) having no target base sequence. In a separation method, the mixed solution is placed in a cell in which a probe having a base sequence complementary to the target base sequence is fixed to at least one of the inner surface of the cell and a solid phase in the cell. And a first region adjusted to a first temperature higher than a dissociation temperature of a hybrid chain formed by a polynucleotide (B) and a probe, and The second regions, which are low and adjusted to a second temperature below the temperature at which the total amount of the hybrid strand formed by the polynucleotide (A) and the probe dissociates, are in contact with each other A method for separating polynucleotides, wherein a polynucleotide (A) is hybridized with the probe and separated from the mixed solution by providing a temperature distribution and continuously moving the temperature distribution in one direction. It is.
[0007]
Further, the second invention of the present invention provides a method for preparing a polynucleotide (A) from a mixed solution containing a polynucleotide (A) having a target base sequence and a polynucleotide (B) having no target base sequence. A device for separating polynucleotides to be separated, wherein a container having a probe having a base sequence complementary to the target base sequence immobilized on an inner surface of a cell in the container and / or a solid phase in the cell. A first region adjusted to a first temperature higher than a dissociation temperature of a hybrid chain formed by a polynucleotide (B) and a probe, in the mixed solution accommodated in the cell in the container; and The temperature at which the second regions adjusted to the second temperature which are lower than one temperature and lower than the temperature at which the total amount of the hybrid strand formed by the polynucleotide (A) and the probe dissociates are in contact with each other. A heat source for imparting fabric, a separator of a polynucleotide characterized in that it comprises a moving means for moving the heat source.
[0008]
The term "separating the polynucleotide (A)" as used in the present invention includes separation, detection, concentration and selective removal of the polynucleotide (A), and is indispensable by separating the polynucleotide (A). The separation, separation and detection, concentration, and selective removal of the polynucleotide (B), which is a polynucleotide component other than the polynucleotide (A), which can be obtained in a targeted manner. Here, “concentration of the polynucleotide (A)” means to increase the ratio of the polynucleotide (A) to all the polynucleotides in the solution.
[0009]
BEST MODE FOR CARRYING OUT THE INVENTION
[Polynucleotide (A), polynucleotide (B)]
A polynucleotide having a target base sequence (hereinafter, referred to as "polynucleotide (A)") and a polynucleotide having no target base sequence (hereinafter, referred to as "polynucleotide (B)") are optional, and may be DNA. , RNA, and other modified polynucleotides. The DNA may be, for example, total DNA, cDNA, a DNA fragment cut with a restriction enzyme, a DNA fragment amplified by polymerase chain reaction (PCR), or the like. Among them, a DNA fragment amplified by a polymerase chain reaction (PCR) or the like is particularly useful and preferable.
[0010]
The length of the polynucleotides (A) and (B) is preferably 10 to 1000 bases, more preferably 15 to 300 bases. This range is preferable because the selectivity can be increased and the separation speed can be increased. When prepared by PCR or restriction enzymes, the number of bases of these polynucleotides can be set by selecting primers and restriction enzymes.
[0011]
The polynucleotide (B) may be different in length from the polynucleotide (A), but preferably have the same length, since the effects of the present invention are exhibited. The polynucleotide (B) preferably has a base sequence that differs from the polynucleotide (A) by only one base in the target base sequence portion of the polynucleotide (A), because the effects of the present invention are exhibited. Such examples include single nucleotide polymorphisms (SNPs) and mutated genes.
Further, the polynucleotide (A) and the polynucleotide (B) may each be single-stranded or double-stranded. Double-stranded is useful and preferred.
[0012]
In the mixed solution of polynucleotides used in the separation method of the present invention, the ratio of the polynucleotide (B) to the total polynucleotides in the mixed solution is arbitrary, but it is 5% by mass or less, and further 2% by mass or less. Is preferred. In the conventional method, it was very difficult to improve the composition ratio of the polynucleotide which is a minor component in the mixed solution. However, when the method of the present invention was applied, the composition ratio of the polynucleotide (B) was 5% by mass. Even in the following cases, it is possible to easily obtain a solution having a high composition ratio of the polynucleotide (B) by separating the polynucleotide (A) which is a majority component.
[0013]
[Target nucleotide sequence]
The target base sequence refers to a characteristic base sequence of the polynucleotide (A) to be separated. For example, when the separation is a separation of a single nucleotide polymorphism (SNP) wild type (normal type) polynucleotide and a mutant type (abnormal type) polynucleotide, a nucleotide sequence containing a portion where a mutation occurs (hot spot) The part is used as a target base sequence. In this case, the target base sequence may be a wild-type base sequence or a mutant base sequence. When isolating a small amount of a mutant polynucleotide mixed with a large amount of a wild-type polynucleotide, the target base sequence is used as a wild-type base sequence, and the wild-type polynucleotide is selectively removed to thereby obtain a mutant-type polynucleotide. Preferably, the polynucleotides are separated.
[0014]
The length of the target base sequence may be, for example, 6 or more bases when the polynucleotide (A) has 10 to 100 bases (pairs). With this length, the probability that the polynucleotide (A) has a nucleotide sequence complementary to the probe by chance is so small that it can be ignored, so that sufficient accuracy and reliability can be obtained. When the number of bases (pairs) of the polynucleotide (A) is more than that, it is preferable to increase the number of bases of the probe according to the number of bases in order to prevent selection errors. For example, when the number of bases (pairs) of the polynucleotide (A) is 100 to 1,000, the length of the target base sequence is preferably 10 or more, and the length of the bases (pairs) of the polynucleotide (A) is preferred. If the number is several thousand or more, it is preferably 15 or more. The upper limit of the number of bases in the target base sequence is arbitrary, and the preferable upper limit depends on the degree of difference between the base sequence of the polynucleotide (B) and the target base sequence. The larger the difference in the sequence is, the larger the upper limit of the number of bases of the target base sequence may be accordingly. For example, the upper limit may be 1000, but is preferably 50 or less, and is preferably 30 or less. More preferred. When the sequence difference is one base, the difference is preferably 30 or less, more preferably 25 or less. When the content is not more than the preferable upper limit, separation can be sped up while maintaining sufficient selectivity.
[0015]
[probe]
The probe may be any compound having a base sequence complementary to the polynucleotide (A) and selectively hybridizing with the polynucleotide (A). Examples of the probe include oligo DNA, oligo RNA, and sugar moiety. Oligo DNA or oligo RNA having a modified or substituted phosphate group or any other compound may be used. Such probes can be synthesized, and are commercially available for microarrays and the like.
The probe is used to determine whether the polynucleotide (A) or the polynucleotide (B) is single-stranded (one of the opposing strands), both opposing strands, or, if both are opposing strands, the opposite strand. It can be designed according to the purpose of separation, such as separating only one or both. For example, when the polynucleotide (A) or the polynucleotide (B) is one of the opposite strands, it is preferable that the single strand is complementary to the opposite strand. The same applies to a case where the polynucleotide (A) or the polynucleotide (B) has both opposing chains, and only one of the opposing chains needs to be separated. In the case where the polynucleotide (A) or the polynucleotide (B) contains both opposing strands, and separates both the opposing strands of the polynucleotide (B) and the opposing strand of the polynucleotide (A), the probe Can be achieved by immobilizing single strands complementary to both opposing strands of the polynucleotide (A) at different positions as described later, and using complementary double strands However, it is preferable to fix each single strand independently because of high separation efficiency.
The probe need not be complementary to the entire sequence of the polynucleotide (A), but may have a base sequence complementary to the target base sequence of the polynucleotide (A). Therefore, the lower limit of the length of the probe is the length of the target base sequence of the polynucleotide (A).
[0016]
The probe may have a portion other than the target base sequence. In this case, only the base sequence portion complementary to the polynucleotide (A) contributes to the hybridization. However, in this case, the base sequence portion that is not complementary to the polynucleotide (A) needs to be not complementary to the polynucleotide (B) due to the number of bases equal to or greater than the target base sequence.
The length of the probe is arbitrary, and it is preferable that the length be suitable for the length of the target base sequence. In order to prevent hybridization selection errors, the oligonucleotide is preferably an oligonucleotide having 15 or more bases. The upper limit of the number of bases is not particularly limited, but is preferably not more than the number of bases of the polynucleotide (A), more preferably not more than 50, and further preferably not more than 30. The separation time can be reduced by setting the content to the preferable upper limit or less. When the polynucleotide (B) has a base sequence that differs from the target base sequence by only one base, the number of bases of the probe is preferably 25 or less, more preferably 22 or less. When the content is equal to or less than the upper limit, selectivity is improved.
The probe may further have a portion unrelated to the nucleotide sequence. For example, a functional group such as an amino group for immobilizing the probe on a solid phase during solid phase synthesis of the probe, an alkylene group or a polyethylene glycol group serving as a spacer between a base sequence portion and the functional group for immobilization, and the like. Can be.
[0017]
The probe is immobilized on at least one of the inner surface of the cell in which the mixed solution of polynucleotides is placed and the solid phase in the cell. The inner surface or solid phase of the cell to which the probe is fixed is, for example, the inner wall surface of the cell, the surface of some structure such as a pillar or a pile formed in the cell, the surface of beads or fibers loaded inside the cell, the cell. It may be a pore surface of a porous body which is formed by adhering to the inner wall surface of the cell and has a shape that blocks or does not block the cross section of the cell. Of these, the inner surface of the cell is preferred. By adopting such a shape, it is easy to produce a minute cell that can be separated even if the amount of the polynucleotide is very small, and the heat transfer rate is high, so that the separation can be speeded up, while the solution is flowing through the cell. In the case of separation, liquid sending becomes easy due to small pressure loss. Further, it is more preferable to form a porous layer on the inner surface of the cell that does not block the cross section of the cell. By adopting such a shape, in addition to the above-mentioned features, it is possible to increase the immobilization density of the probe, so that when isolating the same amount of polynucleotide, the cell can be downsized. The immobilization method is arbitrary, and for example, a known method used for a DNA microarray or the like can be used.
When the probes are both opposing strands, they may be fixed in a mixed state or may be fixed independently of each other, but are preferably fixed to fixed portions independent of each other because the separation efficiency is increased. . The mutually fixed portions are preferably minute regions having a center-to-center distance of 0.1 μm to 0.5 mm, more preferably 1 μm to 0.1 mm.
[0018]
[cell]
A cell is a hollow part formed in a container and dispensing a polynucleotide solution therein, and its shape is arbitrary. Among them, it is preferable that the cell has a capillary shape or a slit-like cross section because the temperature following property of the polynucleotide solution is high and rapid separation is possible, and the cell is preferably long in one direction. Further, in order to increase the processing amount, a structure for increasing the surface area inside the cell, for example, a pile, a column, a plate, a porous body in a shape to block the cross section, a powder or a fiber, etc. Although the filling is formed in a shape that does not block the cross section in the cell, it is preferable to form a porous layer or the like or to fill the filling such as a powdery or fibrous form.
[0019]
Although the size of the cell is also arbitrary, it is preferable that the moving direction of the temperature distribution described later be the longitudinal direction of the cell. The length of the cell is preferably 0.3 to 1000 mm, more preferably 3 to 100 mm. By setting the temperature at or above the lower limit of the preferred range, the formation of a temperature distribution described later becomes easy, and the separation rate can be increased. By setting the temperature at or below the upper limit of the preferred range, the separation time can be shortened. When the "width" direction and the "thickness" direction are perpendicular to each other in a plane perpendicular to the cell length direction, at least one of the width and the thickness is preferably 0.01 to 1 mm, and 0.1 to 1 mm. More preferably, it is 1 to 0.5 mm. By setting the lower limit or more, it is possible to avoid the difficulty of production and to increase the throughput. Further, by adjusting the temperature to be equal to or less than the preferable upper limit, the temperature followability can be enhanced, and the rate of temperature rise and fall of each part in the cell can be increased, so that the separation time can be shortened and the separation efficiency can be improved. The other dimension in the width or thickness direction is arbitrary. The dimensions in the longitudinal direction may be the same as those described above, or may be larger than the dimensions in the longitudinal direction.
[0020]
[container]
The shape of the container having the cell therein is arbitrary, but it is preferable that the container wall is thin in order to increase the followability of the temperature change of the polynucleotide solution disposed in the cell. For this reason, it is preferable that the microfluidic device has an arbitrary shape in which a thin tube or a cell is formed at a position shallow from the surface and parallel to the surface, and particularly a tube or plate.
The material of the container has a heat conduction coefficient of 10 wm^-1・ K^-1Preferably 1 w · m^-1・ K^-1And more preferably 0.3 w · m^-1・ K^-1It is most preferred that: By setting the temperature to be equal to or less than the upper limit, it is easy to give a large temperature gradient to the solution in the cell in the longitudinal direction of the cell, and it is possible to reduce the size of the separation device and to improve the separation efficiency, that is, when the temperature distribution is moved. The rate of concentration increase is high. The lower limit of the heat conduction coefficient is not necessarily limited, although it is naturally limited, but is, for example, 0.01 w · m^-1・ K^-1Can be Examples of the material having such a heat conductivity coefficient include, for example, organic polymers, glass, carbon, ceramics and the like, and these foams and composites may be used. , And easy to manufacture, which is preferable.
[0021]
[Melting temperature, dissociation temperature]
As used herein, the term "dissociation temperature" refers to the lowest temperature at which the dissociation of the polynucleotide and the probe starts when the temperature of the hybrid strand formed by the polynucleotide and the probe is increased at a sufficiently low rate. However, the "minimum temperature at which dissociation starts" as used herein may include a measurement error and has a certain industrial meaning, for example, a temperature at which 1 to 3% is dissociated.
Similarly, when the test polynucleotide in a double-stranded state dissociates into single strands, the lowest temperature at which dissociation starts is referred to as dissociation temperature.
[0022]
“Melting temperature” refers to the temperature at which half of the hybrid strand dissociates when the temperature of the hybrid strand formed by the polynucleotide and the probe is increased at a sufficiently low rate. However, the amount of undissociated hybrid chains described in the next section is excluded. Similarly, when a test polynucleotide in a double-stranded state dissociates into a single strand, the temperature at which half of the polynucleotide dissociates is referred to as the melting temperature.
"The temperature at which the entire amount of the hybrid chain dissociates" refers to the lowest temperature at which the entire amount of the hybrid chain dissociates when the temperature of the hybrid chain formed by the polynucleotide and the probe is raised at a sufficiently low rate. Here, the term “all amounts dissociate” means a temperature at which the temperature does not substantially dissociate even if the temperature is further increased. Polynucleotides hybridized with the probe immobilized on the solid phase may remain undissociated even when the temperature is raised to around 100 ° C., although the reason is unknown, but such undissociated polynucleotides are excluded. Think. Further, "substantially does not dissociate further" may include a measurement error and has a certain industrial meaning, for example, a degree at which 97 to 99% of the dissociation occurs.
[0023]
The melting temperature and dissociation temperature depend on the number of bases in the probe compound, the number of mismatches in the base sequence, the types of bases in the probe compound, the salt concentration of the sample solution, and the concentrations of additives such as EDTA and organic solvents in the sample solution. change. For example, the melting temperature is generally 50-95C.
The above-mentioned dissociation temperature, melting temperature, and the like can be determined, for example, by performing an experiment in which washing is performed while changing the temperature using a so-called DNA microarray, and using a curve of the washing temperature versus the hybrid residual amount.
[0024]
Hereinafter, in the present specification, in order to describe the above-mentioned temperature, the temperature and the temperature range are denoted by reference numerals as described below.
T_LB: Dissociation temperature of hybrid of polynucleotide (B) and probe
T_MB: Melting temperature of hybrid of polynucleotide (B) and probe
T_HB: Temperature at which the total amount of the hybrid of the polynucleotide (B) and the probe dissociates
T_LA: Dissociation temperature of hybrid of polynucleotide (A) and probe
T_MA: Melting temperature of hybrid of polynucleotide (A) and probe
T_HA: Temperature at which the total amount of the hybrid of the polynucleotide (A) and the probe is dissociated
Then T_LB<T_MB<T_HB, T_LA<T_MA<T_HA, T_MB<T_MAThere is a relationship. Also, T_LB, T_MB, T_HBAre in such a relationship that the higher the non-complementarity of the polynucleotide (B) with the target base sequence, the lower the temperature.
[0025]
A quasi-static condition, that is, a hybrid chain of a polynucleotide (A) and a probe (hereinafter referred to as a “probe / (A) hybrid”) when a sample solution is heated at a sufficiently low heating rate (for example, 10 ° C./hour) FIG. 1 and FIG. 2 are schematic diagrams showing melting curves of a hybrid chain of a polynucleotide (B) and a probe (hereinafter sometimes referred to as “probe / (B) hybrid”). Was. 1 and 2, the horizontal axis indicates the temperature of the solution containing the test polynucleotide, and the vertical axis indicates the hybridization rate between the polynucleotide and the probe compound. In each figure, the broken line is the melting curve of the polynucleotide (A), and the solid line is the melting curve of the polynucleotide (B).
[0026]
FIG._HB≤T_LA  (Hereinafter, the melting curve may be referred to as “non-overlapping” in some cases). In this case, the temperature ranges are named as follows.
Temperature range α: Freezing temperature of solution to T_LB
Temperature range β: T_LB~ T_HB
Temperature range δ: T_LA~ T_HA
Temperature range ε: T_HB~ Solution boiling temperature
[0027]
FIG. 2 shows that T_LA≤T_HB  (Hereinafter, the melting curve may be referred to as “superposition”). In this case, the temperature ranges are named as follows.
Temperature range β ': T_LB~ T_LA
Temperature range γ ': T_LA~ T_HB
Temperature range δ ': T_HB~ T_HA
However, in the case of the superimposed temperature relationship in FIG. 2, the temperature ranges α and ε are common to those in FIG.
[0028]
The relationship between the dissociation rates of the polynucleotides (A) and (B) in the solution in each temperature range is shown in Tables 1 and 2 below. Table 1 shows the case where the dissociation curve is non-overlapping, and Table 2 shows the case where it is superimposing.
[0029]
[Table 1]

[0030]
[Table 2]

(However, x and y are each 0 or more and 1 or less, and are values dependent on the dissociation curve of each polynucleotide.)
[0031]
[Separation method]
The separation method of the present invention provides a method for separating a polynucleotide (A) from a mixed solution containing a polynucleotide (A) having a target base sequence and a polynucleotide (B) having no target base sequence. In a separation method, a probe having a base sequence complementary to a target base sequence is arranged on a cell fixed to at least one of the inner surface of the cell or a solid phase in the cell, A mixed solution disposed therein, a first region adjusted to a first temperature higher than the dissociation temperature of a hybrid chain formed by the polynucleotide (B) and the probe, and a polynucleotide lower than the first temperature and the polynucleotide (A) and a second region adjusted to a second temperature lower than the temperature at which the total amount of the hybrid chain formed by the probe dissociates has a temperature distribution in which the second regions are in contact with each other; By moving the degree distribution in one direction continuously, and separating polynucleotide (A) from the mixed solution was hybridized with probes.
[0032]
The first temperature is the dissociation temperature (T.sub.T) of the hybrid strand formed by the polynucleotide (B) and the probe._LB) Higher temperature. First temperature is T_LBIf the temperature is within the range (α), the second temperature lower than the first temperature enters the same region (α), and both the polynucleotide (A) and the polynucleotide (B) in the solution are adsorbed to the solid phase by hybridization. However, there is no dissociated polynucleotide in solution in any region of the cell, and no separation is performed.
[0033]
The second temperature is lower than the first temperature and a temperature (T.sub.T) at which the total amount of the hybrid chain formed by the polynucleotide (A) and the probe is dissociated._HA). The second temperature is set in the temperature range ε, ie, T_HAAs a result, the first temperature higher than the second temperature also falls within the temperature range ε, the polynucleotide (A) and the polynucleotide (B) do not hybridize in the entire region in the cell, and the entire amount is dissolved in the solution. Will be. Therefore, the separation of the present invention is not performed under these conditions.
[0034]
Therefore, in the present invention, the first temperature is set to T_LBAnd the second temperature is lower than the first temperature and T_HATemperature below. Under these conditions, whether the two dissociation curves for polynucleotide (A) and polynucleotide (B) are non-overlapping or superimposing, the total adsorption of both polynucleotides in the first region The amount is less than or equal to that in the second region, and the ratio of the polynucleotide (B) dissolved in the solution in the first region (dissolved amount / total amount) is the polynucleotide (A) is equal to or more than the dissolving ratio (dissolved amount / total amount).
[0035]
Although the separation mechanism of the present invention is not yet clear, it is presumed as follows.
When any part of the cell is heated to the first temperature, at least a part of the polynucleotide (B) is in a dissociated (dehybridized) state. At this time, the first temperature is T_HAIf it is less than 1, at least a part of the polynucleotide (A) forms a hybrid chain with the probe and is adsorbed on the solid phase, so that the composition ratio of the polynucleotide (B) in the solution increases. Therefore, the ratio of the polynucleotide (B) flowing out of the first region in the moving direction or downstream direction of the temperature distribution by diffusion or liquid sending is higher than that of the polynucleotide (A). Thereafter, the probe and the polynucleotide (A) are preferentially hybridized by the temperature of the portion being changed to the second temperature by a continuous temperature change, so that the composition ratio of the polynucleotide (B) in the solution is further increased. To rise. Therefore, also in this region, the proportion of the polynucleotide (B) that flows out of the region in the moving direction or downstream direction of the temperature distribution due to diffusion or liquid sending is higher in the polynucleotide (B) than in the polynucleotide (A). At this time, the first temperature is T_HAEven above, the rate at which the immobilized probe adsorbs the polynucleotide (A) is faster than the rate at which the polynucleotide (B) is adsorbed during a continuous temperature change from the first temperature to the second temperature. Therefore, in a region where the temperature changes, the polynucleotide (B) in the solution is separated by an increase in the composition ratio._HAIt is preferable that the amount is less than the value, since the polynucleotide (A) and the polynucleotide (B) can be more efficiently separated.
[0036]
Further, the first temperature is a temperature at which the total amount of the polynucleotide (B) dissociates (T_HB) And the second temperature is the melting temperature of the polynucleotide (B) (T_MBIt is preferred that: The first temperature is T_HBWith the above, when an arbitrary part in the cell enters the first region, almost all of the polynucleotide (B) is dissociated, so that the composition ratio of the polynucleotide (B) in the solution can be increased. In addition, the amount of movement in the cell is increased, and the separation efficiency, that is, the degree of separation by a single change from the first temperature to the second temperature is improved. Also, if the second temperature is T_MBSince the polynucleotide (B) can be hybridized to the probe at a sufficiently high ratio when the concentration is less than or equal to the above, the concentration difference of the polynucleotide in the solution between the first region and the second region becomes large, and the separation efficiency is improved. I do. At this time, the second temperature is the dissociation temperature T of the polynucleotide (B)._LBThe following (temperature range α) is more preferable for the same reason.
The second temperature is in the temperature range α, and the first temperature is T_MBThe above is preferable because the concentration difference of the polynucleotide in the solution between the first region and the second region is large, and the separation efficiency is improved, and the first temperature is in the temperature range β, γ, β ′, or More preferably, it is at γ ′.
[0037]
Conversely, when any part in the cell changes from the second temperature to the first temperature, contrary to the above case, when any part in the cell is made to the second temperature Then, at least a part of the polynucleotide (A) is adsorbed to the solid phase by hybridization. At this time, the second temperature is T_LBIf it is higher, at least a part of the polynucleotide (B) dissociates from the probe and elutes in the solution, so that the composition ratio of the polynucleotide (B) in the solution increases in the second region. Accordingly, the ratio of the polynucleotide (B) flowing out of the second region in the moving direction or downstream direction of the temperature distribution by diffusion or liquid sending is higher than that of the polynucleotide (A). Thereafter, the temperature of the same portion is changed to the first temperature by a continuous temperature change, so that the hybrid strand of the probe and the polynucleotide (B) is preferentially dissociated, whereby the composition ratio of the polynucleotide (B) in the solution is increased. Further rise. Therefore, also in this region, the ratio of the polynucleotide (B) flowing out of the region in the moving direction or downstream direction of the temperature distribution due to diffusion or liquid sending is higher than that of the polynucleotide (A). Also, if the first temperature is T_LBEven when the temperature is below, the hybridization between the probe and the polynucleotide (B) is preferentially dissociated during a continuous temperature change from the second temperature to the first temperature, so that the temperature change region In, the polynucleotide (B) in the solution is separated by increasing the composition ratio, but the second temperature is T_LBIt is preferable that the amount is less than the value, since the polynucleotide (A) and the polynucleotide (B) can be more efficiently separated.
[0038]
Furthermore, if the first temperature is T_HAAnd the second temperature is T_LBThe rate at which the polynucleotide (A) is adsorbed on the immobilized probe during a continuous temperature change from the first temperature to the second temperature is lower than that at which the polynucleotide (B) is adsorbed. The rate at which the polynucleotide (B) adsorbed on the immobilized probe dissociates during the continuous temperature change from the second temperature to the first temperature is higher than the rate at which the polynucleotide (A) dissociates. By increasing the speed, the composition ratio of the polynucleotide (B) in the solution is increased and separated in the region where the temperature changes.
[0039]
Of course, the above explanation is a conceptual principle, and it is presumed that a more complicated phenomenon occurs in an actual system. For example, a change from the first temperature to the second temperature or vice versa may pass through an intermediate temperature, or a time delay between hybridization and dehybridization after reaching a predetermined temperature. Effect. Therefore, depending on the length in the moving direction of the first region and the second region, the moving speed of the temperature distribution, the length of the probe, the length of the polynucleotide (A) or the polynucleotide (B), the flow rate of the solution, etc. Velocity relationships can change, giving rise to complex aspects.
[0040]
In the prior art (Patent Document 1), a wild-type polynucleotide in a solution is selectively hybridized to an oligonucleotide (probe in the present invention) immobilized on a solid phase. It took a long time, for example, 1 to 10 hours, to hybridize only the mold with sufficient selectivity. On the other hand, in the present invention, the holding time of each of the first temperature and the second temperature may be less than 1 minute, and even in such a short time, sufficient selectivity is exhibited and separation is performed. The reason is that firstly, the degree of selectivity is higher during dehybridization than during hybridization, and secondly, even though the degree of separation at each step is low, the degree of separation is raised to a power by repeating it. It is thought that this is due to a temporary increase.
[0041]
As described above, the movement of the temperature distribution may be performed in any of the first and second regions. In the conventional zone melting method in crystal purification, impurities are extruded into the melt during crystal precipitation, so that the impurities are not purified unless a high-temperature portion is first. On the other hand, in the present invention, the polynucleotide (A ) And the polynucleotide (B) can be purified both when hybridizing with the probe and when dissociating. However, in the present invention, contrary to the conventional zone melting method, it is preferable to move the second region first, because the separation efficiency is improved. By placing the second region first, any location within the cell will be at a second temperature and then at a first temperature. Therefore, the polynucleotide (B) and the polynucleotide (A) are separated when dissociated. The reason why such conditions are preferable is not clear, but it is presumed that the polynucleotide has a higher dissociation rate than the hybridization rate and has higher selectivity at the time of dissociation.
When the separation method of the present invention is carried out in a room temperature atmosphere, the cell is initially at the second temperature unless the polynucleotide (B) is extremely short. (The tip of the second area is considered to be ahead of the edge of the cell).
[0042]
Further, in the present invention, it is preferable that the first region and the second region are alternately and repeatedly moved at least twice, and in that case, the set of the first region and the second region is regarded as one set. Think of repeating it. If the dimensions of the first region and the second region are the same, the first region and the second region alternately pass through any part of the cell, so that the difference between the two is small, By increasing the effect of the order by changing the size of the second region or by providing a difference in the temperature gradient between the portion transitioning from the first region to the second region and the portion transitioning from the second region to the first region. I can do it.
[0043]
Although the separation mechanism of the separation method of the present invention is not yet clear, separation is not performed even when the polynucleotides (A) and (B) are dissociated in all regions of the solution or adsorbed in all regions, And, since the separation is performed even when the first temperature is applied first or the second temperature is applied first, at least one of the difference in the dissociation amount, the difference in the dissociation rate, and the difference in the hybridization rate is the reason. I think that the. For example, when the composition ratio of the polynucleotide (B) in the solution in the first region is high, the ratio of the polynucleotide (B) leaving the first region due to diffusion, liquid sending, electroosmotic flow, electrophoresis, or the like is high. I'm sure you're going to be.
[0044]
Further, in the present invention, it is also preferable to carry out the reaction at a temperature change faster than the quasi-static condition, so that the dissociation rates of the polynucleotide (A) and the polynucleotide (B) seem to be the same. These are also separated due to different conditions due to differences in speed and transition of temperature. For example, when the solution in each part of the cell enters the first region, there is a delay in the time required for the solution to rise in temperature and a delay in the time required for dissociation. Generally, the time required for dissociation is shorter than the time required for hybridization, and the selectivity between polynucleotide (A) and polynucleotide (B) is higher at the time of dissociation. Thus, by setting the first temperature for a short time and the second temperature for the other time, the polynucleotide (B) selectively flows out of the first region by diffusion or by other moving means. Therefore, separation is possible even if the second temperature is in the temperature range α and the first temperature is in the temperature range ε.
[0045]
In the present invention, the temperature distribution where the first region and the second region are in contact with each other is continuously moved in one direction along the cell. The speed of movement is arbitrary, cell size, cell wall thickness, probe length, polynucleotide (A) or polynucleotide (B) length, polynucleotide (A) and polynucleotide (B) The optimum speed can be appropriately selected depending on the arrangement of the cells, whether the solution in the cell is moved or not, and the like. The optimum speed can be found by simple experiments in the system used.
[0046]
For example, when the solution in the cell is not allowed to flow, the diameter or minor axis in the width or depth direction of the cell is 10 to 100 μm, and when there is no filler, the moving speed is 0.1 to 10 mm. / Min is preferred. The separation time can be shortened by setting the lower limit of the preferable range or more, and the separation efficiency can be prevented from being lowered by setting the lower limit of the preferable range or less. If the diameter or minor diameter of the cell in the width or depth direction is greater than this, it is preferable to reduce the speed accordingly. Even if the diameter or minor axis in the width or depth direction of the cell is larger than the above, if the porous body or the filler is filled and the size of the pores is smaller than the above size, the temperature is adjusted. Depending on the method, if the temperature can be adjusted at a sufficiently high temperature rising / falling rate, the temperature can be increased. Further, when the solution in the cell is caused to flow in the moving direction of the temperature distribution, it is preferable to increase the speed.
[0047]
The method of adjusting the temperature of the solution in the cell to the first temperature and the second temperature is optional. For example, movement of the relative position with the contact or close temperature control block, contact with the temperature-controlled airflow and movement of its position, contact with the temperature-controlled liquid and movement of its position, infrared and electromagnetic waves Irradiation and movement of the irradiation position can be mentioned.
[0048]
The temperature distribution where the first region and the second region contact each other has, of course, a temperature gradient therebetween. The temperature gradient at the transition between the first region and the second region is preferably from 1 to 1000 ° C./mm, particularly preferably from 2 to 100 ° C./mm when no forced (ie, other than diffusion) polynucleotide transfer is performed. More preferred. When the polynucleotide is forcibly transferred, the temperature is preferably 0.1 to 100 ° C / mm, more preferably 0.5 to 10 ° C / mm. Within this range, the separation efficiency is high, the size of the separation device can be reduced, and the temperature can be easily adjusted.
[0049]
In order to form such a large temperature gradient, the container having cells has a thermal conductivity of 10 w · m in the moving direction of the temperature distribution.^-1・ K^-1Preferably 1 w · m^-1・ K^-1And more preferably 0.3 w · m^-1・ K^-1It is most preferred that: The lower limit of the thermal conductivity in the above direction does not need to be limited because there is naturally no limit, but there is no inconvenience due to itself. For example, 0.01 w · m^-1・ K^-1Can be In order to make the thermal conductivity in the moving direction of the temperature distribution such a value, a method of forming a material having a corresponding thermal conductivity, a thermal conductivity in the direction by forming a void or foam in the direction. Can be reduced. The thermal conductivity in the direction perpendicular to the temperature distribution is arbitrary, but is preferably higher than the thermal conductivity in the moving direction of the temperature distribution of the container. By doing so, it becomes easy to make the temperature of the first region and the second region of the cell uniform.
[0050]
In the present invention, the polynucleotide is forcedly moved in a cell (that is, by a method other than spontaneous movement by diffusion. However, for the sake of simplicity of description, the following "forced" Is omitted). It is also preferable to carry out the transfer. By transferring the polynucleotide, the efficiency and speed of the separation can be improved. The method of transporting the polynucleotide is arbitrary. For example, the solution is sent (that is, a method in which the polynucleotide and the solvent are simultaneously moved), the polynucleotide is transferred in the solution (that is, the solvent is not moved), and These may be a mixed transfer (that is, a transfer method in which the transfer speeds of the polynucleotide and the solvent are different). Among these, it is preferable to transfer the solution, that is, to move the temperature distribution while flowing the mixed solution of polynucleotides in one direction in the cell, because it can be performed by a simple device. The solution sending method is arbitrary, quantitative sending by extrusion or suction, sending by pressure difference by pressurized gas or depressurized gas, sending by absorption by absorber, intermittent sending by diaphragm pump, etc. Examples include transfer by electromagnetic waves, transfer by vibration and ultrasonic waves. The transfer of the polynucleotide can be exemplified by electrophoresis. Transfers where they are confused can exemplify electroosmotic flow. Of course, a plurality of the above methods may be used in combination.
[0051]
For the sake of simplicity, the transfer of a polynucleotide will be exemplified by the transfer of a solution unless otherwise specified. The direction in which the solution is transferred may be the same or opposite to the direction in which the temperature distribution moves, but the same direction is more effective and preferable. In the case where the solution is transferred in the same direction as the moving direction of the temperature distribution, the moving speed of the temperature distribution is preferably 0.01 to 1.5 times, more preferably 0.1 to 1 times. By transferring at a speed in this range, the efficiency and speed of separation can be increased. In the case of electrophoresis or electroosmotic flow, the applied voltage is adjusted so that the moving speed of the polynucleotide becomes this value.
[0052]
Further, the movement of the temperature distribution is preferably repeated twice or more, more preferably 5 or more times, particularly preferably 10 times or more. By repeating the movement twice or more, the separation efficiency can be further improved.
Further, in performing the above, an operation of moving a set of temperature distributions including the first region and the second region from one end of the cell to the other end may be repeated. It is preferable that the region is repeatedly provided two or more times alternately in the moving direction of the temperature distribution. Further, it is preferable that the solution is repeatedly provided 5 times or more, especially 10 times or more, because the temperature of the solution at an arbitrary position in the cell can be changed many times by one operation. At this time, the temperatures in the first region need not all be the same, and the temperatures in the second region need not be all the same.
[0053]
According to the separation method of the present invention, when the solution is not allowed to flow, the solution at the moving end of the temperature distribution of the cell is collected after the treatment, and a solution having an improved composition ratio of the polynucleotide (B) can be obtained. . When the solution is made to flow, the solution is obtained by collecting the solution flowing out of the cell. Further, by making the moving direction of the temperature distribution and the flowing direction of the solution the same, collecting the solution flowing out from the final end of the flowing direction of the solution and the moving direction of the temperature distribution to improve the composition ratio of the polynucleotide (B). It is preferable to obtain a solution which is not in use.
[0054]
Alternatively, regardless of whether the solution is allowed to flow or not, after the treatment, the polynucleotide solution in the cell is replaced with a washing solution, and the solid solution at the front end or upstream of the movement of the temperature distribution in the cell, preferably in the cell. The separated polynucleotide (A) can also be obtained by eluting and collecting the adsorbed polynucleotide.
[0055]
[Separation device]
The polynucleotide separating device of the present invention separates a polynucleotide (A) from a mixed solution containing a polynucleotide (A) having a target base sequence and a polynucleotide (B) having no target base sequence. A device for separating polynucleotides, wherein a container having a probe having a base sequence complementary to the target base sequence immobilized on an inner surface of a cell in the container and / or a solid phase in the cell, A first region adjusted to a first temperature higher than a dissociation temperature of a hybrid chain formed by a polynucleotide (B) and a probe, in the mixed solution contained in the cell in the container, and the first temperature The second regions, which are lower and adjusted to the second temperature which is lower than the temperature at which the total amount of the hybrid strand formed by the polynucleotide (A) and the probe is dissociated, are in contact with each other. A heat source in order to provide a temperature distribution that is characterized by comprising moving means for moving the heat source.
[0056]
Examples of the heat source for providing the mixed solution in the cell with a temperature distribution in which the first region and the second region are in contact with each other include a temperature control block, a temperature-controlled air flow, a temperature-controlled liquid, irradiation of infrared rays and electromagnetic waves, and the like. No. Of these, it is preferable to use a temperature control block from the viewpoint of simplicity of the device. These heat sources may be installed at any position as long as the solution in the cell can have a desired temperature distribution, and may be in direct contact with the container or only in close proximity to the container.
In addition, examples of the moving means for moving the heat source include a moving mechanism using an electromagnetic motor such as a pulse motor, a DC motor, and an AC motor. The movement of the heat source may be such that the heat source itself is moved, or the position of the heat source may be relatively moved by moving the container.
[0057]
As the container and the cell used in the apparatus of the present invention, those described above can be suitably used. In particular, the cell is preferably a capillary having a cross section perpendicular to the direction of movement of the heat source or a side of 1 μm to 1 mm in diameter. By using a capillary within this diameter range, it is easy to achieve a desired temperature distribution with excellent thermal conductivity, and it is possible to provide a device that is easy to manufacture.
[0058]
【Example】
Hereinafter, examples of the present invention will be described, but the scope of the present invention is not limited to these examples. In the following examples, “parts” and “%” represent “% by mass” and “parts by mass”, respectively, unless otherwise specified.
[0059]
[Composition (X1)]
The method for preparing the ultraviolet-curable composition (X1) used in the production example of the container D1 will be described.
As a polymerizable compound, 60 parts of a trifunctional urethane acrylate oligomer “Unidick V-4263” manufactured by Dainippon Ink and Chemicals, Ltd. having an average molecular weight of about 2000, and “hexane diol diacrylate” “New Frontier HDDA” 30 manufactured by Daiichi Kogyo Seiyaku Co., Ltd. Parts, 10 parts of glycidyl methacrylate manufactured by Wako Pure Chemical Industries, Ltd., 5 parts of 1-hydroxycyclohexyl phenyl ketone “Irgacure 184” manufactured by Ciba Specialty Chemicals as a photopolymerization initiator, and manufactured by Kanto Chemical Co., Ltd. as a polymerization retarder A composition (X1) is prepared by mixing 0.5 part of 2,4-diphenyl-4-methyl-1-pentene.
[0060]
[Composition (X2)]
A method for preparing the ultraviolet-curable composition (X2) used in the production example of the container D2 will be described.
60 parts of the above-mentioned “Unidick V-4263”, 20 parts of the above “New Frontier HDDA”, 20 parts of the above-mentioned glycidyl methacrylate, 5 parts of the above-mentioned “Irgacure 184” as a photopolymerization initiator, and polymerization delay The composition (X2) is prepared by mixing 0.5 parts of the above 2,4-diphenyl-4-methyl-1-pentene as an agent.
[0061]
[Film forming solution (Y)]
A method for preparing the ultraviolet-curable composition (Y) used in the production example of the container D1 will be described.
As a polymerizable compound, 72 parts of the above-mentioned "Unidick V-4263", 18 parts of dicyclopentanyl diacrylate "R-684" manufactured by Nippon Kayaku Co., Ltd., 10 parts of the above-mentioned glycidyl methacrylate, as a poor solvent (R) 120 parts of methyl decanoate manufactured by Wako Pure Chemical Industries, Ltd., 10 parts of acetone as a good volatile solvent, and 13 parts of the above-mentioned "Irgacure 184" as an ultraviolet polymerization initiator were uniformly mixed to prepare a film forming solution (Y). Prepare.
[0062]
[UV irradiation]
The ultraviolet irradiation in the production example can be carried out, for example, by the following apparatus and method.
A UV light intensity at 365 nm of 100 mW / cm was used using a light source unit for a multi-light 200 type exposure apparatus manufactured by Ushio Inc. using a 200 W metal halide lamp as a light source.²Is irradiated at room temperature in a nitrogen atmosphere unless otherwise specified.
[0063]
[Container D1]
3A and 3B show a container having cells in one embodiment of a separation apparatus suitably used in the separation method of the present invention. FIG. 3A is a schematic plan view, and FIG. It is a schematic diagram.
As shown in FIG. 3, the container 100 of this embodiment has a porous layer 2, a first resin layer 3, and a second resin layer 4 sequentially laminated on a flat support 1, and these are integrated. The overall outer shape is a plate having a length of 75 mm, a width of 50 mm, and a thickness of about 1.18 mm. The external shape may be determined arbitrarily according to the amount of the polynucleotide mixed solution to be separated or the like, and is arbitrary.
[0064]
As the support 1, an acrylic plate, a polystyrene plate, a polycarbonate plate, a glass plate, or the like can be used. In the container D1, an acrylic plate is used.
The porous layer 2 is formed by being fixed on the support 1, and is obtained by curing the coating film of the above-mentioned film-forming solution (Y) and removing the poor solvent. The thickness is arbitrary, but is about 5 μm in D1.
The first resin layer 3 is a layer obtained by curing the composition (X1) described above. In the first resin layer 3, a U-shaped thin line-shaped defective portion 5 penetrating from the front surface to the rear surface of the layer is formed, and this is the cell 5 in the container D1. This cell 5 has a linear defect portion 5d having a width of 500 μm, a height of 85 μm, a length of 30 mm, and a length of 30 mm each extending from both ends thereof at an angle of 45 degrees. It is made up of linear cutouts 5c and 5e.
A probe which is a single-stranded DNA having a length of 20 bases is bound to each surface of the inner wall of the cell 5.
[0065]
The second resin layer 4 is obtained by curing the composition (X1), and has a thickness of about 90 μm.
The second resin layer 4 has an inlet 6 and an outlet 7 which are holes penetrating from the front surface to the back surface of the layer. The inflow port 6 communicates with the upstream end 5 a of the cell 5 of the first resin layer 3. The outlet 7 communicates with the downstream end 5 b of the cell 5 of the first resin layer 3.
A thermocouple 8 for measuring the temperature of the cell 5 is sealed near the cell 5d in the center of the container D1, and the lead wire 9 is drawn out of the container.
[0066]
Hereinafter, a method of manufacturing the container D1 will be described.
[Step 1: formation of porous layer]
Using a spin coater, a film forming solution (Y) is coated on a 1 mm-thick acrylic plate support 1, irradiated with ultraviolet rays for 60 seconds, cured, and the phase-separated poor solvent (R) is removed. The porous layer 2 is formed by washing and removing with n-hexane.
[0067]
[Step 2: formation of defective portion (cell)]
The composition (X1) is coated on the porous layer 2 using a bar coater to form an uncured coating film, a portion of the uncured coating film to be formed into the cell 5, a thermocouple 8 mounting portion Ultraviolet rays were irradiated for 10 seconds through a photomask to portions other than the lead wire 9 lead-out portion, and the uncured coating film was semi-cured to form the first resin layer 3. Next, the uncured composition (X1) in the non-irradiated portion is removed with ethanol, and as a defective portion of the first resin layer 3, the cell 5 having the porous layer 2 formed on the bottom surface and the thermocouple mounting portion 8a and A defective portion to be the lead wire lead-out portion 9a is formed.
[0068]
[Step 3: Fixing lid]
An alumel wire and a tochromel wire each having a thickness of 85 μm are prepared by electric welding, and the thermocouple 8 and the lead wire 9 are mounted on the thermocouple mounting portion 8a and the lead wire lead-out portion 9a prepared above.
The composition (X1) is applied using a spin coater on a biaxially oriented polypropylene film “OPP film” manufactured by Nimura Chemical Co., Ltd. After bonding to the first resin layer 2 of the member prepared in the above step 2 and irradiating with ultraviolet rays for 40 seconds to completely cure both the first resin layer 2 and the second resin layer 4, The OPP film was peeled off to produce a container D1 precursor.
[0069]
(Formation of inlet and outlet)
At the positions of both ends 5a and 5b of the cell 5 of the container D1 precursor, holes 6 and 7 having a diameter of 1 mm are drilled in the second resin layer 4 using a drill, and an inlet 6 and an outlet connected to the cell 5 are formed. By forming the container No. 7, the container D1 was manufactured.
[0070]
[DNA immobilization]
A 5% aqueous solution of polyallylamine (Nittobo Co., Ltd.) was injected into the cell from the inflow section 6 and allowed to stand at 50 ° C. for 2 hours. Then, running water was passed through the cell for 15 minutes to wash and remove excess polyallylamine. Then, an amino group is introduced into a solid phase (porous layer and other cell inner walls) inside the flow channel.
Next, a 5% aqueous solution of glutaraldehyde is poured into the cell 5 and allowed to stand at 50 ° C. for 2 hours, and then washed with running water for 10 minutes to remove excess glutaraldehyde, and the porous layer and the inner wall of the cell are dried by passing dry nitrogen. Thus, a formyl group-introduced porous membrane is obtained.
Next, a buffer solution (DNA concentration: 50 μM) of a DNA having a length of 20 bases and having a modified amino group at the 5 ′ end manufactured by ESPEC Oligo Service Co., Ltd. After washing through a buffer, DNA serving as a probe is immobilized on the solid phase in the cell 5 by covalent bonding.
[0071]
[Production of connection part F]
FIG. 4 shows a perspective view of a connecting part F suitably used for connecting the container D1 and the like according to the present invention. As shown in FIG. 4, the connection part F has a through hole 12 having a diameter of 1.6 mm in the center of a plane of a polystyrene plate 11 having a size of 5 mm × 5 mm × thickness 2 mm. 13 are bonded to form a connection member (F). The connecting member (F) is brought into close contact with the inflow port 6 and the outflow port 7 of the container D1 with a clamp (not shown) across the O-ring 14, thereby forming a micro syringe (not shown) or a pump (not shown). ) Etc. can be connected.
[0072]
[Production Example of Container D2]
Next, a tube-shaped container D2 is shown as another example of the container suitably used in the separation device of the present invention.
A hard vinyl chloride pipe having an outer diameter of 6 mm and an inner diameter of 4 mm is drawn by heating to produce a hard vinyl chloride tube (not shown) having an outer diameter of 1.0 mm and an inner diameter of 0.5 mm.
An ultraviolet-curable composition (X2) is injected into the tube having a length of 10 cm, extruded with pressurized nitrogen, applied to the inner surface of the tube, and irradiated with ultraviolet rays for 60 seconds to introduce an epoxy group into the inner surface of the tube. Coat with a cured coating.
Next, a buffer solution (DNA concentration: 50 μM) of a DNA having a length of 20 bases and having a modified amino group at the 5 ′ end manufactured by Espec Oligo Service Co., Ltd. was injected into the tube, and both ends of the tube were heated to obtain polystyrene. After softening and sealing with a clamp and leaving at 60 ° C. for 15 hours, a buffer solution is poured into the tube to remove excess DNA, and DNA serving as a probe is fixed to the inner surface of the tube by covalent bonding. .
As described above, a container D2 in which a probe, which is a single-stranded DNA of 20 bases, is bonded to the inner surface of a tube made of hard vinyl chloride having a length of 10 cm, an outer diameter of 1.0 mm, and an inner diameter of 0.5 mm.
[0073]
[Temperature control block (H)]
FIG. 5 is a block perspective view of a temperature control block H suitably used in the separation apparatus of the present invention.
As shown in FIG. 5, this temperature control block H has eight fins 21b of 10 mm × 20 mm × 1.5 mm in thickness on an aluminum plate portion 21a of 22.5 mm × 20 mm × thickness 3 mm. A plate-like electric heater 22 and a sheath-type thermocouple 23 are mounted on an aluminum member 21 formed at equal intervals of 0.5 mm.
[0074]
[Separation of polynucleotide]
For example, using a microsyringe (not shown), the container D1 or D2 in which the mixed DNA solution has been injected into the cell from the inlet 7 is placed in the air at room temperature, and the two temperature control blocks H adjusted to, for example, 85 ° C. When the fins 21b and the fins 21b 'of the temperature control block H' (hereinafter, the name of each part of the second temperature control block H 'are affixed) are moved between the butt 21b and the fin 21b', the container D1 or The area sandwiched by the fins 21b and 21b 'from both sides of D2 is the first area, and the area sandwiched by the gap 24 between the fins 21b and the gap 24' between the fins 21b 'is the second area.
If necessary, a pump is connected to the inlet 7 or the outlet 8 to move the DNA mixed solution.
As a result, the DNA having the target base sequence is hybridized with the probe and separated, and a DNA solution having an increased composition ratio of the DNA having no target base sequence can be obtained from the outlet 8.
[0075]
【The invention's effect】
As described above, according to the separation method of the present invention, a polynucleotide having a target base sequence can be easily separated from a mixed solution of polynucleotides, and particularly, a solution of a polynucleotide having a low composition ratio to the total polynucleotides The medium composition ratio can be increased. Further, according to the present invention, it is possible to provide a separation device which can be suitably used in the above-mentioned separation method, and which can be easily incorporated into a part of μ-TAS (micro total analytical system).
[Brief description of the drawings]
FIG. 1 is a schematic diagram showing a dissociation curve of a polynucleotide (A) and a polynucleotide (B) in a non-overlapping case.
FIG. 2 is a schematic diagram showing a dissociation curve of a polynucleotide (A) and a polynucleotide (B) in a superimposed case.
FIG. 3 is a schematic plan view and a schematic cross-sectional view of a container D1 suitably used in the manufacturing apparatus of the present invention.
FIG. 4 is a perspective view of a connecting part F suitably used in the manufacturing apparatus of the present invention.
FIG. 5 is a perspective view of a temperature control block H suitably used in the manufacturing apparatus of the present invention.
[Explanation of symbols]
D1 ... container, F ... connecting parts, H ... temperature control block
1. Support, 2. Porous layer, 3. First resin layer, 4. Second resin layer, 5. Cell (defective part), 6. Inflow port, 7. Outflow port, 8. Thermocouple, 9 Lead wire, 11 Polystyrene plate, 12 Through hole, 13 Tube, 14 O-ring, 21 Aluminum member, 21a Plate-shaped part, 21b ..Fins, 22..electric heaters, 23..thermocouples, 24..voids

Claims (16)

A polynucleotide separation method for separating a polynucleotide (A) from a mixed solution containing a polynucleotide (A) having a target base sequence and a polynucleotide (B) not having the target base sequence, A probe having a base sequence complementary to the target base sequence, the cell is fixed to the inner surface of the cell or at least one of the solid phase in the cell, the mixed solution is arranged,
In the mixed solution disposed in the cell, a first region adjusted to a first temperature higher than a dissociation temperature of a hybrid chain formed by a polynucleotide (B) and a probe, and lower than the first temperature, In addition, the second regions adjusted to a second temperature that is lower than the temperature at which the total amount of the hybrid strand formed by the polynucleotide (A) and the probe dissociates have a temperature distribution in contact with each other, and the temperature distribution is continuous in one direction. The polynucleotide (A) is hybridized with the probe and separated from the mixed solution by moving the polynucleotide (A). The polynucleotide separation method according to claim 1, wherein the movement of the temperature distribution is performed by moving the first region first. The polynucleotide according to claim 1 or 2, wherein the first temperature is equal to or higher than a temperature at which the entire amount of the polynucleotide (B) dissociates, and the second temperature is equal to or lower than the dissociation temperature of the polynucleotide (B). Separation method. 2. The polynucleotide according to claim 1, wherein the second temperature is lower than a temperature at which the entire amount of the polynucleotide (B) dissociates, and the movement of the temperature distribution moves the second region first. 3. Separation method. The polynucleotide separation method according to any one of claims 1 to 4, wherein the temperature distribution is moved while flowing the mixed solution in one direction in the cell. The method for separating polynucleotides according to claim 5, wherein the flowing direction of the solution is the same as the moving direction of the temperature distribution. The method for separating polynucleotides according to any one of claims 1 to 6, wherein the cell is a capillary, and the movement of the temperature distribution is a movement in a length direction of the capillary. The polynucleotide separation method according to any one of claims 1 to 7, wherein the movement of the temperature distribution is repeated twice or more. The polynucleotide according to any one of claims 1 to 8, wherein the temperature distribution is provided by alternately repeating the first region and the second region two or more times in a moving direction of the temperature distribution. Method. The polynucleotide separation method according to any one of claims 1 to 9, wherein the content of the polynucleotide (B) is 5% by mass or less based on all the polynucleotides in the mixed solution. The method for separating a polynucleotide according to any one of claims 1 to 10, wherein the polynucleotide (B) has a base sequence that differs from the target base sequence of the polynucleotide (A) by only one base. The polynucleotide separation method according to any one of claims 1 to 11, wherein the concentrated polynucleotide (B) is collected from the solution at the moving end of the temperature distribution. The method for separating a polynucleotide according to any one of claims 1 to 12, wherein the number of bases in the target base sequence is 15 to 30. The method for separating a polynucleotide according to any one of claims 1 to 13, wherein the probe is an oligonucleotide having 15 to 30 bases. A polynucleotide separation device for separating a polynucleotide (A) from a mixed solution containing a polynucleotide (A) having a target base sequence and a polynucleotide (B) having no target base sequence,
A container in which a probe having a base sequence complementary to the target base sequence is fixed on an inner surface of a cell in the container and / or a solid phase in the cell,
A first region adjusted to a first temperature higher than a dissociation temperature of a hybrid chain formed by a polynucleotide (B) and a probe, and the first solution in the mixed solution accommodated in the cell in the container; A heat source for providing a temperature distribution in which the second regions adjusted to a second temperature lower than the temperature and lower than the temperature at which the total amount of the hybrid strand formed by the polynucleotide (A) and the probe dissociates are in contact with each other;
And a moving unit for moving the heat source. The apparatus for separating polynucleotides according to claim 15, wherein the cell is a capillary having a cross section perpendicular to the moving direction of the heat source and having a diameter of 1 µm to 1 mm.

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