JP2004093297A - Gene-diagnostic system and gene diagnostic method - Google Patents

Gene-diagnostic system and gene diagnostic method Download PDF

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JP2004093297A
JP2004093297A JP2002253948A JP2002253948A JP2004093297A JP 2004093297 A JP2004093297 A JP 2004093297A JP 2002253948 A JP2002253948 A JP 2002253948A JP 2002253948 A JP2002253948 A JP 2002253948A JP 2004093297 A JP2004093297 A JP 2004093297A
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dna
separation
electrode
sample
binding substance
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JP4090821B2 (en
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Hideaki Hashimoto
橋本　英明
Takeshi Nishida
西田　毅
Kazuyoshi Mori
森　一芳
Mizuo Maeda
前田　瑞夫
Yoshiki Katayama
片山　佳樹
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Panasonic Holdings Corp
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Matsushita Electric Industrial Co Ltd
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Abstract

<P>PROBLEM TO BE SOLVED: To provide a compact and light-weight gene-diagnostic system and a gene-diagnostic method at a low cost, which can detect gene abnormality of one or more bases rapidly, easily and accurately, and can automate the diagnosis at a very low running cost. <P>SOLUTION: The gene diagnostic system includes a sealed channel, wherein buffer solution contains DNA samples and a DNA bonding substance for separation that is made up of a polymer compound in bonding with a base sequence being hydrogen-bondable to the DNA samples and separates the DNA samples into normal DNAs and abnormal DNAs, based on the difference of their bonding forces. The gene-diagnostic system also includes a selective separating filter, disposed on the side of at least in the electromigration direction in the sealed channel and allowing the DNA samples to pass through it and preventing the DNA bonded substance for separation from passing it. The DNA samples in the sealed channel is subjected to electrophoresis by applying a voltage thereon, and a detecting section measures the amounts of the normal DNAs and/or the abnormal DNAs passing through it. <P>COPYRIGHT: (C)2004,JPO

Description

【０００１】
【発明の属する技術分野】
本発明は、遺伝子異常の有無を簡単、正確に検出できる遺伝子診断装置、及び遺伝子診断方法にあって、特に判定のために必要な分離用ＤＮＡ結合物質を使い捨てすることなく再利用することを可能にする遺伝子診断装置及び遺伝子診断方法に関する。
【０００２】
【従来の技術】
全ての疾患には、遺伝性要因と環境要因が種々の確率で関与しているが、先天性代謝異常症・癌・糖尿病・高血圧・アルツハイマー・自己免疫疾患・アトピー・肥満・アルコール依存などの疾患は、遺伝性要因が非常に大きな割合を占めている。一方、環境要素の寄与が大きい疾患は感染症や外傷の後遺症から誘因される疾患等である。
【０００３】
ところで、近年分子生物学の急速な進展によって、様々な疾患において遺伝的要素、すなわち遺伝子の関与がかなり正確に解明されるようになり、遺伝子をターゲットにした医療に注目が集まるようになってきている。現在、最も注目されているのはＳＮＰｓ（スニップス）と呼ばれるものである。これは、ｓｉｎｇｌｅ　ｎｕｃｌｅｏｔｉｄｅ　ｐｏｌｙｍｏｒｐｈｉｓｍの略で「１塩基多型」と一般に訳されており、個人間の遺伝子における１暗号（１塩基）の違いの総称である。
【０００４】
人を含め地球上の全ての生命体遺伝子（または遺伝子の全集合体を意味するゲノム）は、共通の４つの塩基から成り立っており、この塩基の配列によって様々なタンパク質が作られ、各生物特有の生命活動が行われている。全ての生物に共通する４つの塩基とは、アデニン（Ａと表記される）、グアニン（Ｇと表記される）、チミン（Ｔと表記される）、シトシン（Ｃと表記される）である。人の遺伝子は、約３０〜３２億塩基配列で構成されているといわれているが、各個人で数百から１０００塩基に１ヶ所程度の割合で他の人と１つの塩基が異なっている場所が存在する。通常、この１塩基の変化が、あるヒト集団の全人口中１％以上の頻度で存在しているものを、ＳＮＰｓと呼んでいる。
【０００５】
従って、全遺伝子（ゲノム）中には、３００万〜１０００万のＳＮＰｓが存在しているといわれ、現在世界中でＳＮＰｓの探索が続けられている。ＳＮＰｓが注目されている理由は、ＳＮＰｓの分類により、統計的に各個人の遺伝子が関与しているといわれている多くの疾患に対する罹患率が推測できると考えられているからである。例えば、乳がんを例にとると、乳がんにかかった患者群と正常な群とのＳＮＰｓの比較により、乳がんにかかりやすい人に共通のＳＮＰｓを特定することができる。そして、健康診断時に、遺伝子を調査しそのＳＮＰｓを持った人、すなわち現在は正常でも将来乳がんにかかりやすい体質の人を見つけることが可能になる。
【０００６】
この診断によって、乳がんにかかりやすい体質の人は、頻繁に検査をすることで万一癌に罹患しても、超早期に治療が行え生存の可能性が向上する。それと同様のことが、糖尿病や高血圧などの生活習慣病についても言え、世界中で多くの人が苦しんでいる病気に対して発病の前から食事や生活指導を正確にすることが可能になる。
【０００７】
また、病気の治療に用いている薬剤に関してもＳＮＰｓは重要な役割を期待されている。治療の際に用いられる薬剤は全ての人に均等に効果を示すものではない。一般に薬剤は、ある割合の人には効果があっても他の人には全く効果が無く、かえって副作用等で逆の結果を招くことがあることも広く知られている。因みに、アメリカにおける死亡原因の中で薬剤による副作用が上位に位置しているのは周知のことである。薬剤の効果はその人がもつ体質に深く関与しており、その体質もＳＮＰｓの分類によって区別可能であると言われている。すなわち、ＳＮＰｓの解析による分類で、ある薬剤に対してあらかじめ効果や感受性が予測でき適正な処方をすることが可能になり、患者個々人の体質に合わせた最適な薬剤の投与や副作用の危険性の回避が期待されている。このような医療のことをテーラーメイド医療またはオーダーメイド医療と呼び、将来の実用化が確実視されている。
【０００８】
また癌は、正常な細胞においては重要な役割をする遺伝子上の特定の部位に例えば紫外線や変異原性物質の作用によって突然変異が生じることによって引き起こされることがわかっている。ある特定の遺伝子上の変異を読み取ることで細胞が癌化しているか否かを早い段階から診断できるようになる。そして、犯罪捜査における犯人の特定や曖昧な親子関係の確定さらには本人であるか否かの識別にもＳＮＰｓは、威力を発揮する。前述したように、各個人には３００万〜１０００万のＳＮＰｓが存在しており、両親からそれぞれ別々のＳＮＰｓを引き継ぐため、地球上に親子兄弟といえども全く同じＳＮＰｓをもつ人間は絶対に存在しないと言われている。これが個人の完全な特定を可能にする理由である。
【０００９】
このように、特定の遺伝子中の１塩基に起きた変異を観察することで医療をはじめ様々の事柄に多大な貢献をもたらす可能性がある。しかしながら現在、特定の遺伝子の変異を観察する方法は以下に述べるような非常に複雑な操作あるいは高価な装置を必要とし、ランニングコストも非常に嵩むため、広く利用されるまでには至っていない状況にある。
【００１０】
現在最も一般的に用いられているＳＮＰｓを調べる方法は、ＤＮＡの塩基配列を端から直接読んでいくシーケンシング（塩基配列の決定）と呼ばれている方法である。遺伝子は１種類のタンパク質を形成するための塩基配列情報をもったＤＮＡの単位であるから、塩基配列を端から読んでいけばＳＮＰｓが解明することができる。シーケンシングを行う方法としては、いくつかの報告があるが、最も一般的に行われているのは以下に述べるジデオキシシーケンシング（Ｓａｎｇｅｒ法）である。この方法を含めいずれの方法も、分離能の高い変性ポリアクリルアミドゲル電気泳動かキャピラリー電気泳動によって１塩基長の長さの違いを分離・識別できる技術が基になって成り立っている。
【００１１】
ジデオキシシーケンシング（Ｓａｎｇｅｒ法）は、酵素的シーケンシングとも呼ばれ、１本鎖鋳型ＤＮＡの相補的鎖を合成するためにＤＮＡポリメラーゼを用い、さらに人工的につくった特殊な４種類のジデオキシヌクレオチドを利用するのが特徴である。シーケンシング操作としては、塩基配列を行いたい１本鎖ＤＮＡの３’末端を相補する合成塩基配列をプライマーとして用い、そのプライマーからＤＮＡポリメラーゼと均等に加えられたデオキシヌクレオチドを酵素反応によって伸長させる操作を行うが、この時同時に４つの反応容器を準備しておき、それぞれにＡＴＧＣ４つの塩基の３’末端に水酸基を持たない、従ってこれ以上ＤＮＡ伸長反応を続けることができない塩基アナログであるジデオキシヌクレオチドを別々に少量混入させておく。これにより、伸長中のＤＮＡの末端にジデオキシヌクレオチドが付加された時点でＤＮＡ合成がストップし、それぞれの反応容器中に様々な長さを持った、しかし端は必ず加えた塩基アナログである２本鎖ＤＮＡが形成される。この反応容器にＳ１エンドヌクレアーゼを反応させ、１本鎖ＤＮＡを全て消化し２本鎖ＤＮＡのみとする。こうして得られた４つの反応容器のＤＮＡ鎖をゲル電気泳動またはキャピラリー電気泳動し、分離されたＤＮＡを短い方（速く移動したもの）から順に読めば、鋳型鎖と相補的なＤＮＡの塩基配列がわかる。この際、泳動結果の識別は、例えば加えるジデオキシヌクレオチドのリンまたはイオウを放射性標識したり蛍光を発する化学物質を結合させたりして行う。放射性標識の場合はフィルムへの露光での検出、蛍光化学物質の場合はレーザービームを照射し蛍光を検出する。最近では、Ａ，Ｔ，Ｇ，Ｃの４種類の塩基アナログを、それぞれ４種類の蛍光波長の異なる試薬により標識し、その４色の蛍光を同時に検出する方法も開発されている。
【００１２】
このように、従来は被験者から分離・精製した遺伝子の正確な塩基配列をこのジデオキシシーケンシング（Ｓａｎｇｅｒ法）あるいはその他の塩基配列決定法により決定し、正常あるいは標準的な塩基配列と比較することによってＳＮＰｓの有無や突然変異の有無の確認、個人の識別等を行っている。
【００１３】
【発明が解決しようとする課題】
以上説明したように、従来からの遺伝子配列決定法を利用した、遺伝子診断や遺伝子による個人の識別は、ターゲットとする遺伝子を単離したのち、増幅・精製し、遺伝子の塩基配列決定用装置を用いて、目的遺伝子の塩基配列を読むことによって行っていたため、実験に膨大な作業量と非常に長い時間、さらには多大のランニングコストを要していた。また塩基配列決定のための自動化した装置は、非常に高価で、大きなスペースを占有し、しかも高価な試薬を大量に必要とするものであった。
【００１４】
そこで、従来のこのような問題を解決するため本発明は、一塩基以上の遺伝子異常を短時間、且つ簡単、正確に検出することができ、小型、軽量、安価に、しかも非常に少ないランニングコストで、診断を自動化することができる遺伝子診断装置及び遺伝子診断方法を提供することを目的とする。
【００１５】
加えて本発明は、判定しようとする遺伝子の正常型と異常型を分離・計測するために用いている、分離用ＤＮＡ結合物質の繰返し利用を可能にし、さらに染色体ＤＮＡや血液由来の分子量の大きなノイズ物質を除去することができ、診断装置の簡素化と診断にかかるコストの削減、そして高精度な判定を実現する遺伝子診断装置及び遺伝子診断方法を提供する。
【００１６】
【課題を解決するための手段】
上記課題を解決するために本発明の遺伝子診断装置は、緩衝液を収容し第１電極が浸漬された第１容器と、緩衝液を収容し第２電極が浸漬された第２容器と、第１容器と第２容器間をリニアポリマーとＤＮＡ結合制御剤を含む緩衝液を充たして連絡した密閉流路と、第２電極に正電位または負電位を印加するとともに第１電極に負電位または正電位を印加する電源部と、電源部を制御して第２電極と第１電極間に電圧を印加する制御部と、密閉流路に設けられ、内部を通過するＤＮＡの通過量を検出する検出部を備え、密閉流路には、緩衝液の中に、試料ＤＮＡに水素結合可能な塩基配列と高分子化合物とが結合し、結合力の差から試料ＤＮＡを正常ＤＮＡと異常ＤＮＡに分離する分離用ＤＮＡ結合物質と、試料ＤＮＡとを備え、密閉流路の少なくとも電気泳動方向側に、試料ＤＮＡを通過可能とし、分離用ＤＮＡ結合物質を通過させない選択性分離フィルターを備え、電圧を印加することにより密閉流路内の試料ＤＮＡが電気泳動され、検出部が正常ＤＮＡ及び／または異常ＤＮＡの通過量を測定する構成とした。
【００１７】
これにより、分離用ＤＮＡ結合物質の繰返し利用が可能になったり、染色体等由来のノイズＤＮＡにより測定結果のばらつきを防止することができる。その結果、一塩基以上の遺伝子異常を短時間、且つ簡単、正確に検出することができ、小型、軽量、安価に、しかも非常に少ないランニングコストで、診断を自動化することができる。
【００１８】
【発明の実施の形態】
請求項１に記載の発明は、緩衝液を収容し第１電極が浸漬された第１容器と、緩衝液を収容し第２電極が浸漬された第２容器と、第１容器と第２容器間をリニアポリマーとＤＮＡ結合制御剤を含む緩衝液を充たして連絡した密閉流路と、第２電極に正電位または負電位を印加するとともに第１電極に負電位または正電位を印加する電源部と、電源部を制御して第２電極と第１電極間に電圧を印加する制御部と、密閉流路に設けられ、内部を通過するＤＮＡの通過量を検出する検出部を備え、密閉流路には、緩衝液の中に、試料ＤＮＡに水素結合可能な塩基配列と高分子化合物とが結合し、結合力の差から試料ＤＮＡを正常ＤＮＡと異常ＤＮＡに分離する分離用ＤＮＡ結合物質と、試料ＤＮＡとを備え、密閉流路の少なくとも電気泳動方向側に、試料ＤＮＡを通過可能とし、分離用ＤＮＡ結合物質を通過させない選択性分離フィルターを備え、電圧を印加することにより密閉流路内の試料ＤＮＡが電気泳動され、検出部が正常ＤＮＡ及び／または異常ＤＮＡの通過量を測定することを特徴とする。
【００１９】
このように電気泳動とＤＮＡの水素結合を利用し、試料ＤＮＡと正常ＤＮＡとの分離または非分離を観察することによって試料ＤＮＡの異常の有無を判定でき、さらにフィルターの利用により、分離用ＤＮＡ結合物質の再利用や染色体由来のＤＮＡから発生するノイズ除去が実現できることで、繰返し測定による低ランニングコスト化と、より正確な判定を可能にする装置が提供できる。
【００２０】
そして、電気泳動が終了し結果を判定した後も長時間通電することで、試料ＤＮＡと正常ＤＮＡが全て流れ去り、結果的に電気泳同方向の密閉流路終点近傍に分離用ＤＮＡ結合物質が集中する。この密閉流路に対して測定とは逆の電位を通電することで、分離用ＤＮＡ結合物質を測定前の状態に戻すことができ、分離用ＤＮＡ結合物質を新たに補充することなく繰返し測定を行なうこともできる。
【００２１】
請求項２に記載の発明は、請求項１において、密閉流路に、試料ＤＮＡおよび対照として用いる正常ＤＮＡを通過可能とし、より高分子の物質を通過させず、選択性分離フィルターよりも大量の高分子を捕捉することができる選択性分離プレフィルターを備え、選択性分離フィルターと、選択性分離プレフィルターとで、分離用ＤＮＡ結合物質を挟むことを特徴とする。
【００２２】
よって、測定しようとする試料に、大きな分子量を持つＤＮＡやたんぱく質等の不純物が混入していても、ノイズとして検出されることなく正確な測定を行なうことが可能になる。
【００２３】
請求項３に記載の発明は、緩衝液を収容し第１電極が浸漬された第１容器と、緩衝液を収容し第２電極が浸漬された第２容器と、第１容器と第２容器間をリニアポリマーとＤＮＡ結合制御剤を含む緩衝液を充たして連絡した密閉流路と、第２電極に正電位または負電位を印加するとともに第１電極に負電位または正電位を印加する電源部と、電源部を制御して第２電極と第１電極間に電圧を印加する制御部と、密閉流路に設けられ、内部を通過するＤＮＡの通過量を検出する検出部を備え、密閉流路には、緩衝液の中に、試料ＤＮＡに水素結合可能な塩基配列と高分子化合物とが結合し、結合力の差から試料ＤＮＡを正常ＤＮＡと異常ＤＮＡに分離する分離用ＤＮＡ結合物質と、試料ＤＮＡとを備え、密閉流路の分離用ＤＮＡ結合物質を挟むように、試料ＤＮＡを通過可能とし、分離用ＤＮＡ結合物質を通過させない選択性分離フィルターを備え、電圧を印加することにより密閉流路内の試料ＤＮＡが電気泳動され、検出部が正常ＤＮＡ及び／または異常ＤＮＡの通過量を測定することを特徴とする。
【００２４】
よって、電気泳動が終了し結果を判定した後も長時間通電することで、試料ＤＮＡと正常ＤＮＡが全て流れ去り、結果的に密閉流路終点近傍に分離用ＤＮＡ結合物質が集中する。測定終了後この密閉流路に対して測定とは逆の電位を通電することで、分離用ＤＮＡ結合物質を測定前の状態に戻すことができ、分離用ＤＮＡ結合物質を新たに補充することなく繰返し測定に供することが可能になる。両端にフィルターを装着したことで、分離用ＤＮＡ結合物質の損失を最小限に抑えることが可能になる。なお、逆の電位を通電する操作ではなく、測定終了後、密閉流路のみを取り出し前後を交換することでも、同じ効果を得ることができる。
【００２５】
請求項４に記載の発明は、請求項３において、少なくとも密閉流路の電気泳動方向の逆側に位置する選択性分離フィルターよりも上流側に、試料ＤＮＡおよび対照として用いる正常ＤＮＡを通過可能とし、より高分子の物質を通過させず、選択性分離フィルターよりも大量の高分子を捕捉することができる選択性分離プレフィルターを備えたことを特徴とする。
【００２６】
よって、測定しようとする試料に、大きな分子量を持つＤＮＡやたんぱく質等の不純物が混入していても、ノイズとして検出されることなく正確な測定を行なうことが可能になる。
【００２７】
請求項５に記載の発明は、請求項３において、密閉流路の前記選択性分離フィルターを挟むように、試料ＤＮＡおよび対照として用いる正常ＤＮＡを通過可能とし、より高分子の物質を通過させず、選択性分離フィルターよりも大量の高分子を捕捉することができる選択性分離プレフィルターを備えたことを特徴とする。
【００２８】
よって、測定終了後この密閉流路に対して測定とは逆の電位を通電することで、分離用ＤＮＡ結合物質を測定前の状態に戻すことができ、さらにプレフィルターで捕捉したノイズとなりうる高分子物質を除去することが可能になる。これにより、分離用ＤＮＡ結合物質を新たに補充することなく繰返し測定に供することが可能になる。なお、逆の電位を通電する操作ではなく、測定終了後、密閉流路のみを取り出し前後を交換することでも、同じ効果を得ることができる。
【００２９】
請求項６に記載の発明は、請求項２〜５において、密閉流路の内部を通過するＤＮＡの通過量を検出するための検出窓を少なくとも分離用ＤＮＡ結合物質を挟むように、それぞれ密閉流路の両側に備えたことを特徴とする。
【００３０】
よって、試料ＤＮＡが正常であるか異常であるかの判定を、密閉流路を取り外したり付け替えたりすることなく行なうことができる。
【００３１】
請求項７に記載の発明は、請求項２において、選択性分離フィルターと選択性分離プレフィルターとで挟まれた領域を含む密閉流路部分を着脱自在としたことを特徴とする。
【００３２】
よって、流路の一部をカートリッジ化、チップ化することが可能であり、測定による分離用ＤＮＡ結合物質の流出がないので、複数回の測定も可能となる。
【００３３】
また、測定が終了した後に密閉流路部分をひっくり返して再び判定を行なっても、さらに何度も繰返し測定を行なったとしても、一ヶ所の検出窓のみで通過するＤＮＡ量を測定することもできる。この場合、検出窓は選択性分離フィルターおよび密閉流路を一つのユニットとして、そのユニットの外側に位置し、ユニットを自在に取り替えても測定には何ら影響を受けない。
【００３４】
請求項８に記載の発明は、請求項３〜５において、少なくとも選択性分離フィルターで挟まれた領域を含む密閉流路部分を着脱自在としたことを特徴とする。
【００３５】
よって、流路の一部をカートリッジ化、チップ化することが可能であり、測定による分離用ＤＮＡ結合物質の流出がないので、複数回の測定も可能となる。また、測定が終了した後に密閉流路部分をひっくり返して再び判定を行なっても、さらに何度も繰返し測定を行なったとしても、一ヶ所の検出窓のみで通過するＤＮＡ量を測定することもできる。
【００３６】
請求項９に記載の発明は、請求項８において、選択性分離プレフィルターを含む密閉流路部分を着脱自在としたことを特徴とする。
【００３７】
よって、測定しようとする試料に、大きな分子量を持つＤＮＡやたんぱく質等の不純物が混入していても、ノイズとして検出されることなく正確な測定を行なうことが可能になる。
【００３８】
請求項１０に記載の発明は、緩衝液を収容し第１電極が浸漬される第１容器と、緩衝液を収容し第２電極が浸漬される第２容器と、第１容器と第２容器間をリニアポリマーとＤＮＡ結合制御剤を含む緩衝液を充たして連絡する密閉流路と、第２電極に正電位または負電位を印加するとともに第１電極に負電位または正電位を印加する電源部と、電源部を制御して第２電極と第１電極間に電圧を印加する制御部と、密閉流路に設けられ、内部を通過するＤＮＡの通過量を検出する検出部を備え、密閉流路に着脱自在な部材であって、密閉流路に装着した際に密閉流路の一部を構成する部材内に、試料ＤＮＡに水素結合可能な塩基配列と高分子化合物とが結合し、結合力の差から試料ＤＮＡを正常ＤＮＡと異常ＤＮＡに分離する分離用ＤＮＡ結合物質と、分離用ＤＮＡ結合物質を挟むように、試料ＤＮＡを通過可能とし、分離用ＤＮＡ結合物質を通過させない選択性分離フィルターを備え、密閉流路に試料ＤＮＡを導入し、電圧を印加することにより密閉流路内の試料ＤＮＡが電気泳動され、検出部が正常ＤＮＡ及び／または異常ＤＮＡの通過量を測定することを特徴とする。
【００３９】
よって、流路の一部をカートリッジ化、チップ化することが可能であり、測定による分離用ＤＮＡ結合物質の流出がないので、複数回の測定も可能となる。
【００４０】
請求項１１に記載の発明は、請求項１０において、選択性分離フィルターの一方を試料ＤＮＡおよび対照として用いる正常ＤＮＡを通過可能とし、より高分子の物質を通過させず、選択性分離フィルターよりも大量の高分子を捕捉することができる選択性分離プレフィルターとしたことを特徴とする。
【００４１】
よって、測定しようとする試料に、大きな分子量を持つＤＮＡやたんぱく質等の不純物が混入していても、ノイズとして検出されることなく正確な測定を行なうことが可能になる。
【００４２】
請求項１２に記載の発明は、請求項１０において、選択性分離フィルターを挟むように、試料ＤＮＡおよび対照として用いる正常ＤＮＡを通過可能とし、より高分子の物質を通過させず、選択性分離フィルターよりも大量の高分子を捕捉することができる選択性分離プレフィルターを備えたことを特徴とする。
【００４３】
よって、測定しようとする試料に、大きな分子量を持つＤＮＡやたんぱく質等の不純物が混入していても、ノイズとして検出されることなく正確な測定を行なうことが可能になる。
【００４４】
請求項１３に記載の発明は、密閉流路をキャピラリー管としたことを特徴とし、効率の良い電気泳動が可能となる。
【００４５】
請求項１４に記載の発明は、請求項１３において、キャピラリー管内壁をアクリルアミドでコーティングしたことを特徴とし、電気浸透流を防止できる。
【００４６】
請求項１５に記載の発明は、第１容器に緩衝液を収容して第１電極を浸漬し、第２容器に緩衝液を収容して第２電極を浸漬し、第１容器と第２容器間を連絡する密閉流路であって、密閉流路の少なくとも電気泳動方向側に、試料ＤＮＡを通過可能とし、分離用ＤＮＡ結合物質を通過させない選択性分離フィルターと、試料ＤＮＡに水素結合可能な塩基配列と高分子化合物とが結合し、結合力の差から試料ＤＮＡを正常ＤＮＡと異常ＤＮＡに分離する分離用ＤＮＡ結合物質とを備えた密閉流路に、リニアポリマーとＤＮＡ結合制御剤を含む緩衝液を充たし、密閉流路に試料ＤＮＡを導入し、第１電極と第２電極間に電圧を印加し、密閉流路内の試料ＤＮＡを電気泳動させて、正常ＤＮＡ及び／または異常ＤＮＡを検出することを特徴とする遺伝子診断方法であり、判定の再現性向上およびコストの削減をはかることができる。
【００４７】
請求項１６に記載の発明は、請求項１５において、密閉流路は、試料ＤＮＡおよび対照として用いる正常ＤＮＡを通過可能とし、より高分子の物質を通過させず、選択性分離フィルターよりも大量の高分子を捕捉することができる選択性分離プレフィルターを備え、選択性分離フィルターと、選択性分離プレフィルターとで、分離用ＤＮＡ結合物質が挟まれていることを特徴とする遺伝子診断方法であり、判定の再現性向上およびコストの削減に加え、新たに測定ノイズの低減および分離用ＤＮＡ結合物質を捕捉することを目的としている第一の選択性分離フィルターの寿命を延ばすことができる。
【００４８】
請求項１７に記載の発明は、第１容器に緩衝液を収容して第１電極を浸漬し、第２容器に緩衝液を収容して第２電極を浸漬し、第１容器と第２容器間を連絡する密閉流路であって、試料ＤＮＡに水素結合可能な塩基配列と高分子化合物とが結合し、結合力の差から試料ＤＮＡを正常ＤＮＡと異常ＤＮＡに分離する分離用ＤＮＡ結合物質と、分離用ＤＮＡ結合物質を挟むように、試料ＤＮＡを通過可能とし、分離用ＤＮＡ結合物質を通過させない選択性分離フィルターとを備えた密閉流路に、リニアポリマーとＤＮＡ結合制御剤を含む緩衝液を充たし、密閉流路に試料ＤＮＡを導入し、第１電極と第２電極間に電圧を印加し、密閉流路内の試料ＤＮＡを電気泳動させて、正常ＤＮＡ及び／または異常ＤＮＡを検出することを特徴とする遺伝子診断方法であり、判定の再現性向上およびコストの削減をはかることができる。
【００４９】
請求項１８に記載の発明は、第１容器に緩衝液を収容して第１電極を浸漬し、第２容器に緩衝液を収容して第２電極を浸漬し、第１容器と第２容器間を連絡した密閉流路であり、密閉流路の一部を構成する着脱自在な部材が装着された密閉流路であって、試料ＤＮＡに水素結合可能な塩基配列と高分子化合物とが結合し、結合力の差から試料ＤＮＡを正常ＤＮＡと異常ＤＮＡに分離する分離用ＤＮＡ結合物質と、分離用ＤＮＡ結合物質を挟むように、試料ＤＮＡを通過可能とし、分離用ＤＮＡ結合物質を通過させない選択性分離フィルターとを備えた密閉流路に、リニアポリマーとＤＮＡ結合制御剤を含む緩衝液を充たし、密閉流路に試料ＤＮＡを導入し、第１電極と第２電極間に電圧を印加し、密閉流路内の試料ＤＮＡを電気泳動させて、正常ＤＮＡ及び／または異常ＤＮＡを検出することを特徴とする遺伝子診断方法であり、判定の再現性向上およびコストの削減をはかることができ、カートリッジ化、チップ化された部材を用いることができ、自由度が向上する。
【００５０】
以下、本発明の実施の形態における遺伝子診断装置と遺伝子診断方法について、図面を参照しながら説明する。
【００５１】
（実施の形態１）
図１は本発明の実施の形態１における遺伝子診断装置の外観図、図２は本発明の実施の形態１における遺伝子診断装置の上蓋開放外観図、図３は本発明の実施の形態１における遺伝子診断装置の装置構成図、図４は本発明の実施の形態１における遺伝子診断装置の制御回路要部図、図５は本発明の実施の形態１における遺伝子診断装置の分離用ＤＮＡ結合物質を用い試料ＤＮＡおよび正常ＤＮＡを電気泳動させる本発明における選択性分離フィルターを終点近傍にのみ付属した密閉流路の構造図である。
【００５２】
図１において、１は遺伝子診断装置の電源スイッチ、２は装置の種々の操作を行うための操作ボタン、３は表示パネル、４は上蓋、５は装置の筐体である。図２、図３、図４において、６は正常ＤＮＡと異常ＤＮＡとを分離するための所定温度に調整するとともに、この部分でＤＮＡを正常ＤＮＡと異常ＤＮＡとその他のノイズＤＮＡの３つに分離するために電気泳動を行わせるための密閉流路を有する電気泳動部、７は電気泳動部６を支える支持台、８は電気泳動を行うための制御や後述する吸引ポンプ（１６）、検出部（１０）の制御を行い、検出したデータを演算する基板からなる制御演算部である。９は電源ボックス、９ａは電源ボックス９内に設けられた電源部である。本遺伝子診断装置ではＤＮＡの種類や濃度、処理条件ごとに異なった最適印加電圧値が存在するため、後述の試料ＤＮＡ２１から異常ＤＮＡと正常ＤＮＡを分離するのに最も適した泳動が行えるように、制御演算部８が電源部９ａを制御して最適電圧を印加する。１１は電気泳動部６の温度を調整する温調器である。この電気泳動部６内の構成の詳細については後述するが、測定開始時には、図５に示すように分離用ＤＮＡ結合物質２０が電気泳動部６の密閉流路内にすでにあるように配置する。そして、このような配置をとるととともに電圧制御、濃度調整、温度制御等を行うことにより、電気泳動させながら本遺伝子診断装置は試料ＤＮＡ２１を正常ＤＮＡと異常ＤＮＡとノイズＤＮＡに数〜十数分程度で分離するものである。電気泳動部６では分離用ＤＮＡ結合物質２０が泳動作用を受けながら水素結合の結合力の差によって試料ＤＮＡ２１を正常ＤＮＡと異常ＤＮＡとノイズＤＮＡを分離できるように、１５℃〜８０℃、望ましくは２０℃〜６０℃の温度範囲の所定温度に保つように制御される。正常ＤＮＡと異常ＤＮＡとノイズＤＮＡとを分離するためにはＤＮＡの分離に適したこの所定の温度から±１℃の範囲で制御するのがよい。
【００５３】
１０は電気泳動する試料ＤＮＡの通過量を測定する検出部である。図４に基づいて検出部１０について説明すると、１０ａは紫外線を照射するＤ２ランプ、１０ｂはＤ２ランプ１０ａから出た紫外線を目的の部分に集中させるためのスリット、１０ｃは紫外線を受光するフォトダイオード、１０ｄはフォトダイオード１０ｃが検出した微弱電流を増幅するプリアンプ、１０ｅはデジタル量に変換するＡ／Ｄコンバータである。これらの詳細は後述する。１２は電気泳動のときに正電位を印加する電極、１３は電気泳動のときに負電位を印加する電極である。
【００５４】
図３，図４，図５において、１４は電気泳動させるとき電荷の運搬と試料ＤＮＡのｐＨを安定させるための緩衝液、１４ａは緩衝液１４を収容する陽極側の容器、１４ｂは緩衝液１４を収容する陰極側の容器、１４ｃはＤＮＡ結合制御剤と下記のリニアポリマー１４ｄを緩衝液１４に添加した緩衝液、１４ｄは電気泳動を可能にし、正常ＤＮＡと異常ＤＮＡとノイズＤＮＡとノイズＤＮＡを分離する媒体であるポリアクリルアミドのようなリニアポリマーである。１５は電気泳動のときに試料ＤＮＡ２１を泳動するためのキャピラリー管（密閉流路）、１７は試料ＤＮＡは通過可能であるが試料ＤＮＡより分子量の大きな分離用ＤＮＡ結合物質は通過することができない選択性分離フィルター。１６はキャピラリー管１５の中に試料ＤＮＡ２１やリニアポリマー１４ｄとＤＮＡ結合制御剤などの試薬を注入するための吸引ポンプである。容器１４ａの緩衝液１４の中には電極１２が浸漬され、容器１４ｂの緩衝液１４の中に、電極１３が浸漬される。容器１４ａと容器１４ｂ内の緩衝液１４は、キャピラリー管１５内の緩衝液１４ｃによって連通される。電極１２と電極１３間に電圧を印加すると、キャピラリー管１５内に電気泳動が誘発され、分離用ＤＮＡ結合物質２０と試料ＤＮＡ２１は負に帯電しているため、容器１４ａから容器１４ｂ側へ分離用ＤＮＡ結合物質２０と試料ＤＮＡ２１が移動する。
【００５５】
次に、本発明の遺伝子診断装置で測定を行うために必要な詳細について説明する。本遺伝子診断装置で測定する試料ＤＮＡ２１は、人の細胞や血液等から入手したＤＮＡである。ヒト・ゲノムＤＮＡからＰＣＲ（ポリメラーゼ連鎖反応）などの方法を利用して目的の部分を目的の長さに切り出して測定用に数を増加させる作業が必要である。ただ、このＰＣＲに関しては本遺伝子診断装置の説明に必要がないため具体的な説明を省略する。
【００５６】
ＰＣＲなどで取り出した目的のＤＮＡは塩基数でいうと２０個程度〜数１０００個程度であるが、約３０億塩基対あるといわれるヒト・ゲノムＤＮＡの中に、同じＤＮＡ配列が存在しないと確率的にいえる個数としては、４０個程度以上である。正常ＤＮＡと異常ＤＮＡの分離は、試料ＤＮＡの濃度（μＭ、但し、Ｍ＝モル／リットル）、分離用ＤＮＡ結合物質濃度（モル％）、測定温度、その他と密接な関係があるため、適正な条件にしないと分離が起こらないから、このような条件を設定することができるか否かが重要である。
【００５７】
次に、図３，図４，図５のキャピラリー管１５について説明する。キャピラリー管１５にゲルを入れて電気泳動を行うと、電気浸透流と呼ばれる液の流れが発生してしまう。従ってこの現象が起きないようにするため、キャピラリー管１５の内壁をアクリルアミドなどでコーティングするのがよい。電気浸透流の発生が阻止できるならコーティング方法は他の方法でもよい。例えば、一般に販売されているコーティングキャピラリー管を使用するのでもよい。なお、キャピラリー管１５としては内径５０〜１００μｍのフューズドシリカ製キャピラリー管が、紫外線を９０％以上透過でき、且つ後述するように紫外線を利用してＤＮＡを検出するとき、紫外線の通過量を容易に検出できるから最も適当である。そして、溝のある板と、紫外線が９０％以上透過する板との組み合わせでキャピラリー管１５を構成するのでも、紫外線を９０％以上透過でき、紫外線の通過量を検出することで異常ＤＮＡの検出を容易に行うことができる。なお、溝のある板を紫外線が透過可能な板にした場合は透過光を検出し、溝のある板を紫外線が不透過の板にした場合は、異常ＤＮＡを反射光で検出する。反射光を検出する場合には溝形状を均一な反射面とするため矩形断面にする必要がある。
【００５８】
容器１４ａ，１４ｂの中やキャピラリー管１５に収容する緩衝液１４，１４ｃは、Ｔｒｉｓ系（ｐＨ７．２〜ｐＨ８程度）の緩衝液等を利用するのが適当である。このうちキャピラリー管１５に収容する緩衝液１４ｃに混入するＤＮＡ結合制御剤には、分離用ＤＮＡ結合物質に対するＤＮＡの結合を促進する塩化マグネシウム等の結合促進剤と、離脱を促す尿素等の離脱剤の２種類が存在する。この２種類のＤＮＡ結合制御剤は、２種類の混合割合や、材料物質（例えば、結合促進剤として他の電解質）を選ぶことで、ＤＮＡに対する多様な泳動速度の制御が可能になるものである。分離用ＤＮＡ結合物質２０や試料ＤＮＡ２１等を電気泳動するためのリニアポリマー１４ｄとしては、ポリアクリルアミドを用いることができ、これは分離用ＤＮＡ結合物質２０にも利用されるため、相性がよく適当である。
【００５９】
次に、キャピラリー管１５への充填順序であるが、図５に示すように先ずキャピラリー管１５内にリニアポリマー１４ｄとＤＮＡ結合制御剤を含んだ緩衝液１４ｃを導入し、続いて以下詳述する分離用ＤＮＡ結合物質２０を加える。このときリニアポリマー１４ｄと混合して導入してもよいし、分離用ＤＮＡ結合物質２０導入後にリニアポリマー１４ｄを導入してもよい。続いて、分離用ＤＮＡ結合物質２０と分離状態で緩衝液１４ｃで希釈した試料ＤＮＡ２１を導入して電気泳動する。
【００６０】
本実施の形態１では、分離用ＤＮＡ結合物質２０を作成するのにアクリルアミドとＤＮＡを重合させているため、ＤＮＡとの重量差、構造差は大きく、分離用ＤＮＡ結合物質２０の泳動速度（０．６ｃｍ／分〜０．７ｃｍ／分）は、慣性のためＤＮＡの最適の泳動速度（１３ｃｍ／分〜２０ｃｍ／分）の１／２０〜１／３０程度であって、非常に動きが鈍く相対的に擬似的に固定されているといってもよいような状態が実現される。なお、遺伝子診断装置のキャピラリー管１５に試料ＤＮＡや分離用ＤＮＡ結合物質、リニアポリマーの各溶液を交換的に導入する機構、例えば図３に示した吸引ポンプ１６のほかに、各試料ＤＮＡや分離用ＤＮＡ結合物質、リニアポリマーの各溶液をそれぞれ保管するカラムと、そのカラムを自動的に交換し、それをキャピラリ−管１５に接続する機構を装備するのが望ましい。
【００６１】
さらに、キャピラリー管１５を１本または複数本単位でユニット化し、これを交換用の分離用密閉流路カートリッジとして、電気泳動部６に装着し、温調器１１で温度制御するようにするのも適当である。キャピラリ−管１５内に、予めリニアポリマー１４ｄとＤＮＡ結合制御剤を含む緩衝液１４ｃを充填し、分離用ＤＮＡ結合物質２０と試料ＤＮＡ２１を分離状態で充填して分離用密閉流路カートリッジとして用意しておくことが可能になる。このように構成することで、測定を行うたびに分離用密閉流路カートリッジごと交換するため、分離用密閉流路カートリッジごとに分離用ＤＮＡ結合物質２０、試料ＤＮＡ２１の配置を予め設定できるから、測定が簡単に行え、且つ正確な測定が行える。なお、この分離用密閉流路カートリッジでは、分離用ＤＮＡ結合物質２０と試料ＤＮＡ２１を分離状態にするため、その間にリニアポリマー１４ｄを挟んで充填している。それぞれの中にリニアポリマー１４ｄを混合させて充填するのでもよい。
【００６２】
続いて、分離用ＤＮＡ結合物質２０について説明する。
【００６３】
分離用ＤＮＡ結合物質２０は、判定しようとする試料ＤＮＡ（正常なＤＮＡと比較して１塩基以上の配列の違いがあるか、または正常なＤＮＡと同じ配列のＤＮＡ）および判定しようとするＤＮＡに対応し正常であることが分かっており、試料と同数の塩基を有する正常ＤＮＡと、正常ＤＮＡまたは判定しようとするＤＮＡに相同な塩基配列を有するＤＮＡおよび試料ＤＮＡより分子量の大きな物質とが結合したものである。なお、分離用ＤＮＡ結合物質とは、正常ＤＮＡとそれと同じ塩基数であるが、１塩基以上の配列の違いを有する異常ＤＮＡとを電気泳動法により密閉流路内を同時に移動させたときに、分離用ＤＮＡ結合物質内に存在している、正常ＤＮＡまたは異常ＤＮＡどちらか一方に対してだけ完全に相同な塩基配列との水素結合力の違いを利用して正常または異常ＤＮＡとを分離することのできる物質のことを指す。本物質は、判定しようとするＤＮＡよりも常に大きな分子量を持つことを特徴としている。
【００６４】
ここで、図１１は本発明の実施の形態１における遺伝子診断装置の密閉流路内の分離用ＤＮＡ結合物質と試料ＤＮＡとの関係概念図である。図１１において、２０は分離用ＤＮＡ結合物質である。
【００６５】
ＤＮＡは二本鎖を形成するものと一本鎖のものと存在するが、ＤＮＡのもつＡ，Ｔ，Ｃ，Ｇ４つの塩基は互いにＡとＴ、ＧとＣがそれぞれ水素結合し易い性質をもち、ＤＮＡの二本鎖においてもＡＴ，ＧＣで対をなしている。従って、一方の鎖のＤＮＡがＡＴＣＧＣＧＴＣＴＡＧＣ（配列番号１に記載）と配列されている場合、もう一方の鎖のＤＮＡは、ＴＡＧＣＧＣＡＧＡＴＣＧ（配列番号２に記載）という塩基配列をもっている。この関係は相補的関係と呼ばれるもので、この相補関係を充たす限り、ＡとＴ、ＧとＣがそれぞれ水素結合により結合し、二本鎖を形成する。本発明ではこの関係を利用するために、図１１に示すように分離用ＤＮＡ結合物質２０のＤＮＡ部分には試料ＤＮＡの正常ＤＮＡに相補的な塩基配列を持たせている。従って、試料ＤＮＡの正常ＤＮＡの塩基配列がＡＴＣＧＣＧＴＣＴＡＧＣ（配列番号１に記載）を含み、異常ＤＮＡがＡＴＣＡ＊ＣＧＴＣＴＡＧＣ（配列番号３に記載）で、＊で示した部分で正常ＤＮＡと異常ＤＮＡとノイズＤＮＡの塩基が異なっている場合、分離用ＤＮＡ結合物質２０のＤＮＡ部分の配列をＴＡＧＣＧＣＡＧＡＴＣＧ（配列番号２に記載）とすると、異常ＤＮＡはＡ＊において分離用ＤＮＡ結合物質２０と相補的ではなくなるため水素結合せず、水素結合全体の結合力は試料ＤＮＡの正常ＤＮＡの方が異常ＤＮＡより１塩基分の結合力分だけ大きくなる。その結果、後で説明する電気泳動時に正常ＤＮＡの方が異常ＤＮＡより強い結合力で長い時間分離用ＤＮＡ結合物質２０と結合するため、正常ＤＮＡは異常ＤＮＡより相対的に遅延するようになる。というのは、電気泳動時結合力だけでなく電気泳動による引離力も作用するから、多数のＤＮＡが断続的に結合と離脱を繰り返すような結合状態となるからである。そして、この泳動速度を平均的にみると、長い時間結合する正常ＤＮＡの方が異常ＤＮＡより泳動速度が低下することになる。試料ＤＮＡの中には、２本鎖のＤＮＡのうち測定対象ではない分離した残りの１本鎖ＤＮＡのように正常ＤＮＡと異常ＤＮＡとノイズＤＮＡ以外のＤＮＡも含まれており、このＤＮＡが後で説明する電気泳動時にノイズとして作用するが、このノイズＤＮＡは分離用ＤＮＡ結合物質２０のＤＮＡとはほとんど無反応で結合しないから、最も速い泳動速度をもち分離されることになる。なお、分離用ＤＮＡ結合物質２０の濃度は、試料ＤＮＡの濃度の２０〜６００倍が適当である。
【００６６】
次に、このような分離用ＤＮＡ結合物質２０の作成・合成方法について説明する。ビニル化ＤＮＡを合成するために、ＰＣＲによって、分離用ＤＮＡ結合物質２０用のＤＮＡを切り出す。上述の例では、ＴＡＧＣＧＣＡＧＡＴＣＧ（配列番号２に記載）が分離用ＤＮＡ結合物質２０のＤＮＡである。
【００６７】
続いて、目的の塩基配列を有するＤＮＡの５’末端をアミノ化（通常は、ヘキシル基を介してアミノ化）する。このようにして得られたアミノ化ＤＮＡを２．６ｍＭになるように滅菌した超純水を加えて希釈する。次いで、ＭＯＳＵ（メタクリロイドオキシスクシンイミド）を７１．３８８ｍＭになるようにＤＭＳＯ（ディメチルスルオキシド）で希釈する。そして、このようにして得たアミノ化ＤＮＡとＭＯＳＵを１：５０の比率になるように加えて調整する。さらに、この調整溶液に対して、ｐＨ調整用としてｐＨ９になるように炭酸水素ナトリウムと水酸化ナトリウムで調整した溶液を、アミノ化ＤＮＡの量と等量加える。
【００６８】
そして、得られた溶液を一晩振とうする。その後、ＨＰＬＣ（Ｈｉｇｈ　Ｐｅｒｆｏｒｍａｎｃｅ　Ｌｉｑｕｉｄ　Ｃｈｒｏｍａｔｏｇｒａｐｈｙ：高速液体クロマトグラフィー）を使用して、振とうした溶液中のビニル化ＤＮＡを、アミノ化ＤＮＡ，ＭＯＳＵ，その他と分離する。ビニル化ＤＮＡは溶離液（ＴＥＡＡ；トリエチルアミンー酢酸とアセトニトリルの混合溶液）を含んでいるため、さらに真空乾燥機能を持った遠心エバポレーターで減圧濃縮する。
【００６９】
次いで、重合溶液である１０％のＡＡＭ（アクリルアミド）を５３μｍｏｌ窒素置換する。さらに、重合開始剤である１．３４％のＴＥＭＤ（Ｎ，Ｎ，Ｎ’Ｎ’−テトラメチルエチレンジアミン）を超音波で脱気した滅菌超純水で希釈する。これを重合開始剤である２％のＡＰＳ（過硫酸アンモニウム）を同じく超音波で脱気した滅菌超純水で希釈する。さらに濃縮したビニル化ＤＮＡを滅菌した超純水で希釈する。そして、１００μｌの分離用ＤＮＡ結合物質２０を合成するために、上記のＡＡＭを３４μｌ，上記のＴＥＭＤとＡＰＳを各々５μｌ、ビニル化ＤＮＡがＡＡＭに対して０．０１％モル〜０．０５％モルになるように加え、１００μｌになるように滅菌超純水を追加して、６０分程度放置しておくとアクリルアミド化された分離用ＤＮＡ結合物質２０が得られる。
【００７０】
続いて、本実施の形態１の遺伝子診断装置の動作について説明する。先ず遺伝子診断装置内に、試料ＤＮＡ２１や各溶液を導入したキャピラリー管１５をセットし、図３，図４に示すように両端に容器１４ａ，１４ｂ内の緩衝液１４を浸す。電極１２と電極１３間に電源部９ａによって所定電圧を印加する。印加する電圧の大きさとして望ましくは１０〜２０キロボルトが好ましいが、電解質や電極の状態により１００ボルト〜３０キロボルトでもよい。例えば、低電解質濃度の場合や、電極面積が大きな場合には電圧を低電圧にする。そして、最初から所定電圧を印加するのでなく、定電圧の印加の前に準備用の３０キロボルト以上の高電圧を印加し、ノイズＤＮＡを分離してから所定電圧を印加することで、測定を迅速に行うことができる。
【００７１】
ここで、電極１２と電極１３間に所定の電圧を印加する理由を説明する。分離用ＤＮＡ結合物質２０に対する試料ＤＮＡ２１の結合力と、電気泳動力による引離力との差が、各ＤＮＡの泳動速度差の発現を支配するため、正常ＤＮＡと異常ＤＮＡとノイズＤＮＡの分離が最も効果的に行われる電圧を選んで印加する必要があるからである。すなわち、この電圧は最も泳動しにくい正常ＤＮＡを電気泳動することができるという条件と、高電圧にすると正常ＤＮＡと異常ＤＮＡの分解能が低下するので、分解能を上げるため、できるだけ低電圧でなければならないという条件の２つを充たす電圧である。従って、予め印加する電圧として最適な電圧をＤＮＡごと、条件ごとに調べておき、上述したように当初３０キロボルト程度以上の準備電圧を短時間印加した後、この電圧を印加するようにするのが最適である。なお、キャピラリー管１５内のＤＮＡは負に帯電しており陽極側に進むため、電源９ａは電極１２を正電位、電極１３を負電位になるように印加する必要がある。また、電気泳動に当たっては、泳動時間を長くしすぎると、キャピラリー管１５内で緩衝液１４ｃが乾燥するので、必要以上に長い時間泳動を行うのは避けなければならない。
【００７２】
なお、準備用の３０キロボルト以上の高電圧を印加し、ノイズＤＮＡをいち早く分離し、その後徐々に電圧の絶対値を増加させて、高電圧を印加してもよい。そして、徐々に電圧の絶対値を増加させる場合、規則的或いは不規則に増加させても良いが、特に好ましい方法は、電圧を掃引して印加することである。掃引とは、電圧を単位時間あたり一定値で変化させる事であり、例えば、０ボルトから１キロボルト／秒で掃引を開始すれば、１０秒後には１０キロボルト印加することになる。印加電圧を掃引する理由を説明すると、分離用ＤＮＡ結合物質２０に対するＤＮＡ試料２１の結合力と、電気泳動力による引離力との差が、ＤＮＡの泳動速度や移動差に大きな影響を与えるため、正常ＤＮＡと異常ＤＮＡとノイズＤＮＡの分離が最も効果的に行われる電圧を調整するため掃引するものである。掃引により最も泳動速度に差が生じる電圧が分かったら、この電圧を保てばよい。印加電圧を掃引することで、分解能の高い最適電圧にきわめて簡単に調整できると言う効果が得られる。というのは、ノイズＤＮＡは、掃引電圧が低い段階で一番先に泳動されて検出部で検出されるが、異常ＤＮＡは分離用ＤＮＡ結合物質２０に対して一塩基分弱い結合力を有するため少し高電圧にならないと、これを振り切って離脱できない。さらに、正常ＤＮＡはもっと電圧を高くしないと離脱できない。この付近の電圧を印加しておけば、異常ＤＮＡと正常ＤＮＡは明確に分離される。但し、これより掃印電圧を上げると、正常ＤＮＡと異常ＤＮＡの分解能が低下するため、正常ＤＮＡが泳動を開始する電圧付近に電圧を保つのが最適となる。そして、当初３０キロボルト程度以上の準備電圧を短時間印加した後、掃引開始電圧を簡単に通常０ボルトから開始するのでよいが、分離用ＤＮＡ結合物質２０と試料ＤＮＡ２１の結合条件によっては、３０キロボルト以上を印加した後、５キロボルト〜７キロボルトから掃引を開始してもよい。なお、キャピラリー管１９内のＤＮＡは陽極側に進むため、可変電源部９ａは電極１６を正電位、電極１７を負電位になるように印加する必要がある。電圧が掃引されるとＤＮＡ試料２３は泳動され、分離用ＤＮＡ結合物質２０は正常ＤＮＡと異常ＤＮＡとの結合力に一塩基分の差があり、泳動されていくとき両者間に移動速度差が生じ、ノイズＤＮＡとの間で大きな移動差を生じる。
【００７３】
次に、分離が行われる電気泳動部６の説明を行う。電気泳動部６では、試料ＤＮＡの状態によっても異なるが、分離用ＤＮＡ結合物質２０と試料ＤＮＡ２１の結合力の差に基づいて、正常ＤＮＡや異常ＤＮＡ、あるいはノイズＤＮＡを分離できるように、１５℃〜８０℃、望ましくは２０℃〜６０℃の温度範囲の所定温度に保たなければならない。実施の形態１の遺伝子診断装置では、キャピラリー管１５を覆っている電気泳動部６の下部に、シリコンラバーヒーターやニクロム線等と熱電対やサーマル等の温度センサーを配置し、温調器１１で所定の温度±１℃以下になるように管理する。ただ、環境温度によっては室温が目的の所定温度を超える場合もあり、シリコンラバーヒーターやニクロム線等に代えて、ペルチェ等の暖・冷可能な部品を使うのがよい。
【００７４】
続いて、分離用ＤＮＡ結合物質２０の作用で泳動速度に差を生じ、移動差が生じた正常ＤＮＡと異常ＤＮＡとノイズＤＮＡを、どのようにして検出するか説明する。図１２は、泳動開始からの経過時間を横軸にして吸光度を縦軸にしたときの経過時間と吸光度の関係図である。検出は紫外線の照射が正常ＤＮＡと異常ＤＮＡとノイズＤＮＡ、ノイズＤＮＡによって遮光されたときの吸光度を測定することで行う。実施の形態１においては、図３、図４に示すようにキャピラリー管１５の一部でガラス部を露出させ、Ｄ２ランプ１０ａから波長２６０ｎｍの紫外線を照射し、このとき得られる紫外線照射光をフォトダイオード１０ｃで検出し、吸光度を測定している。制御演算部８によって電源部９ａを制御してＤ２ランプ１０ａを発光させ、フォトダイオード１０ｃで検出した電流はプリアンプ１０ｄで増幅し、Ａ／Ｄコンバータ１０ｅでデジタル量として吸光度に制御演算部８で換算される。制御演算部８はタイマ（図示しない）を内蔵し、泳動開始時間からの経過時間を測定することができる。図７において、経過時間が小さい（時間的に早く通過する）方（Ｉ）が、ＤＮＡコンジュゲートと結合しないノイズＤＮＡ、経過時間が中位の（ＩＩ）が異常ＤＮＡ、経過時間が大きい（ＩＩＩ）が正常ＤＮＡである。
【００７５】
この関係図よりピークの高さと時間とピークの数を読み取れば、ピークの数から試料ＤＮＡに異常ＤＮＡが含まれていることが分かる。すなわち、ピークの数から異常ＤＮＡが存在するか否かが判定できる。なお、印加する電圧やＤＮＡ濃度、ＤＮＡの長さ（塩基の個数）によってはピークの数を異常ＤＮＡとノイズＤＮＡだけにすることができるから、このときは異常ＤＮＡの通過量だけを測定する。また、ピークの高さを比べるのは、同じ条件下で電気泳動されたＤＮＡ中の泳動速度差のある２つの集合の吸光度差を示すから、異常ＤＮＡと正常ＤＮＡの存在比率に相当し、存在比率の判定が可能になる。異常ＤＮＡの存在量の判定は、標準試料ＤＮＡの検出ピーク波形から得られた標準データを制御演算基部８に予め入力しておき、そのデータと測定したデータを比較して換算すればよい。
【００７６】
ここで図５に示すように、この選択性分離フィルター１７は、試料ＤＮＡ２１は通過可能であるが試料ＤＮＡより分子量の大きな分離用ＤＮＡ結合物質２０は通過することができないような選択透過性を持ったフィルターであり、材質として例えば限外ろ過膜やナノろ過膜等が考えられる。
【００７７】
また、選択性分離フィルター１７は、判定しようとしている試料ＤＮＡ２１（及びコントロールとして入れた正常なＤＮＡ）は通過可能であるが、試料ＤＮＡ２１または正常ＤＮＡより分子量の大きな分離用ＤＮＡ結合物質２０は通過することができない選択性を有している。ここで用いる選択性分離フィルター１７は、半透膜（透析膜）あるいは限外濾過膜などのように分子量の違いにより選択透過性を示すものであれば何でもよい。例えば、判定しようとしているＤＮＡおよびコントロールとしての正常ＤＮＡは、２０ｍｅｒから２００ｍｅｒ程度の１本鎖ＤＮＡを想定しており分子量としては約６０００から６万程度のものである。これに対して分離用ＤＮＡ結合物質２０は、常に判定しようとするＤＮＡや正常ＤＮＡよりも分子量が大きな物質であることが必要である。選択性分離フィルター１７は、分子量６０００から６万の分子量を有す試料ＤＮＡ２１および対照として用いる正常ＤＮＡは通過可能であり、より大きな分子量を持つ分離用ＤＮＡ結合物質２０や染色体由来の巨大ＤＮＡは通過できないことを条件として選別される。
【００７８】
図５において、密閉流路に電圧を印加した場合、試料ＤＮＡ２１は陽極に向かって泳動を開始し、分離用ＤＮＡ結合物質２０の中を通過する間に化学結合力の差により分離が行われ、密閉流路の外側にある検出装置により２つのピークとして検出される。試料ＤＮＡ２１を泳動させるために密閉容器の両端に電圧を印加した場合、試料ＤＮＡに比較するとわずかではあるが分子量の大きな分離用ＤＮＡ結合物質２０も陽極に向かって泳動がおきる。同じ密閉流路を用いて複数回泳動を行わせた場合、総泳動時間によっては、分離用ＤＮＡ結合物質２０も密閉流路の外に出てしまい、その後の試料ＤＮＡの分離ができなくなってしまうことが予想される。それを防ぐために選択性分離フィルター１７を設置した。本フィルターにより泳動を何度行っても、分離用ＤＮＡ結合物質２０を失うことはない。フィルターに補足された後は、陽極と陰極を切り替え逆に電流を流すことでまた元の位置に分離用ＤＮＡ結合物質２０を戻すことが可能であり、このことで繰り返し使用ができる。
【００７９】
以上説明したように、本実施の形態１の遺伝子診断装置と遺伝子診断方法によれば、細胞や血液等から取り出したＤＮＡの中で、特定のＤＮＡをＰＣＲなどで取り出した試料ＤＮＡ中に含まれる正常ＤＮＡと異常ＤＮＡとノイズＤＮＡの存在比率等が分かることにより、遺伝子診断、判定ができる。また、分離用ＤＮＡ結合物質が系外に漏出することを防止できるために、繰り返しの操作が可能になり、低コストでの診断・判定ができる。
【００８０】
（実施の形態２）
本発明の実施の形態２における遺伝子診断装置及び遺伝子診断方法について図面に基づいて説明する。なお、実施の形態１と重複する部分、図１〜図４までの説明については実施の形態１に譲り説明を省略する。
【００８１】
図６は本発明の実施の形態２における実施の形態１で説明した選択性分離フィルター１７を密閉流路の両端に装着した図である。この選択性分離フィルター１７は、試料ＤＮＡ２１は通過可能であるが試料ＤＮＡより分子量の大きな分離用ＤＮＡ結合物質２０は通過することができないような選択透過性を持ったフィルターであり、材質として例えば限外ろ過膜やナノろ過膜等が考えられる。
【００８２】
図６において、密閉流路に電圧を印加した場合、試料ＤＮＡ２１は陽極に向かって泳動を開始し、分離用ＤＮＡ結合物質２０の中を通過する間に化学結合力の差により分離が行われ、密閉流路の外側にある検出装置により２つのピークとして検出される。試料ＤＮＡ２１を泳動させるために密閉容器の両端に電圧を印加した場合、試料ＤＮＡ２１に比較するとわずかではあるが分子量の大きな分離用ＤＮＡ結合物質２０も陽極に向かって泳動がおきる。同じ密閉流路を用いて複数回泳動を行わせた場合、総泳動時間によっては、分離用ＤＮＡ結合物質２０も密閉流路の外に出てしまい、その後の試料ＤＮＡ２１の分離ができなくなってしまうことが予想される。それを防ぐために選択性分離フィルター１７を密閉流路両端に設置した。密閉流路終点近傍のフィルターにより泳動を何度行っても、分離用ＤＮＡ結合物質２０を失うことはない。フィルターに補足された後は、陽極と陰極を切り替え逆に電流を流すことでまた元の位置に分離用ＤＮＡ結合物質２０を戻すことが可能であり、また密閉流路始点近傍のフィルターによって逆電流を流しすぎた場合でも、分離用ＤＮＡ結合物質２０を失うことはない。フィルターを両端に装着したことによって、安心して繰り返し使用ができる。
【００８３】
さらに、図６で説明した密閉流路を取り外し可能にしておくことで、何度かの泳動の後、密閉流路ごとひっくり返せば、偏った分離用ＤＮＡ結合物質２０を逆電流によって戻す事なく繰り返し使用する事ができるようになる。
【００８４】
なお、図６においては、試料ＤＮＡ２１が導入された状態を示しているが、少なくとも選択性分離フィルター１７で挟まれた領域を含む密閉流路部分を着脱自在として、カートリッジ化、チップ化した場合、そのカートリッジ、チップ内には、選択性分離フィルター１７で挟まれた領域には試料ＤＮＡ２１は導入されておらず、分離用ＤＮＡ結合物質２０が充填されていることは言うまでもない。
【００８５】
以上説明したように、本実施の形態２の遺伝子診断装置と遺伝子診断方法によれば、細胞や血液等から取り出したＤＮＡの中で、特定のＤＮＡをＰＣＲなどで取り出した試料ＤＮＡ２１中に含まれる正常ＤＮＡと異常ＤＮＡとノイズＤＮＡの存在比率等が分かることにより、遺伝子診断、判定ができる。また、分離用ＤＮＡ結合物質２０が系外に漏出することを防止できるために、繰り返しの操作が可能になり、また、分離用ＤＮＡ結合物質２０が系外に漏出することを完全に防止できるため、長期間正確な測定が可能になる。さらに、選択性分離フィルター１７が両端に装着されているため、密閉流路ごとひっくり返すことで、連続しての泳動操作が可能になり、低コストで連続した診断・判定ができる。
【００８６】
（実施の形態３）
本発明の実施の形態３における遺伝子診断装置及び遺伝子診断方法について図面に基づいて説明する。なお、実施の形態１と重複する部分、図１〜図４までの説明については実施の形態１に譲り説明を省略する。
【００８７】
図７は本発明の実施の形態３における実施の形態１で説明した選択性分離フィルター１７を密閉流路の終点近傍に装着し、実施の形態１、２に記載の選択性分離フィルターよりも大量の高分子を捕捉することができる第２の選択性分離フィルター（プレフィルター）を最上流部装着した図である。ここで選択性分離フィルター１７は、試料ＤＮＡ２１は通過可能であるが試料ＤＮＡ２１より分子量の大きな分離用ＤＮＡ結合物質２０は通過することができないような選択透過性を持ったフィルターであり、第２の選択性分離フィルター（プレフィルター）１８は、試料ＤＮＡ２１および対照として用いる正常ＤＮＡは通過することができるが、より高分子の物質は通過することのできず、実施の形態１，２に記載の選択性分離フィルターよりも大量の高分子を捕捉することができる。両方のフィルターとも材質は同じでも違っていても良く、例えば精密ろ過膜、限外ろ過膜あるいはナノろ過膜等が考えられる。
【００８８】
図７において、密閉流路に電圧を印加した場合、試料ＤＮＡ２１は陽極に向かって泳動を開始し、分離用ＤＮＡ結合物質２０の中を通過する間に化学結合力の差により分離が行われ、密閉流路の外側にある検出装置により２つのピークとして検出されるが、試料ＤＮＡ２１を図７の位置に装着させる場合に、あらかじめ選択性分離プレフィルター１８を通過させることによって試料に混在していた高分子の不純物を除くことができ、正確な検出を行うことができる。さらに選択性分離フィルター１７を密閉流路終点近傍に装着することで、実施の形態１で説明したように、泳動を何度行っても分離用ＤＮＡ結合物質２０を失うことを防止することができる。以上説明したように、本実施の形態３の遺伝子診断装置と遺伝子診断方法によれば、細胞や血液等から取り出したＤＮＡの中で、特定のＤＮＡをＰＣＲなどで取り出した試料ＤＮＡ中に含まれる正常ＤＮＡと異常ＤＮＡとノイズＤＮＡの存在比率等が分かることにより、遺伝子診断、判定ができる。また、泳動開始点近傍のプレフィルターにより不純物をあらかじめ除くことができ、さらに終点近傍のフィルターにより分離用ＤＮＡ結合物質が系外に漏出することを防止できるために、繰り返し正確な検出が可能になり、低コストでの診断・判定ができる。
【００８９】
（実施の形態４）
本発明の実施の形態４における遺伝子診断装置及び遺伝子診断方法について図面に基づいて説明する。なお、実施の形態１と重複する部分、図１〜図４までの説明については実施の形態１に譲り説明を省略する。
【００９０】
図８は本発明の実施の形態４における実施の形態１で説明した選択性分離フィルター１７を密閉流路の両端に装着し、実施の形態３で説明したプレフィルター１８を密閉流路の始点近傍に装着した図である。
【００９１】
これら３つのフィルターを用いることで、どちらの方向に電気泳動を行っても分離用ＤＮＡ結合物質２０を失うことなく、さらに不純物を除くことで長期間正確な測定を行うことができる。
【００９２】
以上説明したように、本実施の形態４の遺伝子診断装置と遺伝子診断方法によれば、細胞や血液等から取り出したＤＮＡの中で、特定のＤＮＡをＰＣＲなどで取り出した試料ＤＮＡ中に含まれる正常ＤＮＡと異常ＤＮＡとノイズＤＮＡの存在比率等が分かることにより、遺伝子診断、判定ができる。また、分離用ＤＮＡ結合物質が系外に漏出することを完全に防止できるために、繰り返しの操作が可能になり、低コストでの診断・判定ができる。
【００９３】
以上説明したように、本実施の形態４の遺伝子診断装置と遺伝子診断方法によれば、細胞や血液等から取り出したＤＮＡの中で、特定のＤＮＡをＰＣＲなどで取り出した試料ＤＮＡ中に含まれる正常ＤＮＡと異常ＤＮＡとノイズＤＮＡの存在比率等が分かることにより、遺伝子診断、判定ができる。また、泳動開始点近傍のプレフィルターにより不純物をあらかじめ除くことができ、さらに両端のフィルターにより分離用ＤＮＡ結合物質が系外に漏出することを完全に防止できるために、繰り返し正確な検出が恒常的に可能になり、低コストでの診断・判定ができる。
【００９４】
（実施の形態５）
本発明の実施の形態５における遺伝子診断装置及び遺伝子診断方法について図面に基づいて説明する。なお、実施の形態１と重複する部分、図１〜図４までの説明については実施の形態１に譲り説明を省略する。
【００９５】
図９は本発明の実施の形態５における実施の形態１で説明した選択性分離フィルター１７を密閉流路の両端に装着し、実施の形態３で説明したプレフィルター１８を密閉流路の両端に装着した選択性分離フィルター１７よりもさらに開始点および終点に近いところにそれぞれ装着した図である。
【００９６】
これら４つのフィルターを用いることで、どちらの方向に電気泳動を行っても分離用ＤＮＡ結合物質２０を失うことなく、不純物も除くことができるため長期間正確な測定を行うことができる。さらに、図９で説明した密閉流路を取り外し可能にしておくことで、何度かの泳動の後、密閉流路ごとひっくり返せば偏った分離用ＤＮＡ結合物質を逆電流によって戻す事なく繰り返し使用する事ができるようになる。さらにプレフィルターにより、不純物が除かれるため長期の連続使用にも用いる事ができる。
【００９７】
以上説明したように、本実施の形態５の遺伝子診断装置と遺伝子診断方法によれば、細胞や血液等から取り出したＤＮＡの中で、特定のＤＮＡをＰＣＲなどで取り出した試料ＤＮＡ中に含まれる正常ＤＮＡと異常ＤＮＡとノイズＤＮＡの存在比率等が分かることにより、遺伝子診断、判定ができる。また、分離用ＤＮＡ結合物質が系外に漏出することを完全に防止でき、プレフィルターによって不純物を除くため、長期間正確な測定が可能になる。さらに、フィルターおよびプレフィルターが両側に装着されているため、密閉流路ごとひっくり返すことで、連続しての泳動操作が可能になり、低コストで連続した診断・判定ができる。
【００９８】
（実施の形態６）
本発明の実施の形態６における遺伝子診断装置及び遺伝子診断方法について図面に基づいて説明する。なお、実施の形態１と重複する部分、図１〜図４までの説明については実施の形態１に譲り説明を省略する。
【００９９】
図１０は本発明の実施の形態６における実施の形態１で説明した選択性分離フィルター１７を密閉流路の両端に装着し、密閉流路の両端に検出のための透明な検出窓１９を設けた図である。ここで、選択性分離フィルター１７と検出窓１９の位置はどちらが外側でもかまわず、両方とも密閉流路の端近傍にあれば良い。
【０１００】
２つの選択性分離フィルターを用いることで、どちらの方向に電気泳動を行っても分離用ＤＮＡ結合物質２０を失うことなく繰り返しの泳動が可能になり、さらに、両方の端に検出窓１９があることで、密閉流路部分を取り外すことなく、電極１２および電極１３の陰極・陽極を変換するだけで繰り返しの泳動・検出ができる。
【０１０１】
以上説明したように、本実施の形態５の遺伝子診断装置と遺伝子診断方法によれば、細胞や血液等から取り出したＤＮＡの中で、特定のＤＮＡをＰＣＲなどで取り出した試料ＤＮＡ中に含まれる正常ＤＮＡと異常ＤＮＡとノイズＤＮＡの存在比率等が分かることにより、遺伝子診断、判定ができる。また、フィルターおよびプレフィルターが両側に装着されているため分離用ＤＮＡ結合物質が系外に漏出することを防止でき、さらに、密閉流路の両端に検出窓１９があることで、密閉流路部分を取り外すことなく、電極の陰陽を変換するだけで繰り返しの泳動・検出ができるため、手間がかからず低コストで連続した診断・判定ができる。
【０１０２】
【発明の効果】
以上のように、本発明によれば、一塩基以上の遺伝子異常を短時間、且つ簡単、正確に検出することができ、小型、軽量、安価に、しかも非常に少ないランニングコストで、診断を自動化することができる遺伝子診断装置及び遺伝子診断方法を提供することできる。
【０１０３】
そして、判定しようとする遺伝子の正常型と異常型を分離・計測するために用いている、分離用ＤＮＡ結合物質の繰返し利用を可能にし、さらに染色体ＤＮＡや血液由来の分子量の大きなノイズ物質を除去することができ、診断装置の簡素化と診断にかかるコストの削減、そして高精度な判定を実現する遺伝子診断装置及び遺伝子診断方法を提供することができる。
【０１０４】
また、請求項１に記載された遺伝子診断装置によれば、本装置内に存在するＤＮＡを分離し試料のＤＮＡが正常型か異常型かを識別するための電気泳動を行うための密閉流路には、分離用ＤＮＡ結合物質を充填しておく必要がある。このとき用いる分離用ＤＮＡ結合物質が最もランニングコストを要する材料である。分離用ＤＮＡ結合物質を使い捨てにする必要が無くなれば、コストの削減に加えて、分離用ＤＮＡ結合物質の濃度や品質の変化に由来する様々な調整が無くなり、測定精度の向上や測定時間の短縮等のメリットも期待できる。このため低ランニングコストで安価かつ高精度な装置とすることができ、診断の自動化がきわめて容易である。
【０１０５】
そして、最もランニングコストを要する分離用ＤＮＡ結合物質が電気泳動および密閉流路物質の吸引あるいは押し出しにより密閉流路外へ流出することを防止できる。
【０１０６】
請求項２に記載された遺伝子診断装置は、請求項１の効果に加え、測定対象以外の不純物をあらかじめ取り除くことで、測定精度の向上と測定の長寿命化を可能にすることができる。
【０１０７】
請求項３に記載された遺伝子診断装置は、最もランニングコストを要する分離用ＤＮＡ結合物質をどちらの方向に移動させても密閉流路外へ流出することを防止できる。
【０１０８】
請求項４に記載された遺伝子診断装置は、請求項３の効果に加え、測定対象以外の不純物をあらかじめ取り除くことで、測定精度の向上と測定の長寿命化を可能にすることができる。
【０１０９】
請求項５に記載された遺伝子診断装置は、請求項３の効果に加え、測定対象以外の不純物をあらかじめ取り除くことで、測定精度の向上と測定の長寿命化を可能にすることができる。
【０１１０】
請求項６に記載された遺伝子診断装置は、請求項２〜５の効果に加え、どちらの方向に電気泳動を行わせても、密閉流路の取替えや移動なしに測定を継続することが可能である。
【０１１１】
請求項７に記載された遺伝子診断装置は、請求項２の効果に加え、密閉流路部を取り替えながら測定を行うことができるので、多検体の試料を連続的に測定することができる。また、一つの検出部で測定を行うことによって、装置を小型化することも可能である
請求項８に記載された遺伝子診断装置は、請求項３〜５の効果に加え、密閉流路部を取り替えながら測定を行うことができるので、多検体の試料を連続的に測定することができる。また、一つの検出部で測定を行うことによって、装置を小型化することも可能である
請求項９に記載された遺伝子診断装置は、請求項８の効果に加え、測定しようとする試料に、大きな分子量を持つＤＮＡやたんぱく質等の不純物が混入していても、ノイズとして検出されることなく正確な測定を行なうことが可能になる。
【０１１２】
請求項１０に記載された遺伝子診断装置は、流路の一部をカートリッジ化、チップ化することが可能であり、測定による分離用ＤＮＡ結合物質の流出がないので、複数回の測定も可能となる。
【０１１３】
請求項１１に記載された遺伝子診断装置は、請求項１０の効果に加え、測定しようとする試料に、大きな分子量を持つＤＮＡやたんぱく質等の不純物が混入していても、ノイズとして検出されることなく正確な測定を行なうことが可能になる。
【０１１４】
請求項１２に記載された遺伝子診断装置は、請求項１０の効果に加え、測定しようとする試料に、大きな分子量を持つＤＮＡやたんぱく質等の不純物が混入していても、ノイズとして検出されることなく正確な測定を行なうことが可能になる。
【０１１５】
請求項１３に記載に記載された遺伝子診断装置は、請求項１０〜１２の効果に加え、効率の良い電気泳動が可能となる。
【０１１６】
請求項１４に記載された遺伝子診断装置は、請求項１３の効果に加え、電気浸透流を防止できる。
【０１１７】
請求項１５に記載された遺伝子診断方法は、判定の再現性向上およびコストの削減をはかることができる。
【０１１８】
請求項１６に記載された遺伝子診断方法は、請求項１５の効果に加え、新たに測定ノイズの低減および分離用ＤＮＡ結合物質を捕捉することを目的としている選択性分離フィルターの寿命を延ばすことができる。
【０１１９】
請求項１７に記載された遺伝子診断方法は、判定の再現性向上およびコストの削減をはかることができる。
【０１２０】
請求項１８に記載された遺伝子診断方法は、判定の再現性向上およびコストの削減をはかることができ、カートリッジ化、チップ化された部材を用いることができ、自由度が向上する。
【０１２１】
【配列表】

Figure 2004093297
Figure 2004093297

【図面の簡単な説明】
【図１】本発明の実施の形態１における遺伝子診断装置の外観図
【図２】本発明の実施の形態１における遺伝子診断装置の上蓋開放外観図
【図３】本発明の実施の形態１における遺伝子診断装置の装置構成図
【図４】本発明の実施の形態１における遺伝子診断装置の制御回路要部図
【図５】本発明の実施の形態１における遺伝子診断装置の分離用ＤＮＡ結合物質を用い試料ＤＮＡおよび正常ＤＮＡを電気泳動させる本発明における選択性分離フィルターを終点近傍にのみ付属した密閉流路の構造図
【図６】本発明の実施の形態２における実施の形態１で説明した選択性分離フィルターを密閉流路の両端に装着した図
【図７】本発明の実施の形態３における実施の形態１で説明した選択性分離フィルターを密閉流路の終点近傍に装着し、実施の形態１、２に記載の選択性分離フィルターよりも大量の高分子を捕捉することができる第２の選択性分離フィルター（プレフィルター）を最上流部装着した図
【図８】本発明の実施の形態４における実施の形態１で説明した選択性分離フィルターを密閉流路の両端に装着し、実施の形態３で説明したプレフィルターを密閉流路の始点近傍に装着した図
【図９】本発明の実施の形態５における実施の形態１で説明した選択性分離フィルターを密閉流路の両端に装着し、実施の形態３で説明したプレフィルターを密閉流路の両端に装着した選択性分離フィルターよりもさらに開始点および終点に近いところにそれぞれ装着した図
【図１０】本発明の実施の形態６における実施の形態１で説明した選択性分離フィルターを密閉流路の両端に装着し、密閉流路の両端に検出のための透明な検出窓を設けた図
【図１１】本発明の実施の形態１における遺伝子診断装置の密閉流路内の分離用ＤＮＡ結合物質と試料ＤＮＡとの関係概念図
【図１２】経過時間と吸光度の関係図
【符号の説明】
１　電源スイッチ
２　操作ボタン
３　表示パネル
４　上蓋
５　筐体
６　電気泳動部
７　支持台
８　制御演算部
９　電源ボックス
９ａ　電源部
１０　検出部
１０ａ　Ｄ２ランプ
１０ｂ　スリット
１０ｃ　フォトダイオード
１５ｄ　プリアンプ
１０ｅ　Ａ／Ｄコンバータ
１１　温調器
１２，１３　電極
１４，１４ｃ　緩衝液
１４ａ，１４ｂ　容器
１４ｄ　リニアポリマー
１５　キャピラリー管
１６　吸引ポンプ
１７　選択性分離フィルター
１８　選択性分離プレフィルター
１９　検出窓
２０　分離用ＤＮＡ結合物質
２１　試料ＤＮＡ[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a gene diagnosis apparatus and a gene diagnosis method capable of simply and accurately detecting the presence or absence of a gene abnormality, and in particular, it is possible to reuse a DNA binding substance for separation required for determination without disposable. And a genetic diagnosis method.
[0002]
[Prior art]
In all diseases, genetic and environmental factors are involved with various probabilities, but diseases such as congenital dysbolism, cancer, diabetes, hypertension, Alzheimer's, autoimmune diseases, atopy, obesity, and alcohol dependence. Has a very large percentage of hereditary factors. On the other hand, diseases that greatly contribute to environmental factors include diseases caused by infectious diseases and sequelae of trauma.
[0003]
By the way, in recent years, with the rapid progress of molecular biology, the genetic element, that is, the involvement of genes in various diseases has been clarified fairly accurately, and attention has been drawn to medical treatment targeting genes. I have. At present, what is attracting the most attention are those called SNPs (snips). This is an abbreviation of single nucleotide polymorphism and is generally translated as "single nucleotide polymorphism", and is a general term for the difference of one code (one nucleotide) in a gene between individuals.
[0004]
All living organism genes on the earth, including humans (or the genome, which means the entire collection of genes), consist of four common bases, and the sequence of these bases produces various proteins, which are unique to each organism. Life activities are taking place. The four bases common to all organisms are adenine (denoted as A), guanine (denoted as G), thymine (denoted as T), and cytosine (denoted as C). It is said that human genes are composed of about 3 to 3.2 billion base sequences, but each individual has a base that differs from others by about one in hundreds to 1,000 bases. Exists. Usually, SNPs in which this single base change is present at a frequency of 1% or more in the total population of a certain human population are referred to as SNPs.
[0005]
Therefore, it is said that 3 to 10 million SNPs exist in all genes (genomes), and the search for SNPs is currently being continued worldwide. The reason why SNPs are attracting attention is that it is considered that the classification of SNPs can statistically estimate the prevalence of many diseases that are said to involve the genes of each individual. For example, taking breast cancer as an example, by comparing SNPs between a group of patients having breast cancer and a normal group, it is possible to identify SNPs common to those who are likely to have breast cancer. Then, at the time of a medical examination, it becomes possible to investigate a gene and find a person who has the SNPs, that is, a person who is normal but is likely to contract breast cancer in the future.
[0006]
With this diagnosis, those who are predisposed to breast cancer can be treated very early to improve the chances of survival if they become susceptible to cancer by frequent examinations. The same can be said for lifestyle-related diseases such as diabetes and high blood pressure, and it becomes possible to accurately adjust diet and living guidance before the onset of diseases that many people suffer from all over the world.
[0007]
SNPs are also expected to play an important role in drugs used for treating diseases. Drugs used in treatment are not equally effective for everyone. In general, it is widely known that a drug is effective for a certain percentage of people but has no effect for other people, and may rather cause adverse results due to side effects. By the way, it is well known that drug-related side effects rank high among the causes of death in the United States. It is said that the effect of the drug is deeply related to the constitution of the person, and that the constitution can be distinguished by the classification of SNPs. In other words, it is possible to predict the effects and sensitivities of a certain drug in advance and to make an appropriate prescription based on the classification based on the analysis of SNPs, and to administer the optimum drug to the individual constitution of the patient and the risk of side effects. Avoidance is expected. Such medical treatment is called tailor-made medical treatment or tailor-made medical treatment, and its practical application in the future is surely considered.
[0008]
It has also been found that cancer is caused by mutation in a specific site on a gene that plays an important role in normal cells, for example, by the action of ultraviolet light or a mutagen. By reading a mutation on a specific gene, it becomes possible to diagnose at an early stage whether or not a cell is cancerous. The SNPs are also effective in identifying a criminal in a criminal investigation, determining an ambiguous parent-child relationship, and identifying whether or not the person is a person. As mentioned above, each individual has 3 million to 10 million SNPs, and since each individual inherits a different SNP from their parents, there are absolutely no humans on the planet who have the exact same SNPs, even if they are parents and siblings. It is said not to. This is why it allows complete identification of the individual.
[0009]
As described above, observing a mutation occurring at one base in a specific gene may greatly contribute to medical treatment and various other matters. However, at present, methods for observing mutations in specific genes require extremely complicated operations or expensive equipment as described below, and the running costs are extremely high. is there.
[0010]
The most commonly used method for examining SNPs at present is a method called sequencing (determination of base sequence) in which the base sequence of DNA is read directly from the end. Since a gene is a unit of DNA having nucleotide sequence information for forming one type of protein, SNPs can be understood by reading the nucleotide sequence from the end. There are several reports on the method of performing sequencing, but the most commonly performed method is dideoxy sequencing (Sanger method) described below. All of these methods, including this method, are based on a technique capable of separating and discriminating a difference in the length of one base length by denaturing polyacrylamide gel electrophoresis or capillary electrophoresis having high separation ability.
[0011]
Dideoxy sequencing (Sanger method), also called enzymatic sequencing, uses a DNA polymerase to synthesize the complementary strand of a single-stranded template DNA, and further synthesizes four specially made dideoxynucleotides. The feature is to use. As a sequencing operation, a synthetic base sequence complementary to the 3 ′ end of the single-stranded DNA whose base sequence is to be sequenced is used as a primer, and a deoxynucleotide added evenly to the DNA polymerase from the primer is extended by an enzyme reaction. At this time, four reaction vessels were prepared at the same time, and each of the four bases of ATGC was substituted with a dideoxynucleotide, which is a base analog having no hydroxyl group at the 3 'end of the four bases, and therefore unable to continue the DNA extension reaction. Mix separately in small quantities. This stops DNA synthesis when the dideoxynucleotide is added to the terminus of the growing DNA, and has various lengths in each reaction vessel, but ends with two base analogs that are always added. Strand DNA is formed. The reaction vessel is reacted with S1 endonuclease to digest all the single-stranded DNA to make only double-stranded DNA. The DNA strands of the four reaction vessels thus obtained are subjected to gel electrophoresis or capillary electrophoresis, and the separated DNAs are read in order from the shorter one (the one that moved faster), and the base sequence of the DNA complementary to the template strand is found. Understand. At this time, the result of the electrophoresis is identified by, for example, radiolabeling the phosphorus or sulfur of the added dideoxynucleotide or binding a fluorescent substance. In the case of a radioactive label, detection is performed by exposure to a film, and in the case of a fluorescent chemical substance, a laser beam is irradiated to detect fluorescence. Recently, a method has been developed in which four types of base analogs A, T, G, and C are labeled with four types of reagents having different fluorescence wavelengths, and the four colors of fluorescence are simultaneously detected.
[0012]
As described above, conventionally, the exact nucleotide sequence of a gene isolated and purified from a subject is determined by this dideoxy sequencing (Sanger method) or other nucleotide sequencing methods, and is compared with a normal or standard nucleotide sequence. Confirmation of the presence or absence of SNPs or mutation, identification of individuals, etc. are performed.
[0013]
[Problems to be solved by the invention]
As explained above, using a conventional gene sequencing method, gene diagnosis and identification of an individual by a gene are performed by isolating a target gene, amplifying and purifying the target gene, and using an apparatus for determining the base sequence of the gene. The experiment was performed by reading the base sequence of the target gene, so that the experiment required an enormous amount of work, a very long time, and a large running cost. Automated equipment for base sequence determination is very expensive, occupies a large space, and requires a large amount of expensive reagents.
[0014]
Therefore, in order to solve such a conventional problem, the present invention is capable of detecting a gene abnormality of one or more bases in a short time, easily and accurately, and is small, light, inexpensive, and has very low running cost. Accordingly, an object of the present invention is to provide a gene diagnosis apparatus and a gene diagnosis method capable of automating diagnosis.
[0015]
In addition, the present invention enables the repeated use of a DNA binding substance for separation, which is used for separating and measuring the normal type and the abnormal type of the gene to be determined, and has a large molecular weight derived from chromosomal DNA or blood. Provided are a gene diagnosis apparatus and a gene diagnosis method that can remove a noise substance, simplify a diagnosis apparatus, reduce the cost for diagnosis, and realize highly accurate determination.
[0016]
[Means for Solving the Problems]
In order to solve the above problems, a gene diagnostic apparatus of the present invention includes a first container containing a buffer and a first electrode immersed therein, a second container containing a buffer and a second electrode immersed therein, A closed flow path that connects the first container and the second container with each other by filling a buffer containing a linear polymer and a DNA binding control agent, applying a positive potential or a negative potential to the second electrode, and applying a negative potential or a positive potential to the first electrode; A power supply unit for applying a potential, a control unit for controlling the power supply unit to apply a voltage between the second electrode and the first electrode, and a detection unit provided in a closed flow path for detecting a passing amount of DNA passing through the inside In the closed channel, a base sequence capable of hydrogen bonding to the sample DNA and a high molecular compound are combined in a buffer solution, and the sample DNA is separated into normal DNA and abnormal DNA based on a difference in binding force. A DNA binding substance for separation and a sample DNA; At least, in the electrophoresis direction, a selective separation filter that allows the sample DNA to pass through and does not allow the DNA binding substance for separation to pass through is provided. Is configured to measure the amount of passing normal DNA and / or abnormal DNA.
[0017]
This makes it possible to repeatedly use the DNA binding substance for separation, and to prevent variation in measurement results due to noise DNA derived from chromosomes and the like. As a result, abnormalities of one or more nucleotides can be detected in a short time, easily and accurately, and the diagnosis can be automated at a small size, light weight, low cost, and at a very low running cost.
[0018]
BEST MODE FOR CARRYING OUT THE INVENTION
The first aspect of the present invention provides a first container containing a buffer solution and immersed in a first electrode, a second container containing a buffer solution and immersed in a second electrode, and a first container and a second container. A sealed flow path that is filled and connected with a buffer containing a linear polymer and a DNA binding control agent, and a power supply unit that applies a positive or negative potential to the second electrode and applies a negative or positive potential to the first electrode A control unit that controls a power supply unit to apply a voltage between the second electrode and the first electrode; and a detection unit that is provided in the closed channel and detects a passing amount of DNA passing through the inside. In the channel, a base sequence capable of hydrogen bonding to the sample DNA and a polymer compound are bound in a buffer solution, and a DNA binding substance for separation that separates the sample DNA into normal DNA and abnormal DNA based on a difference in binding strength. , Sample DNA, and at least on the electrophoresis direction side of the closed channel. A selective separation filter that allows the sample DNA to pass therethrough and does not allow the DNA binding substance for separation to pass through is provided, and when a voltage is applied, the sample DNA in the closed channel is electrophoresed, and the detection unit detects normal DNA and / or abnormal DNA. It is characterized by measuring the amount of passing.
[0019]
As described above, the presence or absence of abnormality in the sample DNA can be determined by observing the separation or non-separation of the sample DNA from the normal DNA by using the electrophoresis and the hydrogen bonding of the DNA. Since the substance can be reused and noise generated from chromosome-derived DNA can be realized, it is possible to provide a device that can reduce running cost by repeated measurement and can perform more accurate determination.
[0020]
After the electrophoresis is completed and the result is determined, the power is supplied for a long time, so that the sample DNA and the normal DNA all flow away, and as a result, the DNA binding substance for separation is located near the end of the closed channel in the same direction as the electrophoresis. concentrate. By applying a potential opposite to that of the measurement to this closed channel, the DNA binding substance for separation can be returned to the state before the measurement, and the measurement can be repeated without replenishing the DNA binding substance for separation. You can do it.
[0021]
The invention according to claim 2 is the method according to claim 1, wherein a sample DNA and normal DNA used as a control are allowed to pass through the closed channel, a higher-molecular substance is not passed, and a larger amount than a selective separation filter is used. A selective separation pre-filter capable of capturing a polymer is provided, and the selective binding pre-filter and the selective separation pre-filter sandwich a DNA binding substance for separation.
[0022]
Therefore, even if the sample to be measured contains impurities such as DNA or protein having a large molecular weight, accurate measurement can be performed without being detected as noise.
[0023]
According to a third aspect of the present invention, there is provided a first container containing a buffer solution and immersed in a first electrode, a second container containing a buffer solution and immersed in a second electrode, and a first container and a second container. A sealed flow path that is filled and connected with a buffer containing a linear polymer and a DNA binding control agent, and a power supply unit that applies a positive or negative potential to the second electrode and applies a negative or positive potential to the first electrode A control unit that controls a power supply unit to apply a voltage between the second electrode and the first electrode; and a detection unit that is provided in the closed channel and detects a passing amount of DNA passing through the inside. In the channel, a base sequence capable of hydrogen bonding to the sample DNA and a polymer compound are bound in a buffer solution, and a DNA binding substance for separation that separates the sample DNA into normal DNA and abnormal DNA based on a difference in binding strength. , A sample DNA and sandwiching a DNA binding substance for separation in a closed channel. Thus, a selective separation filter that allows the sample DNA to pass therethrough and does not allow the DNA binding substance for separation to pass through is provided, and when a voltage is applied, the sample DNA in the closed channel is electrophoresed, and the detection unit is normal DNA and / or The method is characterized in that the amount of abnormal DNA passed is measured.
[0024]
Therefore, even after the electrophoresis is completed and the result is determined, the energization is performed for a long time, so that the sample DNA and the normal DNA all flow away, and as a result, the DNA binding substance for separation concentrates near the end of the closed flow path. By applying a potential opposite to that of the measurement to the closed channel after the measurement is completed, the DNA binding substance for separation can be returned to the state before the measurement, without replenishing the DNA binding substance for separation newly. It can be used for repeated measurement. By attaching filters at both ends, it becomes possible to minimize the loss of the DNA binding substance for separation. It should be noted that the same effect can be obtained by taking out only the closed flow path after the measurement is completed and exchanging before and after, instead of the operation of applying the opposite potential.
[0025]
According to a fourth aspect of the present invention, in the third aspect, the sample DNA and the normal DNA used as a control can be passed at least upstream of the selective separation filter located on the opposite side of the closed channel in the electrophoresis direction. And a selective separation pre-filter capable of trapping a larger amount of polymer than a selective separation filter without allowing a higher molecular substance to pass therethrough.
[0026]
Therefore, even if the sample to be measured contains impurities such as DNA or protein having a large molecular weight, accurate measurement can be performed without being detected as noise.
[0027]
According to a fifth aspect of the present invention, in the third aspect, the sample DNA and the normal DNA used as a control are allowed to pass through the selective separation filter in the closed channel, and the higher molecular weight substance is not passed through. And a selective separation pre-filter capable of capturing a larger amount of macromolecules than the selective separation filter.
[0028]
Therefore, by applying a potential opposite to that of the measurement to the closed flow path after the measurement is completed, the DNA binding substance for separation can be returned to the state before the measurement, and furthermore, a noise that may become noise captured by the pre-filter can be obtained. It becomes possible to remove molecular substances. This makes it possible to repeatedly perform the measurement without newly replenishing the DNA binding substance for separation. It should be noted that the same effect can be obtained by taking out only the closed flow path after the measurement is completed and exchanging before and after, instead of the operation of applying the opposite potential.
[0029]
According to a sixth aspect of the present invention, in the second to fifth aspects, the detection windows for detecting the amount of DNA passing through the inside of the closed flow path are respectively sealed so as to sandwich at least the DNA binding substance for separation. It is provided on both sides of the road.
[0030]
Therefore, it can be determined whether the sample DNA is normal or abnormal without removing or replacing the closed flow path.
[0031]
A seventh aspect of the present invention is characterized in that, in the second aspect, a closed channel portion including a region sandwiched between the selective separation filter and the selective separation prefilter is detachable.
[0032]
Therefore, a part of the flow path can be formed into a cartridge or a chip, and since there is no outflow of the DNA binding substance for separation due to measurement, measurement can be performed a plurality of times.
[0033]
Even after the measurement is completed, the sealed flow path portion is turned over and the determination is made again, and even if the measurement is repeated many times, the amount of DNA passing through only one detection window can be measured. it can. In this case, the detection window is located outside the unit with the selective separation filter and the closed channel as one unit, and the measurement is not affected at all even if the unit is freely replaced.
[0034]
An eighth aspect of the present invention is characterized in that, in any of the third to fifth aspects, at least a closed channel portion including a region sandwiched by the selective separation filters is detachable.
[0035]
Therefore, a part of the flow path can be formed into a cartridge or a chip, and since there is no outflow of the DNA binding substance for separation due to measurement, measurement can be performed a plurality of times. Even after the measurement is completed, the sealed flow path portion is turned over and the determination is made again, and even if the measurement is repeated many times, the amount of DNA passing through only one detection window can be measured. it can.
[0036]
A ninth aspect of the present invention is characterized in that, in the eighth aspect, the closed channel portion including the selective separation prefilter is detachable.
[0037]
Therefore, even if the sample to be measured contains impurities such as DNA or protein having a large molecular weight, accurate measurement can be performed without being detected as noise.
[0038]
According to a tenth aspect of the present invention, there is provided a first container containing a buffer solution and immersed in a first electrode, a second container containing a buffer solution and immersed in a second electrode, and a first container and a second container. A sealed flow path for connecting the space between the linear polymer and a buffer containing a DNA binding control agent by filling the space, and a power supply unit for applying a positive or negative potential to the second electrode and applying a negative or positive potential to the first electrode A control unit that controls a power supply unit to apply a voltage between the second electrode and the first electrode; and a detection unit that is provided in the closed channel and detects a passing amount of DNA passing through the inside. A base sequence capable of hydrogen bonding to a sample DNA and a polymer compound are bonded to a member that is detachable to a channel and forms a part of the closed channel when attached to the closed channel. Separation DNA binding to separate sample DNA into normal DNA and abnormal DNA from difference in force A selective separation filter that allows the sample DNA to pass through and sandwiches the DNA binding substance for separation, and that does not allow the DNA binding substance for separation to pass through. Thus, the sample DNA in the closed flow path is electrophoresed, and the detection unit measures the passing amount of normal DNA and / or abnormal DNA.
[0039]
Therefore, a part of the flow path can be formed into a cartridge or a chip, and since there is no outflow of the DNA binding substance for separation due to measurement, measurement can be performed a plurality of times.
[0040]
The invention according to claim 11 is the invention according to claim 10, wherein one of the selective separation filters is allowed to pass a sample DNA and a normal DNA used as a control, and is not allowed to pass a higher-molecular substance than the selective separation filter. It is characterized in that it is a selective separation prefilter capable of capturing a large amount of polymer.
[0041]
Therefore, even if the sample to be measured contains impurities such as DNA or protein having a large molecular weight, accurate measurement can be performed without being detected as noise.
[0042]
According to a twelfth aspect of the present invention, there is provided the selective separation filter according to the tenth aspect, wherein a sample DNA and a normal DNA used as a control are allowed to pass therethrough, and a higher-molecular substance is not passed through the selective separation filter. A selective separation pre-filter capable of capturing a larger amount of macromolecules is provided.
[0043]
Therefore, even if the sample to be measured contains impurities such as DNA or protein having a large molecular weight, accurate measurement can be performed without being detected as noise.
[0044]
According to a thirteenth aspect of the present invention, the closed channel is a capillary tube, and efficient electrophoresis can be performed.
[0045]
According to a fourteenth aspect, in the thirteenth aspect, the inner wall of the capillary tube is coated with acrylamide, and the electroosmotic flow can be prevented.
[0046]
According to a fifteenth aspect of the present invention, the first container contains a buffer solution and immerses the first electrode, and the second container contains a buffer solution and immerses the second electrode. A selective separation filter that allows passage of sample DNA and does not allow passage of a DNA binding substance for separation, at least on the electrophoresis direction side of the closed channel, and a hydrogen-bondable sample DNA A linear polymer and a DNA-binding control agent are contained in a sealed flow path provided with a DNA binding substance for separation in which a base sequence and a high-molecular compound are bound and a sample DNA is separated into a normal DNA and an abnormal DNA based on a difference in binding strength. A buffer is filled, sample DNA is introduced into the closed channel, a voltage is applied between the first electrode and the second electrode, and the sample DNA in the closed channel is electrophoresed to remove normal DNA and / or abnormal DNA. Genetics characterized by detection A diagnostic method, it is possible to achieve improved reproducibility and cost reduction of the determination.
[0047]
The invention according to claim 16 is the method according to claim 15, wherein the closed channel allows passage of a sample DNA and normal DNA used as a control, does not allow passage of higher-molecular substances, and has a larger amount than a selective separation filter. A gene diagnosis method comprising a selective separation prefilter capable of capturing a polymer, wherein a DNA binding substance for separation is sandwiched between the selective separation filter and the selective separation prefilter. In addition to improving the reproducibility of the determination and reducing the cost, it is possible to extend the life of the first selective separation filter which aims at newly reducing the measurement noise and capturing the DNA binding substance for separation.
[0048]
The invention according to claim 17, wherein the first container contains a buffer and the first electrode is immersed, and the second container contains a buffer and the second electrode is immersed in the first container and the second container. A DNA-binding substance that separates a sample DNA into a normal DNA and an abnormal DNA based on a difference in binding strength, wherein a base sequence capable of hydrogen bonding to the sample DNA is bonded to a polymer compound. And a buffer containing a linear polymer and a DNA binding control agent in a closed flow path provided with a selective separation filter that allows the sample DNA to pass therethrough so as to sandwich the DNA binding substance for separation and does not allow the DNA binding substance for separation to pass through. A liquid is filled, sample DNA is introduced into the closed channel, a voltage is applied between the first electrode and the second electrode, and the sample DNA in the closed channel is electrophoresed to detect normal DNA and / or abnormal DNA. Genetic diagnosis characterized by A method, it is possible to achieve improved reproducibility and cost reduction of the determination.
[0049]
According to the present invention, the first electrode is immersed in the first container containing the buffer solution, and the second electrode is immersed in the second container containing the buffer solution. This is a closed flow path that connects between them, and is a closed flow path equipped with a detachable member that constitutes a part of the closed flow path. And a separating DNA-binding substance for separating the sample DNA into normal DNA and abnormal DNA based on a difference in binding force, and allowing the sample DNA to pass through the separating DNA-binding substance, but not allowing the separating DNA-binding substance to pass therethrough. A closed flow path provided with a selective separation filter is filled with a buffer solution containing a linear polymer and a DNA binding control agent, sample DNA is introduced into the closed flow path, and a voltage is applied between the first electrode and the second electrode. Electrophoresing the sample DNA in the closed channel, A gene diagnosis method characterized by detecting normal DNA and / or abnormal DNA, which can improve reproducibility of judgment and reduce cost, and can use a cartridge-shaped and chip-shaped member, The degree of freedom is improved.
[0050]
Hereinafter, a gene diagnosis apparatus and a gene diagnosis method according to an embodiment of the present invention will be described with reference to the drawings.
[0051]
(Embodiment 1)
FIG. 1 is an external view of the genetic diagnostic device according to the first embodiment of the present invention, FIG. 2 is an external view of the top open of the genetic diagnostic device according to the first embodiment of the present invention, and FIG. 3 is a gene according to the first embodiment of the present invention. FIG. 4 is a diagram showing a main part of a control circuit of the gene diagnosis apparatus according to the first embodiment of the present invention, and FIG. 5 is a diagram showing the use of a DNA binding substance for separation of the gene diagnosis apparatus according to the first embodiment of the present invention. FIG. 2 is a structural diagram of a closed channel in which a selective separation filter according to the present invention for electrophoresing a sample DNA and a normal DNA is attached only near an end point.
[0052]
In FIG. 1, reference numeral 1 denotes a power switch of the gene diagnosis apparatus, 2 denotes operation buttons for performing various operations of the apparatus, 3 denotes a display panel, 4 denotes an upper lid, and 5 denotes a housing of the apparatus. In FIGS. 2, 3 and 4, reference numeral 6 denotes a predetermined temperature for separating normal DNA and abnormal DNA, and at this portion, the DNA is separated into three of normal DNA, abnormal DNA and other noise DNA. Electrophoresis unit having a closed flow channel for performing electrophoresis, 7 is a support for supporting the electrophoresis unit 6, 8 is a control for performing electrophoresis and a suction pump (16) to be described later, a detection unit This is a control calculation unit including a board that performs the control of (10) and calculates the detected data. Reference numeral 9 denotes a power supply box, and 9a denotes a power supply unit provided in the power supply box 9. In the present gene diagnosis apparatus, there are different optimum applied voltage values for each type, concentration, and processing condition of DNA, so that electrophoresis most suitable for separating abnormal DNA and normal DNA from the sample DNA 21 described below can be performed. The control operation unit 8 controls the power supply unit 9a to apply an optimum voltage. Reference numeral 11 denotes a temperature controller for adjusting the temperature of the electrophoresis unit 6. Although the details of the configuration inside the electrophoresis unit 6 will be described later, at the start of the measurement, the DNA binding substance for separation 20 is arranged so as to be already in the closed channel of the electrophoresis unit 6 as shown in FIG. By performing such an arrangement and performing voltage control, concentration adjustment, temperature control, and the like, the present gene diagnostic apparatus converts the sample DNA 21 into normal DNA, abnormal DNA, and noise DNA for several to several tens of minutes while performing electrophoresis. It is separated by degree. In the electrophoresis section 6, the sample DNA 21 can be separated from normal DNA, abnormal DNA and noise DNA by a difference in hydrogen bonding force while the separation DNA binding substance 20 is subjected to an electrophoresis action. It is controlled so as to maintain a predetermined temperature in a temperature range of 20 ° C to 60 ° C. In order to separate normal DNA, abnormal DNA, and noise DNA, it is preferable to control the temperature within a range of ± 1 ° C. from a predetermined temperature suitable for DNA separation.
[0053]
Reference numeral 10 denotes a detection unit that measures the amount of the sample DNA to be electrophoresed. The detection unit 10 will be described with reference to FIG. 4. 10a is a D2 lamp for irradiating ultraviolet rays, 10b is a slit for concentrating ultraviolet rays emitted from the D2 lamp 10a on a target portion, 10c is a photodiode for receiving ultraviolet rays, Reference numeral 10d denotes a preamplifier that amplifies a weak current detected by the photodiode 10c, and 10e denotes an A / D converter that converts the current into a digital amount. Details of these will be described later. Reference numeral 12 denotes an electrode for applying a positive potential during electrophoresis, and reference numeral 13 denotes an electrode for applying a negative potential during electrophoresis.
[0054]
3, 4 and 5, reference numeral 14 denotes a buffer for carrying charge and stabilizing the pH of the sample DNA during electrophoresis, 14a denotes an anode-side container containing the buffer 14, and 14b denotes a buffer 14 The container on the cathode side for containing, 14c is a buffer solution obtained by adding a DNA binding control agent and the following linear polymer 14d to the buffer solution 14, and 14d enables electrophoresis and converts normal DNA, abnormal DNA, noise DNA, and noise DNA. It is a linear polymer such as polyacrylamide, which is a separation medium. 15 is a capillary tube (closed flow path) for migrating the sample DNA 21 during electrophoresis, and 17 is a selection that allows the sample DNA to pass but does not allow the separation DNA binding substance having a higher molecular weight than the sample DNA to pass. Gender separation filter. Reference numeral 16 denotes a suction pump for injecting a reagent such as the sample DNA 21 or the linear polymer 14d and a DNA binding control agent into the capillary tube 15. The electrode 12 is immersed in the buffer solution 14 of the container 14a, and the electrode 13 is immersed in the buffer solution 14 of the container 14b. The buffer 14 in the container 14a and the buffer 14b in the container 14b are communicated by the buffer 14c in the capillary tube 15. When a voltage is applied between the electrode 12 and the electrode 13, electrophoresis is induced in the capillary tube 15, and the DNA binding substance for separation 20 and the sample DNA 21 are negatively charged. The DNA binding substance 20 and the sample DNA 21 move.
[0055]
Next, details required for performing measurement with the gene diagnosis apparatus of the present invention will be described. The sample DNA 21 measured by the present gene diagnostic apparatus is DNA obtained from human cells, blood, or the like. It is necessary to cut out a target portion to a target length from a human genomic DNA by using a method such as PCR (polymerase chain reaction) to increase the number for measurement. However, since this PCR is not necessary for the description of the present gene diagnosis apparatus, a specific description is omitted.
[0056]
The number of target DNAs extracted by PCR or the like is about 20 to several thousand in terms of the number of bases, but the probability that the same DNA sequence does not exist in human genomic DNA, which is said to have about 3 billion base pairs, More specifically, the number is about 40 or more. The separation of normal DNA from abnormal DNA is closely related to the concentration of the sample DNA (μM, where M = mol / liter), the concentration of the DNA binding substance for separation (mol%), the measurement temperature, and other factors. Since separation does not occur unless conditions are set, it is important to be able to set such conditions.
[0057]
Next, the capillary tube 15 shown in FIGS. 3, 4, and 5 will be described. When the gel is put in the capillary tube 15 and electrophoresed, a liquid flow called an electroosmotic flow is generated. Therefore, in order to prevent this phenomenon from occurring, the inner wall of the capillary tube 15 is preferably coated with acrylamide or the like. As long as the generation of the electroosmotic flow can be prevented, another coating method may be used. For example, a commercially available coated capillary tube may be used. As the capillary tube 15, a fused silica capillary tube having an inner diameter of 50 to 100 μm can transmit 90% or more of ultraviolet rays, and when detecting DNA using ultraviolet rays as described later, the amount of ultraviolet rays to be passed easily. It is most appropriate because it can be detected. Even if the capillary tube 15 is formed of a combination of a plate having a groove and a plate that transmits 90% or more of ultraviolet rays, it can transmit 90% or more of ultraviolet rays, and detects abnormal DNA by detecting the amount of ultraviolet rays that have passed. Can be easily performed. When the plate with a groove is a plate through which ultraviolet light can pass, transmitted light is detected. When the plate with a groove is a plate through which ultraviolet light is not transmitted, abnormal DNA is detected by reflected light. When detecting reflected light, it is necessary to form a rectangular section in order to make the groove shape a uniform reflecting surface.
[0058]
As the buffers 14 and 14c contained in the containers 14a and 14b and in the capillary tube 15, it is appropriate to use a Tris-based (pH 7.2 to pH 8) buffer or the like. Among these, the DNA binding control agent mixed into the buffer solution 14c contained in the capillary tube 15 includes a binding promoter such as magnesium chloride which promotes the binding of DNA to the DNA binding substance for separation, and a release agent such as urea which promotes the separation. There are two types: These two types of DNA binding control agents can control various electrophoretic speeds of DNA by selecting two kinds of mixing ratios and selecting a material (for example, another electrolyte as a binding promoter). . Polyacrylamide can be used as the linear polymer 14d for electrophoresing the DNA binding substance for separation 20 or the sample DNA 21, and the like is used for the DNA binding substance for separation 20. is there.
[0059]
Next, regarding the order of filling the capillary tube 15, a buffer 14c containing a linear polymer 14d and a DNA binding control agent is first introduced into the capillary tube 15, as shown in FIG. The DNA binding substance for separation 20 is added. At this time, the linear polymer 14d may be mixed and introduced, or the linear polymer 14d may be introduced after the separation DNA binding substance 20 is introduced. Subsequently, the sample DNA 21 diluted with the buffer solution 14c in a separated state from the DNA binding substance for separation 20 is introduced and subjected to electrophoresis.
[0060]
In the first embodiment, since acrylamide and DNA are polymerized to prepare the DNA binding substance for separation 20, the difference in weight and structure between the DNA and the DNA is large, and the migration speed of the DNA binding substance for separation 20 (0 (0.6 cm / min to 0.7 cm / min) is about 1/20 to 1/30 of the optimal migration speed of DNA (13 cm / min to 20 cm / min) due to inertia, and the movement is extremely slow. Thus, a state that can be said to be pseudo-fixed is realized. In addition to the mechanism for exchanging each solution of the sample DNA, the DNA binding substance for separation, and the linear polymer into the capillary tube 15 of the genetic diagnosis apparatus, for example, the suction pump 16 shown in FIG. It is preferable to equip a column for storing each solution of the DNA binding substance for use and the linear polymer, and a mechanism for automatically exchanging the column and connecting it to the capillary tube 15.
[0061]
Further, the capillary tube 15 may be unitized in units of one or more tubes, and this may be mounted on the electrophoresis unit 6 as a sealed cartridge for exchange for separation, and the temperature may be controlled by the temperature controller 11. Appropriate. The capillary tube 15 is previously filled with the buffer solution 14c containing the linear polymer 14d and the DNA binding control agent, and the separation DNA binding substance 20 and the sample DNA 21 are filled in a separated state to prepare a sealed closed channel cartridge. It is possible to keep. With this configuration, each time the measurement is performed, the entire closed channel cartridge for separation is exchanged. Therefore, the arrangement of the DNA binding substance for separation 20 and the sample DNA 21 can be set in advance for each closed channel cartridge for separation. Can be easily performed, and accurate measurement can be performed. In the closed channel cartridge for separation, the linear polymer 14d is interposed between the separation DNA-binding substance 20 and the sample DNA 21 in order to make the separation state. The linear polymer 14d may be mixed and filled in each.
[0062]
Next, the DNA binding substance for separation 20 will be described.
[0063]
The DNA-binding substance 20 for separation contains a sample DNA to be determined (having a sequence difference of at least one base compared to normal DNA or a DNA having the same sequence as normal DNA) and a DNA to be determined. Correspondingly known to be normal, a normal DNA having the same number of bases as the sample, a DNA having a base sequence homologous to the normal DNA or the DNA to be determined and a substance having a higher molecular weight than the sample DNA were bound. Things. In addition, the DNA binding substance for separation means that when normal DNA and abnormal DNA having a sequence difference of one or more bases are simultaneously moved in a closed flow channel by electrophoresis, the number of bases is the same as that of normal DNA. Separation from normal or abnormal DNA utilizing the difference in hydrogen bonding force with the base sequence completely homologous to either normal DNA or abnormal DNA existing in the DNA binding substance for separation Refers to substances that can be used. The substance is characterized in that it has a molecular weight always larger than the DNA to be determined.
[0064]
Here, FIG. 11 is a conceptual diagram showing the relationship between the DNA binding substance for separation and the sample DNA in the closed flow channel of the genetic diagnostic apparatus according to Embodiment 1 of the present invention. In FIG. 11, reference numeral 20 denotes a DNA binding substance for separation.
[0065]
There are two types of DNA, one that forms a double strand and the other that is a single strand. The four bases A, T, C, and G of DNA have the property that A, T, and G and C are easily hydrogen bonded to each other. Also, the double strand of DNA is paired with AT and GC. Therefore, when the DNA of one strand is sequenced as ATCGCGTCTAGC (described in SEQ ID NO: 1), the DNA of the other strand has a base sequence of TAGCGCAGATCG (described in SEQ ID NO: 2). This relationship is called a complementary relationship, and as long as the complementary relationship is satisfied, A and T, and G and C are each bonded by a hydrogen bond to form a double strand. In the present invention, in order to utilize this relationship, as shown in FIG. 11, the DNA portion of the DNA binding substance for separation 20 has a base sequence complementary to the normal DNA of the sample DNA. Therefore, the base sequence of the normal DNA of the sample DNA contains ATCGGCTCTAGC (described in SEQ ID NO: 1), and the abnormal DNA is ATCA * In CGTCTAGC (described in SEQ ID NO: 3), when the bases of the normal DNA, the abnormal DNA, and the noise DNA are different in the portion indicated by *, the sequence of the DNA portion of the DNA binding substance for separation 20 is changed to TAGCGCAGATCCG (SEQ Described), the abnormal DNA is A * In this case, hydrogen bonding is not performed because the compound is no longer complementary to the DNA binding substance 20 for separation, and the binding force of the entire hydrogen bond in the sample DNA is higher than that in the abnormal DNA by one base. As a result, during the electrophoresis described later, the normal DNA binds to the separation DNA binding substance 20 with a stronger binding force than the abnormal DNA for a longer time, so that the normal DNA is relatively delayed from the abnormal DNA. This is because not only the binding force at the time of electrophoresis but also the separation force by electrophoresis acts, so that a large number of DNAs are in a binding state in which binding and detachment are repeated intermittently. When this migration speed is averaged, the migration speed of normal DNA that binds for a longer time is lower than that of abnormal DNA. The sample DNA contains DNA other than normal DNA, abnormal DNA, and noise DNA, such as the remaining single-stranded DNA that is not the target of measurement among double-stranded DNA. The noise DNA acts as a noise during the electrophoresis described in (1). However, since the noise DNA hardly reacts with and binds to the DNA of the DNA binding substance for separation 20, the DNA is separated with the fastest migration speed. The concentration of the DNA binding substance for separation 20 is suitably 20 to 600 times the concentration of the sample DNA.
[0066]
Next, a method for preparing and synthesizing such a DNA binding substance for separation 20 will be described. In order to synthesize the vinylated DNA, the DNA for the DNA binding substance for separation 20 is cut out by PCR. In the above example, TAGCGCAGATCCG (described in SEQ ID NO: 2) is the DNA of the DNA binding substance for separation 20.
[0067]
Subsequently, the 5 ′ end of the DNA having the target base sequence is aminated (usually aminated via a hexyl group). The aminated DNA thus obtained is diluted by adding ultrapure water sterilized to 2.6 mM. Next, MOSU (methacryloid oxysuccinimide) is diluted with DMSO (dimethyl sulfoxide) to a concentration of 71.388 mM. Then, the aminated DNA and MOSU thus obtained are added and adjusted so as to have a ratio of 1:50. Further, to this adjusted solution, a solution adjusted with sodium hydrogen carbonate and sodium hydroxide so as to have a pH of 9 for pH adjustment is added in an amount equal to the amount of the aminated DNA.
[0068]
Then, the obtained solution is shaken overnight. Thereafter, the vinylated DNA in the shaken solution is separated from aminated DNA, MOSU, and the like by using HPLC (High Performance Liquid Chromatography). Since the vinylated DNA contains an eluent (TEAA; a mixed solution of triethylamine-acetic acid and acetonitrile), it is further concentrated under reduced pressure by a centrifugal evaporator having a vacuum drying function.
[0069]
Next, a polymerization solution of 10% AAM (acrylamide) is replaced with 53 μmol of nitrogen. Further, 1.34% of TEMD (N, N, N'N'-tetramethylethylenediamine) as a polymerization initiator is diluted with sterilized ultrapure water deaerated by ultrasonic waves. This is diluted with sterilized ultrapure water of 2% APS (ammonium persulfate), which is a polymerization initiator, also degassed by ultrasonic waves. Further, the concentrated vinylated DNA is diluted with sterilized ultrapure water. Then, in order to synthesize 100 μl of the DNA binding substance 20 for separation, 34 μl of the above-mentioned AAM, 5 μl of each of the above TEMD and APS, and 0.01% to 0.05% of vinylated DNA with respect to the AAM. Then, sterile ultrapure water is added to 100 μl, and the mixture is allowed to stand for about 60 minutes to obtain an acrylamidated DNA binding substance for separation 20.
[0070]
Next, the operation of the gene diagnosis apparatus according to the first embodiment will be described. First, the capillary tube 15 into which the sample DNA 21 and each solution are introduced is set in the gene diagnosis apparatus, and the buffer solution 14 in the containers 14a and 14b is immersed at both ends as shown in FIGS. A predetermined voltage is applied between the electrode 12 and the electrode 13 by the power supply unit 9a. The magnitude of the applied voltage is preferably 10 to 20 kV, but may be 100 to 30 kV depending on the state of the electrolyte and the electrodes. For example, when the electrolyte concentration is low or the electrode area is large, the voltage is set to a low voltage. Then, instead of applying a predetermined voltage from the beginning, a high voltage of 30 kV or more for preparation is applied before the application of a constant voltage, and a predetermined voltage is applied after separating a noise DNA, thereby quickly measuring. Can be done.
[0071]
Here, the reason why a predetermined voltage is applied between the electrode 12 and the electrode 13 will be described. Since the difference between the binding force of the sample DNA 21 to the separation DNA binding substance 20 and the separation force due to the electrophoretic force governs the expression of the difference in the migration speed of each DNA, the separation of the normal DNA, the abnormal DNA, and the noise DNA is not performed. This is because it is necessary to select and apply the most effective voltage. In other words, this voltage must be as low as possible in order to allow electrophoresis of normal DNA, which is most difficult to migrate, and to reduce the resolution of normal DNA and abnormal DNA when a high voltage is used. Is a voltage that satisfies two of the conditions. Therefore, the optimal voltage to be applied in advance is checked for each DNA and each condition, and as described above, a preliminary voltage of about 30 kV or more is initially applied for a short time, and then this voltage is applied. Optimal. Since the DNA in the capillary tube 15 is negatively charged and proceeds to the anode side, it is necessary to apply the power supply 9a so that the electrode 12 has a positive potential and the electrode 13 has a negative potential. In the electrophoresis, if the electrophoresis time is too long, the buffer solution 14c is dried in the capillary tube 15, so that it is necessary to avoid performing electrophoresis for an unnecessarily long time.
[0072]
Note that a high voltage of 30 kV or more for preparation may be applied to quickly separate noise DNA, and then the absolute value of the voltage may be gradually increased to apply the high voltage. When the absolute value of the voltage is gradually increased, the voltage may be increased regularly or irregularly, but a particularly preferable method is to sweep and apply the voltage. The sweep means changing the voltage at a constant value per unit time. For example, if the sweep is started from 0 volt to 1 kilovolt / sec, 10 kilovolts will be applied after 10 seconds. Explaining the reason for sweeping the applied voltage, the difference between the binding force of the DNA sample 21 to the DNA binding substance for separation 20 and the separation force due to the electrophoretic force greatly affects the migration speed and the movement difference of DNA. Sweep is performed to adjust a voltage at which normal DNA, abnormal DNA, and noise DNA are most effectively separated. If the voltage at which the difference in the migration speed is the most caused by the sweep is known, this voltage may be maintained. By sweeping the applied voltage, it is possible to obtain an effect that the voltage can be easily adjusted to an optimum voltage having a high resolution. This is because the noise DNA is first electrophoresed at the stage where the sweep voltage is low and detected by the detection unit, but the abnormal DNA has a weak binding force of one base to the separation DNA binding substance 20. Unless it gets a little high voltage, you can't leave it. In addition, normal DNA cannot be released without further increasing the voltage. If a voltage around this is applied, abnormal DNA and normal DNA are clearly separated. However, if the sweep voltage is increased from this, the resolution of the normal DNA and the abnormal DNA decreases, so that it is optimal to keep the voltage near the voltage at which normal DNA starts to migrate. Then, after a preliminary voltage of about 30 kV or more is initially applied for a short time, the sweep start voltage may be simply started from 0 volts. However, depending on the binding conditions between the DNA binding substance for separation 20 and the sample DNA 21, 30 kV may be used. After applying the above, the sweep may be started from 5 kV to 7 kV. Since the DNA in the capillary tube 19 proceeds to the anode side, the variable power supply unit 9a needs to apply the electrode 16 to the positive potential and the electrode 17 to the negative potential. When the voltage is swept, the DNA sample 23 is electrophoresed, and the DNA binding substance 20 for separation has a difference of one base in the binding force between normal DNA and abnormal DNA. And a large movement difference between the DNA and the noise DNA.
[0073]
Next, the electrophoresis unit 6 where the separation is performed will be described. In the electrophoresis section 6, although it differs depending on the state of the sample DNA, it is set at 15 ° C. so that normal DNA, abnormal DNA, or noise DNA can be separated based on the difference in the binding strength between the DNA binding substance for separation 20 and the sample DNA 21. The temperature must be maintained at a predetermined temperature in the range of ８０80 ° C., preferably 20 ° C.-60 ° C. In the gene diagnosis apparatus of the first embodiment, a silicon rubber heater, a nichrome wire, and a temperature sensor such as a thermocouple or a thermal are arranged below the electrophoresis unit 6 covering the capillary tube 15. The temperature is controlled so as to be a predetermined temperature ± 1 ° C or less. However, depending on the environmental temperature, the room temperature may exceed the intended predetermined temperature, and it is preferable to use a heatable / coolable component such as a Peltier instead of a silicon rubber heater or a nichrome wire.
[0074]
Next, a description will be given of how to detect a normal DNA, an abnormal DNA, and a noise DNA having a migration difference due to the effect of the separation DNA binding substance 20 due to the action of the DNA binding substance 20. FIG. 12 is a relationship diagram between the elapsed time and the absorbance when the elapsed time from the start of electrophoresis is set on the horizontal axis and the absorbance is set on the vertical axis. The detection is carried out by measuring the absorbance when the ultraviolet irradiation is shielded by normal DNA, abnormal DNA, noise DNA, and noise DNA. In the first embodiment, as shown in FIGS. 3 and 4, the glass part is exposed at a part of the capillary tube 15, and ultraviolet light having a wavelength of 260 nm is irradiated from the D2 lamp 10a. The light is detected by the diode 10c and the absorbance is measured. The control operation unit 8 controls the power supply unit 9a to emit light from the D2 lamp 10a, the current detected by the photodiode 10c is amplified by the preamplifier 10d, and converted into a digital amount by the A / D converter 10e to be converted into an absorbance by the control operation unit 8. Is done. The control operation unit 8 has a built-in timer (not shown) and can measure the elapsed time from the electrophoresis start time. In FIG. 7, noise DNA not binding to the DNA conjugate (I) has a shorter elapsed time (passing earlier in time), abnormal DNA has a middle elapsed time (II), and has a longer elapsed time (III). ) Is normal DNA.
[0075]
When the height, time, and number of peaks are read from this relationship diagram, it can be seen from the number of peaks that the sample DNA contains abnormal DNA. That is, whether or not abnormal DNA is present can be determined from the number of peaks. The number of peaks can be limited to abnormal DNA and noise DNA depending on the voltage to be applied, the DNA concentration, and the length of DNA (the number of bases). In this case, only the passing amount of abnormal DNA is measured. Also, comparing the peak heights indicates the difference in absorbance between two sets having a migration speed difference in DNA electrophoresed under the same conditions, and thus corresponds to the abundance ratio of abnormal DNA to normal DNA. The ratio can be determined. The determination of the abundance of abnormal DNA may be performed by inputting standard data obtained from the detected peak waveform of the standard sample DNA into the control calculation base unit 8 in advance, comparing the measured data with the measured data, and converting the data.
[0076]
As shown in FIG. 5, the selective separation filter 17 has a selective permeability such that the sample DNA 21 can pass through, but the separation DNA binding substance 20 having a higher molecular weight than the sample DNA cannot pass through. And an ultrafiltration membrane or a nanofiltration membrane can be considered as a material.
[0077]
In addition, the selective separation filter 17 can pass the sample DNA 21 to be determined (and normal DNA inserted as a control), but passes the sample DNA 21 or the separation DNA binding substance 20 having a higher molecular weight than the normal DNA. Have selectivity that cannot be achieved. The selective separation filter 17 used here may be any filter such as a semipermeable membrane (dialysis membrane) or an ultrafiltration membrane as long as it exhibits selective permeability due to a difference in molecular weight. For example, the DNA to be determined and the normal DNA as a control are single-stranded DNAs of about 20 to 200 mer, and have a molecular weight of about 6,000 to 60,000. On the other hand, the DNA binding substance for separation 20 needs to be a substance having a molecular weight larger than that of DNA or normal DNA to be always determined. The selective separation filter 17 can pass the sample DNA 21 having a molecular weight of 6,000 to 60,000 and the normal DNA used as a control, and can pass the DNA binding substance 20 for separation having a larger molecular weight and the giant DNA derived from the chromosome. We are screened on condition that we cannot do it.
[0078]
In FIG. 5, when a voltage is applied to the closed channel, the sample DNA 21 starts to migrate toward the anode, and is separated by a difference in chemical bonding force while passing through the DNA binding substance 20 for separation. It is detected as two peaks by a detection device outside the closed channel. When a voltage is applied to both ends of the closed container to cause the sample DNA 21 to migrate, the separation DNA binding substance 20 having a small but large molecular weight as compared with the sample DNA also migrates toward the anode. When the electrophoresis is performed a plurality of times using the same closed channel, depending on the total electrophoresis time, the DNA binding substance for separation 20 also goes out of the closed channel, and the subsequent separation of the sample DNA becomes impossible. It is expected that. To prevent this, a selective separation filter 17 was provided. No matter how many times the electrophoresis is performed by the present filter, the DNA binding substance for separation 20 is not lost. After being captured by the filter, it is possible to return the separation DNA binding substance 20 to the original position by switching the anode and the cathode and passing a current in the opposite direction, so that it can be used repeatedly.
[0079]
As described above, according to the genetic diagnostic apparatus and the genetic diagnostic method of the first embodiment, specific DNA is included in sample DNA extracted by PCR or the like among DNA extracted from cells, blood, and the like. Genetic diagnosis and determination can be performed by knowing the ratio of the presence of normal DNA, abnormal DNA, and noise DNA. In addition, since the DNA binding substance for separation can be prevented from leaking out of the system, repetitive operations can be performed, and diagnosis / determination can be performed at low cost.
[0080]
(Embodiment 2)
A gene diagnosis apparatus and a gene diagnosis method according to Embodiment 2 of the present invention will be described with reference to the drawings. Note that the description of the same parts as those of the first embodiment, that is, the description of FIGS.
[0081]
FIG. 6 is a diagram in which the selective separation filter 17 described in Embodiment 1 of Embodiment 2 of the present invention is attached to both ends of a closed channel. The selective separation filter 17 is a filter having selective permeability such that the sample DNA 21 can pass therethrough but cannot pass the separation DNA binding substance 20 having a higher molecular weight than the sample DNA. Ultrafiltration membranes and nanofiltration membranes are conceivable.
[0082]
In FIG. 6, when a voltage is applied to the closed channel, the sample DNA 21 starts to migrate toward the anode, and is separated by a difference in chemical bonding force while passing through the DNA binding substance 20 for separation. It is detected as two peaks by a detection device outside the closed channel. When a voltage is applied to both ends of the closed container to cause the sample DNA 21 to migrate, the separation DNA binding substance 20 having a small but large molecular weight as compared with the sample DNA 21 also migrates toward the anode. When the electrophoresis is performed a plurality of times using the same closed flow path, the DNA binding substance for separation 20 also goes out of the closed flow path depending on the total migration time, and the subsequent separation of the sample DNA 21 becomes impossible. It is expected that. To prevent this, the selective separation filters 17 were installed at both ends of the closed channel. No matter how many times the electrophoresis is performed by the filter near the end point of the closed channel, the DNA binding substance for separation 20 is not lost. After being captured by the filter, it is possible to return the DNA binding substance for separation 20 to the original position by switching the anode and the cathode and passing a current in reverse. Does not lose the separating DNA binding substance 20. By installing filters on both ends, you can use it repeatedly with confidence.
[0083]
Further, by making the closed flow path described in FIG. 6 detachable, if the closed flow path is turned over after several times of electrophoresis, the biased DNA binding substance for separation 20 can be repeated without returning by a reverse current. You can use it.
[0084]
Although FIG. 6 shows a state in which the sample DNA 21 has been introduced, at least a sealed channel portion including a region sandwiched by the selective separation filters 17 is made detachable, and is formed into a cartridge or chip. It goes without saying that the sample DNA 21 is not introduced into the region sandwiched between the selective separation filters 17 in the cartridge and chip, and the separation DNA-binding substance 20 is filled.
[0085]
As described above, according to the genetic diagnostic apparatus and the genetic diagnostic method of the second embodiment, specific DNA is included in the sample DNA 21 extracted by PCR or the like among the DNA extracted from cells, blood, and the like. Genetic diagnosis and determination can be performed by knowing the ratio of the presence of normal DNA, abnormal DNA, and noise DNA. In addition, since the separation of the DNA binding substance 20 for separation can be prevented from leaking out of the system, the operation can be repeated, and the separation of the DNA binding substance 20 for separation can be completely prevented from leaking out of the system. Thus, accurate measurement can be performed for a long period of time. Furthermore, since the selective separation filter 17 is mounted at both ends, the electrophoresis operation can be continuously performed by turning over the closed flow path, and continuous diagnosis and judgment can be performed at low cost.
[0086]
(Embodiment 3)
A gene diagnosis apparatus and a gene diagnosis method according to Embodiment 3 of the present invention will be described with reference to the drawings. Note that the description of the same parts as those of the first embodiment, that is, the description of FIGS.
[0087]
FIG. 7 shows a state in which the selective separation filter 17 described in the first embodiment in the third embodiment of the present invention is mounted near the end point of the closed channel, and has a larger amount than the selective separation filters described in the first and second embodiments. FIG. 4 is a diagram in which a second selective separation filter (pre-filter) capable of capturing a polymer is mounted on the most upstream part. Here, the selective separation filter 17 is a filter having selective permeability such that the sample DNA 21 can pass through, but the separation DNA binding substance 20 having a higher molecular weight than the sample DNA 21 cannot pass through. The selective separation filter (pre-filter) 18 can pass the sample DNA 21 and the normal DNA used as a control, but cannot pass the higher molecular substances, and the selection according to the first and second embodiments. It can capture a larger amount of macromolecules than a sex separation filter. The materials of both filters may be the same or different, and for example, a microfiltration membrane, an ultrafiltration membrane, a nanofiltration membrane, or the like can be considered.
[0088]
In FIG. 7, when a voltage is applied to the closed flow path, the sample DNA 21 starts to migrate toward the anode, and is separated by a difference in chemical bonding force while passing through the DNA binding substance for separation 20; It is detected as two peaks by the detection device outside the closed channel, but when the sample DNA 21 is mounted at the position shown in FIG. 7, the sample DNA 21 is mixed in the sample by passing through the selective separation pre-filter 18 in advance. High molecular impurities can be removed, and accurate detection can be performed. Further, by mounting the selective separation filter 17 in the vicinity of the end of the closed channel, it is possible to prevent the DNA binding substance for separation 20 from being lost no matter how many times the electrophoresis is performed, as described in the first embodiment. . As described above, according to the genetic diagnostic apparatus and the genetic diagnostic method of the third embodiment, specific DNA is included in sample DNA extracted by PCR or the like among DNA extracted from cells, blood, and the like. Genetic diagnosis and determination can be performed by knowing the ratio of the presence of normal DNA, abnormal DNA, and noise DNA. In addition, impurities can be removed in advance by a pre-filter near the start point of electrophoresis, and a filter near the end point can prevent the DNA binding substance for separation from leaking out of the system. Diagnosis and judgment can be performed at low cost.
[0089]
(Embodiment 4)
A gene diagnosis apparatus and a gene diagnosis method according to Embodiment 4 of the present invention will be described with reference to the drawings. Note that the description of the same parts as those of the first embodiment, that is, the description of FIGS.
[0090]
FIG. 8 shows that the selective separation filter 17 described in the first embodiment in the fourth embodiment of the present invention is attached to both ends of the closed channel, and the prefilter 18 described in the third embodiment is near the starting point of the closed channel. FIG.
[0091]
By using these three filters, accurate measurement can be performed for a long period of time without losing the DNA binding substance for separation 20 and removing impurities even when electrophoresis is performed in either direction.
[0092]
As described above, according to the genetic diagnostic apparatus and the genetic diagnostic method of the fourth embodiment, specific DNA is included in sample DNA extracted by PCR or the like, among DNA extracted from cells, blood, and the like. Genetic diagnosis and determination can be performed by knowing the ratio of the presence of normal DNA, abnormal DNA, and noise DNA. Further, since the DNA binding substance for separation can be completely prevented from leaking out of the system, repetitive operations can be performed, and diagnosis / determination can be performed at low cost.
[0093]
As described above, according to the genetic diagnostic apparatus and the genetic diagnostic method of the fourth embodiment, specific DNA is included in sample DNA extracted by PCR or the like, among DNA extracted from cells, blood, and the like. Genetic diagnosis and determination can be performed by knowing the ratio of the presence of normal DNA, abnormal DNA, and noise DNA. In addition, impurities can be removed in advance by a pre-filter near the starting point of electrophoresis, and the filters at both ends can completely prevent the DNA binding substance for separation from leaking out of the system. And diagnosis / judgment can be performed at low cost.
[0094]
(Embodiment 5)
A gene diagnosis apparatus and a gene diagnosis method according to Embodiment 5 of the present invention will be described with reference to the drawings. Note that the description of the same parts as those of the first embodiment, that is, the description of FIGS.
[0095]
FIG. 9 shows that the selective separation filter 17 described in the first embodiment in the fifth embodiment of the present invention is mounted on both ends of the closed channel, and the pre-filter 18 described in the third embodiment is mounted on both ends of the closed channel. FIG. 5 is a view in which the selective separation filter is attached at a position closer to the start point and the end point than the attached selective separation filter 17.
[0096]
By using these four filters, the impurities can be removed without losing the DNA binding substance for separation 20 regardless of the direction of electrophoresis, so that accurate measurement can be performed for a long period of time. Further, by making the closed channel described in FIG. 9 detachable, after several times of electrophoresis, if the closed channel is turned over, the biased DNA binding substance for separation is repeatedly used without returning by a reverse current. Will be able to do things. Furthermore, since the impurities are removed by the pre-filter, it can be used for long-term continuous use.
[0097]
As described above, according to the genetic diagnostic apparatus and the genetic diagnostic method of the fifth embodiment, specific DNA is included in sample DNA extracted by PCR or the like, among DNA extracted from cells, blood, or the like. Genetic diagnosis and determination can be performed by knowing the ratio of the presence of normal DNA, abnormal DNA, and noise DNA. In addition, the DNA binding substance for separation can be completely prevented from leaking out of the system, and impurities can be removed by the prefilter, so that accurate measurement can be performed for a long time. Further, since the filter and the pre-filter are mounted on both sides, the electrophoresis operation can be continuously performed by turning over the sealed flow path, thereby enabling continuous diagnosis and judgment at low cost.
[0098]
(Embodiment 6)
A gene diagnosis apparatus and a gene diagnosis method according to Embodiment 6 of the present invention will be described with reference to the drawings. Note that the description of the same parts as those of the first embodiment, that is, the description of FIGS.
[0099]
FIG. 10 shows that the selective separation filter 17 described in the first embodiment in the sixth embodiment of the present invention is mounted at both ends of the closed channel, and transparent detection windows 19 for detection are provided at both ends of the closed channel. FIG. Here, the position of the selective separation filter 17 and the position of the detection window 19 may be on the outside, and both may be located near the end of the closed channel.
[0100]
By using two selective separation filters, repetitive electrophoresis can be performed without losing the DNA binding substance for separation 20 regardless of the direction of electrophoresis, and the detection windows 19 are provided at both ends. Thus, it is possible to perform repeated electrophoresis / detection simply by changing the cathode / anode of the electrode 12 and the electrode 13 without removing the closed channel portion.
[0101]
As described above, according to the genetic diagnostic apparatus and the genetic diagnostic method of the fifth embodiment, specific DNA is included in sample DNA extracted by PCR or the like, among DNA extracted from cells, blood, or the like. Genetic diagnosis and determination can be performed by knowing the ratio of the presence of normal DNA, abnormal DNA, and noise DNA. Further, since the filter and the pre-filter are mounted on both sides, it is possible to prevent the DNA binding substance for separation from leaking out of the system. Since the electrophoresis and detection can be performed repeatedly by simply changing the yin and yang of the electrode without removing, the continuous diagnosis and judgment can be performed at low cost without any trouble.
[0102]
【The invention's effect】
As described above, according to the present invention, it is possible to detect a gene abnormality of one or more bases in a short time, easily, and accurately, and to automate diagnosis at a small, light, inexpensive, and extremely low running cost. It is possible to provide a gene diagnosis device and a gene diagnosis method that can perform the method.
[0103]
It enables the repeated use of the DNA binding substance for separation, which is used to separate and measure the normal and abnormal forms of the gene to be determined, and removes chromosomal DNA and blood-derived noise substances having a large molecular weight. Therefore, it is possible to provide a gene diagnosis apparatus and a gene diagnosis method that can simplify the diagnosis apparatus, reduce the cost for diagnosis, and realize highly accurate determination.
[0104]
Further, according to the gene diagnostic apparatus as set forth in claim 1, a sealed flow path for separating DNA present in the apparatus and performing electrophoresis for discriminating whether the DNA of the sample is normal or abnormal. Must be filled with a DNA binding substance for separation. The DNA binding substance for separation used at this time is the material requiring the highest running cost. Eliminating the need for disposable DNA binding substances for separation eliminates various adjustments resulting from changes in the concentration and quality of the DNA binding substances for separation, in addition to cost reduction, thereby improving measurement accuracy and reducing measurement time. The benefits such as can be expected. Therefore, a low-cost and high-precision device can be realized at low running cost, and diagnosis can be easily automated.
[0105]
Further, it is possible to prevent the DNA binding substance for separation requiring the highest running cost from flowing out of the closed flow channel by electrophoresis and suction or extrusion of the closed flow channel material.
[0106]
According to the gene diagnostic apparatus of the second aspect, in addition to the effect of the first aspect, by removing impurities other than the object to be measured in advance, it is possible to improve the measurement accuracy and extend the life of the measurement.
[0107]
The gene diagnostic apparatus according to the third aspect can prevent the DNA binding substance for separation requiring the highest running cost from flowing out of the closed flow channel regardless of the direction in which the substance is moved.
[0108]
According to the gene diagnostic apparatus of the fourth aspect, in addition to the effect of the third aspect, by removing impurities other than the object to be measured in advance, it is possible to improve the measurement accuracy and extend the life of the measurement.
[0109]
According to the gene diagnostic apparatus of the fifth aspect, in addition to the effect of the third aspect, by removing impurities other than the object to be measured in advance, it is possible to improve the measurement accuracy and extend the life of the measurement.
[0110]
The gene diagnostic apparatus according to claim 6 has the effects of claims 2 to 5, and can continue measurement without replacement or movement of a closed flow channel, regardless of which direction electrophoresis is performed. It is.
[0111]
According to the gene diagnostic apparatus of the seventh aspect, in addition to the effect of the second aspect, since the measurement can be performed while replacing the closed flow path section, a multi-sample sample can be continuously measured. In addition, it is also possible to reduce the size of the device by performing measurement with one detection unit.
The genetic diagnostic apparatus according to the eighth aspect has the effects of the third to fifth aspects, and in addition, can perform measurement while replacing the closed flow path, so that multiple samples can be continuously measured. . In addition, it is also possible to reduce the size of the device by performing measurement with one detection unit.
In the gene diagnosis apparatus according to the ninth aspect, in addition to the effect of the eighth aspect, even if a sample to be measured contains impurities such as DNA or protein having a large molecular weight, it is detected as noise. It is possible to perform accurate measurement without any problem.
[0112]
In the gene diagnosis apparatus according to claim 10, a part of the flow path can be formed into a cartridge or a chip, and since there is no outflow of the DNA binding substance for separation by measurement, measurement can be performed a plurality of times. Become.
[0113]
According to the gene diagnosis apparatus described in claim 11, in addition to the effect of claim 10, even if impurities such as DNA or protein having a large molecular weight are mixed in a sample to be measured, the apparatus is detected as noise. It is possible to perform accurate measurement without any problem.
[0114]
According to the gene diagnostic apparatus of the present invention, in addition to the effect of the present invention, even if impurities such as DNA or protein having a large molecular weight are mixed in a sample to be measured, it is detected as noise. It is possible to perform accurate measurement without any problem.
[0115]
The gene diagnostic apparatus according to the thirteenth aspect enables efficient electrophoresis in addition to the effects of the tenth to twelfth aspects.
[0116]
The gene diagnostic apparatus according to claim 14 can prevent electroosmotic flow in addition to the effect of claim 13.
[0117]
The genetic diagnosis method according to claim 15 can improve the reproducibility of the judgment and reduce the cost.
[0118]
According to the method for gene diagnosis described in claim 16, in addition to the effect of claim 15, the life of a selective separation filter aimed at newly reducing measurement noise and capturing a DNA binding substance for separation can be extended. it can.
[0119]
The genetic diagnosis method according to claim 17 can improve the reproducibility of the determination and reduce the cost.
[0120]
According to the gene diagnosis method of the eighteenth aspect, the reproducibility of the determination can be improved and the cost can be reduced, and a member made into a cartridge and a chip can be used, and the degree of freedom is improved.
[0121]
[Sequence list]
Figure 2004093297
Figure 2004093297

[Brief description of the drawings]
FIG. 1 is an external view of a gene diagnosis apparatus according to Embodiment 1 of the present invention.
FIG. 2 is an external view of an open top cover of the gene diagnosis apparatus according to Embodiment 1 of the present invention.
FIG. 3 is a device configuration diagram of the gene diagnosis device according to the first embodiment of the present invention.
FIG. 4 is a main part of a control circuit of the gene diagnosis apparatus according to the first embodiment of the present invention.
FIG. 5 is a diagram showing a closed flow path provided with a selective separation filter according to the present invention for electrophoresing a sample DNA and a normal DNA using a DNA binding substance for separation in the genetic diagnostic apparatus according to the first embodiment of the present invention only near the end point; Structural drawing
FIG. 6 is a diagram in which the selective separation filter described in Embodiment 1 in Embodiment 2 of the present invention is attached to both ends of a closed channel.
FIG. 7 shows a state in which the selective separation filter described in the first embodiment in the third embodiment of the present invention is mounted near the end point of the closed channel, and is larger than the selective separation filters described in the first and second embodiments. Of a second selective separation filter (pre-filter) capable of capturing a large number of macromolecules attached to the uppermost stream
FIG. 8 shows that the selective separation filter described in the first embodiment in the fourth embodiment of the present invention is attached to both ends of the closed channel, and the prefilter described in the third embodiment is located near the starting point of the closed channel. Figure attached
FIG. 9 shows that the selective separation filter described in the first embodiment in the fifth embodiment of the present invention is mounted on both ends of the closed channel, and the pre-filter described in the third embodiment is mounted on both ends of the closed channel. Figure attached to the start and end points further than the selective separation filter
FIG. 10 shows that the selective separation filter described in the first embodiment in the sixth embodiment of the present invention is attached to both ends of a closed channel, and transparent detection windows for detection are provided at both ends of the closed channel. Figure
FIG. 11 is a conceptual diagram showing the relationship between a DNA binding substance for separation and a sample DNA in a closed flow channel of the genetic diagnostic device according to the first embodiment of the present invention.
FIG. 12 is a relationship diagram between elapsed time and absorbance.
[Explanation of symbols]
1 Power switch
2 Operation buttons
3 Display panel
4 Upper lid
5 Case
6 Electrophoresis section
7 Support
8 Control operation unit
9 Power supply box
9a Power supply section
10 Detector
10a D 2 lamp
10b slit
10c photodiode
15d preamplifier
10e A / D converter
11 Temperature controller
12, 13 electrodes
14,14c buffer
14a, 14b container
14d linear polymer
15 Capillary tube
16 Suction pump
17 Selective separation filter
18 Selective separation pre-filter
19 Detection window
20 DNA binding substances for separation
21 Sample DNA

Claims (18)

緩衝液を収容し第１電極が浸漬された第１容器と、
緩衝液を収容し第２電極が浸漬された第２容器と、
前記第１容器と前記第２容器間をリニアポリマーとＤＮＡ結合制御剤を含む緩衝液を充たして連絡した密閉流路と、
前記第２電極に正電位または負電位を印加するとともに前記第１電極に負電位または正電位を印加する電源部と、
前記電源部を制御して前記第２電極と前記第１電極間に電圧を印加する制御部と、
前記密閉流路に設けられ、内部を通過するＤＮＡの通過量を検出する検出部を備え、
前記密閉流路には、前記緩衝液の中に、
試料ＤＮＡに水素結合可能な塩基配列と高分子化合物とが結合し、結合力の差から前記試料ＤＮＡを正常ＤＮＡと異常ＤＮＡに分離する分離用ＤＮＡ結合物質と、
試料ＤＮＡとを備え、
前記密閉流路の少なくとも電気泳動方向側に、前記試料ＤＮＡを通過可能とし、前記分離用ＤＮＡ結合物質を通過させない選択性分離フィルターを備え、
前記電圧を印加することにより前記密閉流路内の試料ＤＮＡが電気泳動され、前記検出部が正常ＤＮＡ及び／または異常ＤＮＡの通過量を測定することを特徴とする遺伝子診断装置。A first container containing a buffer solution and immersed in the first electrode;
A second container containing a buffer solution and immersed in the second electrode;
A closed flow path between the first container and the second container, which is filled with a buffer solution containing a linear polymer and a DNA binding control agent to communicate with each other;
A power supply unit for applying a positive potential or a negative potential to the second electrode and applying a negative potential or a positive potential to the first electrode;
A control unit that controls the power supply unit to apply a voltage between the second electrode and the first electrode;
A detection unit is provided in the closed channel and detects a passing amount of DNA passing through the inside,
In the closed channel, in the buffer,
A DNA binding substance for separation in which a base sequence capable of hydrogen bonding to a sample DNA and a high molecular compound are bound, and the sample DNA is separated into a normal DNA and an abnormal DNA based on a difference in binding strength;
Sample DNA,
At least on the electrophoresis direction side of the closed flow path, a selective separation filter that allows the sample DNA to pass therethrough and does not allow the separation DNA binding substance to pass therethrough,
A genetic diagnostic device, wherein the sample DNA in the closed flow path is electrophoresed by applying the voltage, and the detection unit measures a passing amount of normal DNA and / or abnormal DNA. 前記密閉流路に、前記試料ＤＮＡおよび対照として用いる正常ＤＮＡを通過可能とし、より高分子の物質を通過させず、前記選択性分離フィルターよりも大量の高分子を捕捉することができる選択性分離プレフィルターを備え、前記選択性分離フィルターと、前記選択性分離プレフィルターとで、前記分離用ＤＮＡ結合物質を挟むことを特徴とする請求項１記載の遺伝子診断装置。Selective separation that allows the sample DNA and the normal DNA used as a control to pass through the closed channel, does not allow a higher-molecular substance to pass through, and can capture a larger amount of polymers than the selective separation filter. The gene diagnosis apparatus according to claim 1, further comprising a prefilter, wherein the DNA binding substance for separation is sandwiched between the selective separation filter and the selective separation prefilter. 緩衝液を収容し第１電極が浸漬された第１容器と、
緩衝液を収容し第２電極が浸漬された第２容器と、
前記第１容器と前記第２容器間をリニアポリマーとＤＮＡ結合制御剤を含む緩衝液を充たして連絡した密閉流路と、
前記第２電極に正電位または負電位を印加するとともに前記第１電極に負電位または正電位を印加する電源部と、
前記電源部を制御して前記第２電極と前記第１電極間に電圧を印加する制御部と、
前記密閉流路に設けられ、内部を通過するＤＮＡの通過量を検出する検出部を備え、
前記密閉流路には、前記緩衝液の中に、
試料ＤＮＡに水素結合可能な塩基配列と高分子化合物とが結合し、結合力の差から前記試料ＤＮＡを正常ＤＮＡと異常ＤＮＡに分離する分離用ＤＮＡ結合物質と、
試料ＤＮＡとを備え、
前記密閉流路の前記分離用ＤＮＡ結合物質を挟むように、前記試料ＤＮＡを通過可能とし、前記分離用ＤＮＡ結合物質を通過させない選択性分離フィルターを備え、
前記電圧を印加することにより前記密閉流路内の試料ＤＮＡが電気泳動され、前記検出部が正常ＤＮＡ及び／または異常ＤＮＡの通過量を測定することを特徴とする遺伝子診断装置。A first container containing a buffer solution and immersed in the first electrode;
A second container containing a buffer solution and immersed in the second electrode;
A closed flow path between the first container and the second container, which is filled with a buffer solution containing a linear polymer and a DNA binding control agent to communicate with each other;
A power supply unit for applying a positive potential or a negative potential to the second electrode and applying a negative potential or a positive potential to the first electrode;
A control unit that controls the power supply unit to apply a voltage between the second electrode and the first electrode;
A detection unit is provided in the closed channel and detects a passing amount of DNA passing through the inside,
In the closed channel, in the buffer,
A DNA binding substance for separation in which a base sequence capable of hydrogen bonding to a sample DNA and a high molecular compound are bound, and the sample DNA is separated into a normal DNA and an abnormal DNA based on a difference in binding strength;
Sample DNA,
A selective separation filter that allows the sample DNA to pass therethrough and does not allow the separation DNA binding substance to pass therethrough so as to sandwich the separation DNA binding substance in the closed channel,
A genetic diagnostic device, wherein the sample DNA in the closed flow path is electrophoresed by applying the voltage, and the detection unit measures a passing amount of normal DNA and / or abnormal DNA. 少なくとも前記密閉流路の電気泳動方向の逆側に位置する前記選択性分離フィルターよりも上流側に、前記試料ＤＮＡおよび対照として用いる正常ＤＮＡを通過可能とし、より高分子の物質を通過させず、前記選択性分離フィルターよりも大量の高分子を捕捉することができる選択性分離プレフィルターを備えたことを特徴とする請求項３記載の遺伝子診断装置。At least upstream of the selective separation filter located on the opposite side to the electrophoresis direction of the closed flow path, allowing the normal DNA used as the sample DNA and the control to pass, and not allowing the passage of a higher polymer substance, The gene diagnosis apparatus according to claim 3, further comprising a selective separation pre-filter capable of capturing a larger amount of a polymer than the selective separation filter. 前記密閉流路の前記選択性分離フィルターを挟むように、前記試料ＤＮＡおよび対照として用いる正常ＤＮＡを通過可能とし、より高分子の物質を通過させず、前記選択性分離フィルターよりも大量の高分子を捕捉することができる選択性分離プレフィルターを備えたことを特徴とする請求項３記載の遺伝子診断装置。The sample DNA and the normal DNA used as a control are allowed to pass through so as to sandwich the selective separation filter of the closed flow path, and do not allow a higher-molecular substance to pass therethrough. The gene diagnostic apparatus according to claim 3, further comprising a selective separation pre-filter capable of capturing a DNA. 前記密閉流路の内部を通過するＤＮＡの通過量を検出するための検出窓を少なくとも前記分離用ＤＮＡ結合物質を挟むように、それぞれ密閉流路の両側に備えたことを特徴とする請求項２〜５いずれか１項に記載の遺伝子診断装置。3. A detection window for detecting a passing amount of DNA passing through the inside of the closed flow path is provided on both sides of the closed flow path so as to sandwich at least the DNA binding substance for separation. The gene diagnostic apparatus according to any one of claims 1 to 5. 前記選択性分離フィルターと前記選択性分離プレフィルターとで挟まれた領域を含む密閉流路部分を着脱自在としたことを特徴とする請求項２記載の遺伝子診断装置。The gene diagnosis apparatus according to claim 2, wherein a sealed channel portion including an area sandwiched between the selective separation filter and the selective separation prefilter is detachable. 少なくとも前記選択性分離フィルターで挟まれた領域を含む密閉流路部分を着脱自在としたことを特徴とする請求項３〜５いずれか１項に記載の遺伝子診断装置。The gene diagnosis apparatus according to any one of claims 3 to 5, wherein at least a sealed channel portion including a region sandwiched by the selective separation filters is detachable. 前記選択性分離プレフィルターを含む密閉流路部分を着脱自在としたことを特徴とする請求項８記載の遺伝子診断装置。9. The genetic diagnostic apparatus according to claim 8, wherein a closed channel portion including the selective separation prefilter is detachable. 緩衝液を収容し第１電極が浸漬される第１容器と、緩衝液を収容し第２電極が浸漬される第２容器と、前記第１容器と前記第２容器間をリニアポリマーとＤＮＡ結合制御剤を含む緩衝液を充たして連絡する密閉流路と、前記第２電極に正電位または負電位を印加するとともに前記第１電極に負電位または正電位を印加する電源部と、前記電源部を制御して前記第２電極と前記第１電極間に電圧を印加する制御部と、前記密閉流路に設けられ、内部を通過するＤＮＡの通過量を検出する検出部を備え、
前記密閉流路に着脱自在な部材であって、前記密閉流路に装着した際に前記密閉流路の一部を構成する部材内に、試料ＤＮＡに水素結合可能な塩基配列と高分子化合物とが結合し、結合力の差から試料ＤＮＡを正常ＤＮＡと異常ＤＮＡに分離する分離用ＤＮＡ結合物質と、前記分離用ＤＮＡ結合物質を挟むように、試料ＤＮＡを通過可能とし、前記分離用ＤＮＡ結合物質を通過させない選択性分離フィルターを備え、
前記密閉流路に試料ＤＮＡを導入し、前記電圧を印加することにより前記密閉流路内の試料ＤＮＡが電気泳動され、前記検出部が正常ＤＮＡ及び／または異常ＤＮＡの通過量を測定することを特徴とする遺伝子診断装置。A first container containing a buffer solution and immersed in a first electrode; a second container containing a buffer solution and immersed in a second electrode; and a linear polymer and DNA bond between the first container and the second container. A closed flow path that is filled with a buffer containing a control agent to communicate therewith, a power supply unit that applies a positive potential or a negative potential to the second electrode and applies a negative potential or a positive potential to the first electrode, and the power supply unit A control unit that controls the second electrode and applies a voltage between the first electrode and a detection unit that is provided in the closed flow path and detects a passing amount of DNA passing through the inside,
A base sequence and a polymer compound that can be hydrogen-bonded to the sample DNA in a member that is detachably attached to the closed channel and forms a part of the closed channel when attached to the closed channel. Binds, and separates the sample DNA into normal DNA and abnormal DNA based on the difference in the binding strength, and allows the sample DNA to pass through the separating DNA binding substance so as to sandwich the separating DNA binding substance. Equipped with a selective separation filter that does not allow the passage of substances,
It is assumed that the sample DNA is introduced into the closed flow path and the voltage is applied, whereby the sample DNA in the closed flow path is electrophoresed, and the detection unit measures the passing amount of the normal DNA and / or the abnormal DNA. Characteristic genetic diagnostic device. 前記選択性分離フィルターの一方を前記試料ＤＮＡおよび対照として用いる正常ＤＮＡを通過可能とし、より高分子の物質を通過させず、前記選択性分離フィルターよりも大量の高分子を捕捉することができる選択性分離プレフィルターとしたことを特徴とする請求項１０記載の遺伝子診断装置。Selection that allows one of the selective separation filters to pass through the sample DNA and normal DNA used as a control, does not allow the passage of higher molecular substances, and captures a larger amount of macromolecules than the selective separation filter. The gene diagnostic apparatus according to claim 10, wherein the apparatus is a sex separation prefilter. 前記選択性分離フィルターを挟むように、前記試料ＤＮＡおよび対照として用いる正常ＤＮＡを通過可能とし、より高分子の物質を通過させず、前記選択性分離フィルターよりも大量の高分子を捕捉することができる選択性分離プレフィルターを備えたことを特徴とする請求項１０記載の遺伝子診断装置。By sandwiching the selective separation filter, the sample DNA and the normal DNA used as a control can be passed, and a larger amount of macromolecules than the selective separation filter can be captured without passing higher molecular substances. The genetic diagnostic device according to claim 10, further comprising a selectable separation pre-filter. 前記密閉流路をキャピラリー管としたことを特徴とする請求項１０〜１２いずれか１項に記載の遺伝子診断装置。The gene diagnosis apparatus according to any one of claims 10 to 12, wherein the closed channel is a capillary tube. 前記キャピラリー管内壁をアクリルアミドでコーティングしたことを特徴とする請求項１３記載の遺伝子診断装置。14. The apparatus according to claim 13, wherein an inner wall of the capillary tube is coated with acrylamide. 第１容器に緩衝液を収容して第１電極を浸漬し、第２容器に緩衝液を収容して第２電極を浸漬し、
前記第１容器と前記第２容器間を連絡する密閉流路であって、前記密閉流路の少なくとも電気泳動方向側に、試料ＤＮＡを通過可能とし、分離用ＤＮＡ結合物質を通過させない選択性分離フィルターと、試料ＤＮＡに水素結合可能な塩基配列と高分子化合物とが結合し、結合力の差から前記試料ＤＮＡを正常ＤＮＡと異常ＤＮＡに分離する分離用ＤＮＡ結合物質とを備えた密閉流路に、リニアポリマーとＤＮＡ結合制御剤を含む緩衝液を充たし、
前記密閉流路に試料ＤＮＡを導入し、
前記第１電極と前記第２電極間に電圧を印加し、前記密閉流路内の試料ＤＮＡを電気泳動させて、
正常ＤＮＡ及び／または異常ＤＮＡを検出することを特徴とする遺伝子診断方法。A first container containing a buffer and immersing the first electrode; a second container containing a buffer and immersing the second electrode;
A closed channel connecting the first container and the second container, wherein at least an electrophoresis direction side of the closed channel allows sample DNA to pass therethrough and does not allow a DNA binding substance for separation to pass therethrough; A sealed channel comprising a filter, a base sequence capable of hydrogen bonding to the sample DNA, and a polymer compound, and a separating DNA binding substance for separating the sample DNA into normal DNA and abnormal DNA based on a difference in binding force; Was filled with a buffer containing a linear polymer and a DNA binding control agent,
Introducing the sample DNA into the closed channel,
Applying a voltage between the first electrode and the second electrode, electrophoresing the sample DNA in the closed channel,
A gene diagnosis method comprising detecting normal DNA and / or abnormal DNA. 前記密閉流路は、前記試料ＤＮＡおよび対照として用いる正常ＤＮＡを通過可能とし、より高分子の物質を通過させず、前記選択性分離フィルターよりも大量の高分子を捕捉することができる選択性分離プレフィルターを備え、前記選択性分離フィルターと、前記選択性分離プレフィルターとで、前記分離用ＤＮＡ結合物質が挟まれていることを特徴とする請求項１５記載の遺伝子診断方法。The closed flow path allows the sample DNA and normal DNA used as a control to pass therethrough, does not allow the passage of higher-molecular substances, and can capture a larger amount of polymers than the selective separation filter. The method according to claim 15, further comprising a prefilter, wherein the DNA binding substance for separation is sandwiched between the selective separation filter and the selective separation prefilter. 第１容器に緩衝液を収容して第１電極を浸漬し、第２容器に緩衝液を収容して第２電極を浸漬し、
前記第１容器と前記第２容器間を連絡する密閉流路であって、試料ＤＮＡに水素結合可能な塩基配列と高分子化合物とが結合し、結合力の差から前記試料ＤＮＡを正常ＤＮＡと異常ＤＮＡに分離する分離用ＤＮＡ結合物質と、前記分離用ＤＮＡ結合物質を挟むように、前記試料ＤＮＡを通過可能とし、前記分離用ＤＮＡ結合物質を通過させない選択性分離フィルターとを備えた密閉流路に、リニアポリマーとＤＮＡ結合制御剤を含む緩衝液を充たし、
前記密閉流路に試料ＤＮＡを導入し、
前記第１電極と前記第２電極間に電圧を印加し、前記密閉流路内の試料ＤＮＡを電気泳動させて、
正常ＤＮＡ及び／または異常ＤＮＡを検出することを特徴とする遺伝子診断方法。A first container containing a buffer and immersing the first electrode; a second container containing a buffer and immersing the second electrode;
A sealed flow path communicating between the first container and the second container, wherein a base sequence capable of hydrogen bonding to the sample DNA and a polymer compound bind to each other, and the difference between the binding forces makes the sample DNA a normal DNA. A sealed flow comprising: a DNA binding substance for separation to be separated into abnormal DNA; and a selective separation filter that allows the sample DNA to pass through and sandwiches the DNA binding substance for separation so as to sandwich the DNA binding substance for separation. The road is filled with a buffer containing a linear polymer and a DNA binding control agent,
Introducing the sample DNA into the closed channel,
Applying a voltage between the first electrode and the second electrode, electrophoresing the sample DNA in the closed channel,
A gene diagnosis method comprising detecting normal DNA and / or abnormal DNA. 第１容器に緩衝液を収容して第１電極を浸漬し、第２容器に緩衝液を収容して第２電極を浸漬し、
前記第１容器と前記第２容器間を連絡した密閉流路であり、前記密閉流路の一部を構成する着脱自在な部材が装着された密閉流路であって、試料ＤＮＡに水素結合可能な塩基配列と高分子化合物とが結合し、結合力の差から試料ＤＮＡを正常ＤＮＡと異常ＤＮＡに分離する分離用ＤＮＡ結合物質と、前記分離用ＤＮＡ結合物質を挟むように、試料ＤＮＡを通過可能とし、前記分離用ＤＮＡ結合物質を通過させない選択性分離フィルターとを備えた密閉流路に、リニアポリマーとＤＮＡ結合制御剤を含む緩衝液を充たし、
前記密閉流路に試料ＤＮＡを導入し、
前記第１電極と前記第２電極間に電圧を印加し、前記密閉流路内の試料ＤＮＡを電気泳動させて、
正常ＤＮＡ及び／または異常ＤＮＡを検出することを特徴とする遺伝子診断方法。A first container containing a buffer and immersing the first electrode; a second container containing a buffer and immersing the second electrode;
A sealed flow passage communicating between the first container and the second container, the closed flow passage having a detachable member constituting a part of the closed flow passage, and capable of hydrogen bonding to the sample DNA; A DNA binding substance that separates a sample DNA into a normal DNA and an abnormal DNA based on a difference in binding strength between a base sequence and a polymer compound, and passes through the sample DNA so as to sandwich the DNA binding substance for separation. Enabled, and filled a buffer containing a linear polymer and a DNA binding control agent in a closed channel provided with a selective separation filter that does not allow the DNA binding substance for separation to pass therethrough,
Introducing the sample DNA into the closed channel,
Applying a voltage between the first electrode and the second electrode, electrophoresing the sample DNA in the closed channel,
A gene diagnosis method comprising detecting normal DNA and / or abnormal DNA.
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