JP2005006504A

JP2005006504A - Apparatus for amplifying chemically modified dna fragment and method for amplifying dna fragment with the same apparatus

Info

Publication number: JP2005006504A
Application number: JP2003170815A
Authority: JP
Inventors: Hisashi Hagiwara; 久萩原; Toshiji Shiraishi; 利治白石; Yoshiyuki Sakaki; 佳之榊
Original assignee: Aida Engineering Ltd
Current assignee: Aida Engineering Ltd
Priority date: 2003-06-16
Filing date: 2003-06-16
Publication date: 2005-01-13
Anticipated expiration: 2023-06-16
Also published as: JP4069024B2

Abstract

<P>PROBLEM TO BE SOLVED: To provide a small-sized apparatus for amplifying a DNA fragment by chemical modification. <P>SOLUTION: The apparatus for amplifying a chemically modified DNA fragment is characterized as follows. The apparatus is equipped with a first channel extended in a prescribed direction for carrying out an amplification reaction of the DNA fragment by the chemical modification, a second channel extended in the prescribed direction for feeding an alkali modifying reagent solution, a third channel extended in the prescribed direction for feeding a reagent solution for a neutralization reaction, a plurality of fourth channels for weighing out a prescribed amount of the alkali modifying reagent solution and a plurality of fifth channels for weighing out a prescribed amount of the reagent solution for the neutralization reaction. The fourth channels have sixth channels which make the fourth channels communicate with the first channel and are narrower than the channels. The fifth channels have seventh channels which make the fifth channels communicate with the first channel and are narrower than the channels. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

【０００１】
【発明の属する技術分野】
本発明はＤＮＡの増幅装置に関する。更に詳細には、本発明は化学変性によりＤＮＡを増幅するための小型装置に関する。
【０００２】
【従来の技術】
最近、ヒトや動植物などの生命現象を遺伝子レベルで解明するための研究が活発に続けられている。このような研究の中心は遺伝子操作技術又は遺伝子組換え技術の開発に向けられている。ヒトや動植物などの生物の遺伝情報は遺伝子ＤＮＡに格納されている。従って、このＤＮＡに格納されている塩基配列が遺伝情報の本質である。このような塩基配列の決定を行うために、被分析サンプルのＤＮＡを多量に増幅する必要性が生じた。
【０００３】
特定のＤＮＡ領域を挟んだ２種類のプライマーとＤＮＡ合成酵素によるＤＮＡ合成反応を繰り返すことにより、その特定領域を数十万倍に増幅する方法としてポリメラーゼ連鎖反応（ＰＣＲ（ＰｏｌｙｍｅｒａｓｅＣｈａｉｎＲｅａｃｔｉｏｎ））法が１９８５年にマリス（Ｍｕｌｌｉｓ）らにより開発された。
【０００４】
この方法は、その後、耐熱性酵素Ｔａｑポリメラーゼを利用した方法に改良されたことで、効率の良いＰＣＲ法として確立された。ＰＣＲ法の原理は、例えば、非特許文献１に詳細に説明されている。このＰＣＲ法は基本的に、▲１▼変性ステップ、▲２▼アニーリングステップ及び▲３▼伸張ステップの３つのステップを順に繰り返すことにより特定のＤＮＡのコピーを増やしていく。通常サイクルを２５〜３０回繰り返す。この反応によってｎサイクル後には理論的には約２^ｎ倍に増幅されることになる。しかし、増幅が進むとともに、増幅物同士のアニーリングが起こるため、反応が頭打ちになり、実際は数十万倍程度の増幅となる。
【０００５】
実際的には、ＰＣＲ法は、▲１▼先ず、９４℃で７分間加熱し、２本鎖ＤＮＡを熱変性により一本鎖に変性し、その後、Ｔａｑポリメラーゼを加える。▲２▼続いて、５４℃、３０秒間で一本鎖になったＤＮＡとプライマーとのアニーリングを行う。▲３▼次に、７２℃、１分間でＴａｑポリメラーゼによりＤＮＡ鎖を伸長させる。▲４▼次に再びＤＮＡ加熱変性を行うが、２回目以降のＤＮＡ加熱変性は９４℃で１分間で行う。この後、以上のステップ▲２▼〜▲３▼〜▲４▼のサイクルを２９回繰り返すが、最後のサイクルではステップ▲３▼を７２℃、７分間としＰＣＲを終了する。
【０００６】
しかし、加熱変性式のＰＣＲ法では、９４℃、５４℃及び７２℃の３種類の温度を繰り返し必要とするので、温度制御が難しく、また、マイクロチャネル内には数ピコリットルという量の試薬及びＤＮＡ断片溶液が含まれていることから、局部的な加熱や冷却によりチャネル内を勝手に移動してしまう可能性も考えられる。更に、チップの特定部位を各ＰＣＲステップの温度に対応させるには幾つかの方式が考えられているが、温度精度の点、総反応時間の点、反応効率に疑問がある。これは、現在扱われているＰＣＲのように５０℃〜７０℃の一点のアニーリング温度が正確に試料に伝達されているか疑問だからである。特に、９０℃〜９８℃の一点に解離（ディネーチャー）作業からアニーリング作業は連続工程であり、サンプルにより異なる温度領域がそれぞれ干渉してはならないからである。
【０００７】
加熱変性式のＰＣＲ法の別法として、化学変性式のＰＣＲ法が提案されている。二本鎖ＤＮＡは周囲のｐＨを１０以上に傾けると解離し、一本鎖に変性する。この方法はアルカリ変性と呼ばれ、当業者に公知である。次に、ｐＨを中性域の７近くに戻すと再対合（アニーリング）する（中和反応）。この中和再対合も当業者に公知である。この原理を３０回繰り返せば、現在行われている加熱変性式のＤＮＡ増幅方法（ＰＣＲ法）と同等のことができると考えられる。しかし、一般的には正確な試薬の量を秤量して添加していく手間などを考えると、小型の自動装置化に不向きであり、実際には装置化されていない。
【０００８】
【非特許文献１】
服巻保行、ＰＣＲ法の原理と実際、実験医学第９巻第１０号「ＤＮＡ診断と疾患の分子生物学」、１９９１（２８−３４）
【０００９】
【発明が解決しようとする課題】
従って、本発明の目的は化学変性によりＤＮＡ断片を増幅するための小型装置を提供することである。
【００１０】
【課題を解決するための手段】
前記課題は、化学変性によりＤＮＡ断片を増幅するための装置であって、化学変性によりＤＮＡ断片の増幅反応を行わせるための所定の方向に延長される第１の流路と、前記第一の流路の一方の側に並列に配置された、アルカリ変性試薬溶液を供給するための所定の方向に延長される第二の流路と、前記前記第一の流路の他方の側に並列に配置された、中和反応試薬溶液を供給するための所定の方向に延長される第三の流路を有し、前記第二の流路は該流路壁に開口する、所定量のアルカリ変性試薬溶液を秤取するための複数本の第四の流路を有し、前記第三の流路は該流路壁に開口する、所定量の中和反応試薬溶液を秤取するための複数本の第五の流路を有し、前記第四の流路は、該流路と前記第一の流路とを連通するこれらの流路よりも細い第六の流路を有し、かつ、前記第五の流路は該流路と前記第一の流路とを連通するこれらの流路よりも細い第七の流路を有し、前記第六の流路と第七の流路とは前記第一の流路の両側に相対峙しないように離間されて互い違いに配置されていることを特徴とする化学変性ＤＮＡ断片増幅装置により解決される。
【００１１】
【発明の実施の形態】
以下、添付の図面を参照しながら本発明による化学変性ＤＮＡ断片増幅装置を具体的に説明する。
【００１２】
図１は本発明による化学変性ＤＮＡ断片増幅装置の一例の部分概要平面図である。図１に示されるように、本発明の化学変性ＤＮＡ断片増幅装置１は基本的に、化学変性反応を行わせるための所定の方向に延長される第一の流路３と、この第一の流路３の一方の側に並列に配置された、アルカリ変性試薬を供給するための所定の方向に延長される第二の流路５と、この前記第一の流路の他方の側に並列に配置された、中和反応試薬を供給するための所定の方向に延長される第三の流路７を有する。第二の流路５は該流路壁に開口する、所定量のアルカリ変性試薬を秤取するための複数本の第四の流路９を有する。第四の流路９は例えば、３０本配設することができる。この本数はＰＣＲ増幅反応の回数と一致させることが好ましい。また、第三の流路７も、該流路壁に開口する、所定量の中和反応試薬を秤取するための複数本の第五の流路１１を有する。第五の流路１１の本数は前記第四の流路９の本数と同一であることが好ましい。アルカリ変性試薬秤取用の第四の流路９は第六の流路１３により前記第一の流路３と連通されている。第六の流路１３は前記第一の流路３，第二の流路５及び第四の流路９の何れよりも細く、濡れ難い又は相対的に毛管引力が働き難い性質を有することが好ましい。同様に、中和反応試薬秤取用の第五の流路１１は第七の流路１５により前記第一の流路３と連通されている。第七の流路１５は前記第一の流路３，第三の流路７及び第五の流路１１の何れよりも細く、濡れ難い又は相対的に毛管引力が働き難い性質を有することが好ましい。
【００１３】
第一の流路３、第二の流路５及び第三の流路７はそれぞれ、それらの両端に、流路内に液体を出し入れするための大気に向かって開放されたポート１７ａと１７ｂ、１９ａと１９ｂ及び２１ａと２１ｂを有する。例えば、ポート１７ａ、１９ａ及び２１ａには流路内の液体を圧送するための加圧手段（図示されていない）を接続することができる。
【００１４】
第六の流路１３と第七の流路１５は、第一の流路の両側に相対峙しないように離間されて互い違いに配置することが好ましい。これは、第四の流路９内のアルカリ変性試薬が第六の流路１３を介して第１の流路３内に押し出されることにより形成される液滴と、第五の流路１１内の中和反応試薬が第七の流路１５を介して第１の流路３内に押し出されることにより形成される液滴とが接触して不用意に反応してしまうことを防止するためである。従って、第六の流路１３と第七の流路１５との間隔はこれらの流路から押し出されて形成される液滴のサイズ及び化学変性ＤＮＡ断片増幅反応の進行につれて徐々に増量していく反応液の容量を考慮して決定することができる。
【００１５】
図２は図１におけるＩＩ−ＩＩ線に沿った断面図である。図示されているように、本発明の化学変性ＤＮＡ断片増幅装置１は、基本的に、高分子材料（例えば、ポリジメチルシロキサン（ＰＤＭＳ）、ポリメチルメタクリレート（ＰＭＭＡ）、シリコン樹脂、）、ガラスなど素材から形成された上側基板２３と下側基板２５の２枚の平板状基板を重ねて接合させることにより構成することができる。図２（ａ）では、第一の流路３，第二の流路５、第三の流路７、第四の流路９及び第五の流路１１と各ポート１７ａ〜２１ｂが上側基板２３内に形成され、第六の流路１３及び第七の流路１５が下側基板２５内に形成されている。これにより、各流路は封止され、ポートだけが大気に開放されている。別法として、図２（ｂ）に示されるように、全ての流路及びポートを上側基板２３内に形成し、これら流路を下側基板２５で封止することもできる。基板２３及び２５は透明でも、あるいは不透明でもよい。
【００１６】
第一の流路３，第二の流路５、第三の流路７、第四の流路９及び第五の流路１１の深さ（又は高さ）は特に限定されない。一般的に、１〜３００μｍの範囲内で適宜選択することができる。また、その幅も１００〜３００μｍの範囲内で適宜選択することができる。第一の流路３を第二の流路５及び第三の流路７よりも幅広にすることもできる。第四の流路９及び第五の流路１１の高さ及び幅はこの流路で秤取すべきアルカリ変性試薬量及び中和液量を考慮して決定することができる。第六の流路１３及び第七の流路１５の幅は数μｍ〜数十μｍの範囲内であることが好ましい。
【００１７】
基板内に流路及びポートを形成する技法としては例えば、光リソグラフィー法により鋳型を作製し、この鋳型からレプリカを作製することからなる、当業者に公知慣用の技法を使用することができる。その他、金型注型などの技術を用いて作製することもできる。
【００１８】
図３は第四の流路９及び第五の流路１１により所定量のアルカリ変性試薬又は中和液が秤取される原理を説明する図である。ここでは、説明の便宜上、第三の流路７と第五の流路で中和液を秤取する態様について説明する。ポート２１ａ（図１参照）から中和液を注入し、ポート２１ｂ（図１参照）に達するまで第三の流路７内全体に中和液を満たさせる。第三の流路７に中和液１００を導入した場合、第三の流路７の流路壁ａａにおいて開口する第五の流路１１の開口部ｃ１を介して、中和液１００を第五の流路１１に導入することができる。第三の流路７および第五の流路１１が濡れやすい流路壁を有する場合は、第三の流路７より第五の流路１１を細くしておけば、中和液１００はより強い毛管引力により第三の流路７の流路壁ａａに開口する第五の流路１１の開口部ｃ１を介して第五の流路１１に自発的に引き込まれる。第三の流路７および第五の流路１１が濡れにくい流路壁を有する場合は、ポート２１ａより中和液１００に適当な圧力を付与することにより第五の流路１１に中和液１００を導入することができる（図３（ａ）参照）。
【００１９】
この際、第七の流路１５（ｃ２とｄ１の間の流路区間）の第五の流路１１側の端面ｃ２まで到達した中和液１００は、濡れにくい流路壁を有する第七の流路１５の毛管斥力によってせき止められ、第七の流路１５に入り込むことはない。第五の流路１１が濡れにくい流路壁の場合にも、第五の流路１１の毛管斥力よりも第七の流路１５の毛管斥力の方が強いため、第七の流路１５に中和液１００が入り込むことはない（図３（ａ）参照）。
【００２０】
続いて、第三の流路７内に残留する中和液１００を、例えば、ポート２１ａ、２１ｂ（図１参照）の両端部に適当な圧力差を生起させてより低圧側に移動させるなどし、第三の流路７内から取り除く。この際、第五の流路１１内の中和液１００は、通常、第三の流路７内に戻って入り込むようなことはない。その結果、第五の流路１１内の中和液１００の両端面たる端面１００ａと端面１００ｂとが、第五の流路１１の開口部ｃ１ならびに第三の流路７に繋がる側の端面ｃ２に位置するようになり、第五の流路１１のｃ１とｃ２の区間の容積に応じた体積の中和液１００の秤取が可能となる（図３（ｂ）参照）。
【００２１】
さらに、第五の流路１１内に秤取された中和液１００は、第三の流路７の圧力が第一の流路３の圧力より僅かに大きくなるように、ポート２１ｂを閉じてポート２１ａから圧力を加えるなどして、第七の流路１５およびその開口部ｄ１を介して、第一の流路３内に導入することができる。この際、中和液１００を第一の流路３内に全て押し出してしまわず、第七の流路１５内に一部残すと中和液１００を第七の流路１５の位置に留めておくことができる（図３（ｃ）参照）。
【００２２】
図４は本発明の化学変性ＤＮＡ断片増幅装置１によりＤＮＡ断片が増幅される原理を説明する概要図である。ポート１９ａ（図１参照）から反応液２００を第一の流路３内に注入し、該流路内を矢線の方向に移動させる。反応液２００は例えば、ＰＣＲ用試薬、ＤＮＡサンプル、プライマー、酵素などが混合されている。反応液２００は第四の流路９で秤取され第六の流路１３を介して第１の流路３内に押し出されたアルカリ変性試薬液３００と接触し、ｐＨ１０以上となりサンプルのＤＮＡ２本鎖は解離する。このｐＨ１０以上に変性された反応液２００は更に移動され、中和液１００と接触し、ｐＨ７付近に戻されアニーリング（再対合）し、ＤＮＡサンプルが増幅する。本発明の化学変性ＤＮＡ断片増幅装置１により、このアルカリ変性と中和を所定回数（例えば、３０回）繰り返すことによりＤＮＡサンプルは次々と増幅される。増幅反応生成物は第一の流路３の他端にあるポート１９ｂ（図１参照）から取り出すことができる。
【００２３】
本発明の化学変性ＤＮＡ断片増幅装置１によりＤＮＡサンプルを効率的に増幅するために、本発明の化学変性ＤＮＡ断片増幅装置１をアニーリング反応に好適な５４℃付近に保つことが好ましい。このような目的のために、例えば、加熱手段（図示されていない）を下側基板２５側に配置し、５４℃付近に温度制御するか、又は、本発明の化学変性ＤＮＡ断片増幅装置１を５４℃付近の温度に維持された恒温槽内に配置して増幅反応を実施させる。
【００２４】
アルカリ変性反応と中和反応が交互に進行するにつれて反応液２００の容量が徐々に増大する。従って、図５に示されるように、第一の流路３の幅が反応液２００の進行方向に向けて逓増するように構成することもできる。
【００２５】
本発明の化学変性ＤＮＡ断片増幅装置１は図１に示されるような矩形状（例えば、長方形状）に造形することもできるが、図６（ａ）に示されるような渦巻き状又は図６（ｂ）に示されるような同心円状などの形状にして円盤上に造形することもできる。
【００２６】
以上、本発明の化学変性ＤＮＡ断片増幅装置１について好ましい実施態様を挙げて具体的に説明してきたが、本発明は前記実施態様にのみ限定されるものではない。例えば、第一の流路３の反応液注入ポート１９ａを図７に示されるように変更することもできる。この実施態様では、ポート２３ａとポート２３ｂとを繋ぐ第八の流路２５が存在し、この第八の流路２５は該流路壁に開口する、所定量の反応液を秤取するための第九の流路２７を有する。この第九の流路２７は細い第十の流路２９を介して第１の流路３と連通されている。このような構成により、常に一定量の反応液を容易に第一の流路３内に送り出すことができる。
【００２７】
【実施例】
以下、実施例により本発明の化学変性ＤＮＡ断片増幅装置１によるＤＮＡ増幅反応を具体的に例証する。
【００２８】
実施例１
図１に示されるような化学変性ＤＮＡ断片増幅装置１を用いてＤＮＡ増幅反応を実施した。図１の装置は、アルカリ変性試薬を秤取する第四の流路及び中和液を秤取する第五の流路をそれぞれ３０本有していた。各第四の流路９及び第五の流路の容量はそれぞれ４０ｐｌ（ピコリットル）であった。化学変性ＤＮＡ断片増幅装置１の下部に加熱手段を配置し、装置１を５４℃の温度に維持した。
先ず、ポート１７ａからアルカリ変性試薬として０．１Ｍの水酸化ナトリウム（ＮａＯＨ）溶液を第二の流路５に注入し、各第四の流路９内にアルカリ変性試薬液を充填した。第二の流路５内の余ったアルカリ変性試薬はポート１７ｂから排出した。その後、ポート１７ｂを閉じ、ポート１７ａから空気圧を利用して各第四の流路９内のアルカリ変性試薬溶液を第六の流路１３を介して第１の流路３内に押し出した。この際、第六の流路１３内にアルカリ変性試薬溶液が一部残存するようにした。これにより、アルカリ変性試薬溶液を第六の流路１３の位置に定置させておくことができる。
同様に、ポート２１ａから中和液として０．１Ｍの塩酸溶液を第三の流路７に注入し、各第五の流路１１内に中和液を充填した。第三の流路７内の余った中和液はポート２１ｂから排出した。その後、ポート２１ｂを閉じ、ポート２１ａから空気圧を利用して各第五の流路１１内の中和液を第七の流路１５を介して第１の流路３内に押し出した。この際、第七の流路１５内にアルカリ変性試薬溶液が一部残存するようにした。これにより、中和液を第七の流路１５の位置に定置させておくことができる。
次いで、５０ミリモルのＫＣｌ、１０ミリモルのトリス−ＨＣｌ（ｐＨ８．３）、１．５ミリモルのＭｇＣｌ_２、０．１％トライトンＸ−１００、４種類のｄＮＴＰ（デオキシリボヌクレオチド三燐酸）各２００マイクロモル、５０〜１０ｎｇのゲノムＤＮＡ、ＴａｑＤＮＡポリメラーゼ０．２単位と２種類のプライマー各１０ピコモルからなる組成を有する反応液を２ｎｌ（ナノリットル）使用し、この反応液をポート１９ａから第１の流路３の移動開始点に注入した。その後、ポート１９ａから空気圧を利用して反応液を移動前進させ、最初の第六の流路１３の位置に定置されたアルカリ変性試薬溶液と接触させることによりＤＮＡを変性させ、ＤＮＡ相補鎖を分離しランダム構造に変化させた。次いで、この変性された反応液を更に前進させ、最初の第七の流路１５の位置に定置された中和液と接触させることにより、分離していた相補鎖を再対合（アニーリング）させ元の２本鎖に戻してＤＮＡを増幅させた。この増幅された反応液を次ぎのアルカリ変性試薬溶液と接触させて再び変性させ、次いで、中和して再対合させ、以後この変性−中和の反応を最後の３０番目の第六の流路１３及び第七の流路１５まで繰り返した。増幅反応生成物をポート１９ｂから取り出した。この生成物を電気泳動分析したところ、分析可能な量まで十分に増幅されていることが確認された。
【００２９】
【発明の効果】
以上説明したように、従来の加熱変性式のＰＣＲ法では、９４℃、５４℃及び７２℃の３種類の温度を繰り返し必要とし、温度制御が非常に煩雑かつ困難であったが、本発明の化学変性ＤＮＡ断片増幅装置によれば、アニーリングに必要な温度だけでＤＮＡサンプルを増幅することができる。その結果、本発明の化学変性ＤＮＡ断片増幅装置は従来の加熱変性式ＤＮＡ断片増幅装置に比べてサイズが著しく小型化できると共に、同時に、分析時間を短縮することもできる。
【図面の簡単な説明】
【図１】本発明の化学変性ＤＮＡ断片増幅装置の一例の部分平面図である。
【図２】図１におけるＩＩ−ＩＩ線に沿った断面図である。
【図３】図１に示した装置でアルカリ変性試薬溶液又は中和液を秤取する原理を示す概念図である。
【図４】図１に示した装置で化学変性ＤＮＡ断片増幅反応を実施する状態を示す概念図である。
【図５】本発明の化学変性ＤＮＡ断片増幅装置の別の例の部分平面図である。
【図６】本発明の化学変性ＤＮＡ断片増幅装置の更に別の例の概要平面図である。
【図７】図１に示した装置を部分的に変更した例の部分平面図である。
【符号の説明】
１本発明の化学変性ＤＮＡ断片増幅装置
３第一の流路
５第二の流路
７第三の流路
９第四の流路
１１第五の流路
１３第六の流路
１５第七の流路
１７ａ，１７ｂ，１９ａ，１９ｂポート
２１ａ，２１ｂ，２３ａ，２３ｂポート
２５第八の流路
２７第九の流路
２９第十の流路
１００中和液
２００反応液
３００アルカリ変性試薬溶液[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a DNA amplification apparatus. More particularly, the present invention relates to a small apparatus for amplifying DNA by chemical denaturation.
[0002]
[Prior art]
Recently, research to elucidate life phenomena such as humans and animals and plants at the gene level has been actively carried out. The center of such research is directed to the development of gene manipulation techniques or gene recombination techniques. Genetic information of organisms such as humans and animals and plants is stored in genetic DNA. Therefore, the base sequence stored in this DNA is the essence of genetic information. In order to determine such a base sequence, it has become necessary to amplify a large amount of DNA of the sample to be analyzed.
[0003]
As a method for amplifying a specific region several hundred thousand times by repeating a DNA synthesis reaction with two kinds of primers sandwiching a specific DNA region and a DNA synthase, a polymerase chain reaction (PCR (Polymerase Chain Reaction)) method is used. Developed in 1985 by Mullis et al.
[0004]
This method was subsequently established as an efficient PCR method by improving the method using a thermostable enzyme Taq polymerase. The principle of the PCR method is described in detail in Non-Patent Document 1, for example. This PCR method basically increases the number of copies of a specific DNA by sequentially repeating three steps: (1) denaturation step, (2) annealing step, and (3) extension step. Repeat normal cycle 25-30 times. This reaction results in a theoretical amplification of about ²ⁿ times after n cycles. However, as amplification progresses, annealing between amplified products occurs, so that the reaction reaches its peak, and the amplification is actually several hundred thousand times.
[0005]
Actually, the PCR method is as follows: (1) First, heat at 94 ° C. for 7 minutes to denature double-stranded DNA into single strands by thermal denaturation, and then add Taq polymerase. {Circle around (2)} Subsequently, annealing is performed on the DNA and the primer that have become a single strand at 54 ° C. for 30 seconds. (3) Next, the DNA strand is elongated with Taq polymerase at 72 ° C. for 1 minute. {Circle around (4)} Next, DNA heat denaturation is carried out again, but the second and subsequent DNA heat denaturation is carried out at 94 ° C. for 1 minute. Thereafter, the above steps (2) to (3) to (4) are repeated 29 times. In the last cycle, step (3) is set at 72 ° C. for 7 minutes, and the PCR is terminated.
[0006]
However, the heat-denaturing PCR method requires three kinds of temperatures of 94 ° C., 54 ° C. and 72 ° C. repeatedly, so that the temperature control is difficult, and there are several picoliters of reagent and Since the DNA fragment solution is contained, there is a possibility that it may move inside the channel by local heating or cooling. Furthermore, several methods have been considered to make the specific part of the chip correspond to the temperature of each PCR step, but there are doubts about the temperature accuracy, the total reaction time, and the reaction efficiency. This is because it is doubtful that a single annealing temperature of 50 ° C. to 70 ° C. is accurately transmitted to the sample as in currently handled PCR. In particular, the process from dissociation (denier) to annealing at one point of 90 ° C. to 98 ° C. is a continuous process, and different temperature regions should not interfere with each other.
[0007]
As an alternative to the heat-denatured PCR method, a chemically-denatured PCR method has been proposed. Double-stranded DNA dissociates and denatures into single strands when the surrounding pH is tilted to 10 or more. This method is called alkali modification and is known to those skilled in the art. Next, when the pH is returned to near 7 in the neutral range, re-pairing (annealing) occurs (neutralization reaction). This neutralization re-pairing is also known to those skilled in the art. It is considered that if this principle is repeated 30 times, it can be equivalent to the heat-denaturing DNA amplification method (PCR method) currently performed. However, in general, considering the trouble of weighing and adding an accurate amount of reagent, it is not suitable for making a small automatic device, and is not actually made into a device.
[0008]
[Non-Patent Document 1]
Y. Hatamaki, Principles and Practice of PCR Method, Experimental Medicine Vol. 9, No. 10, "DNA diagnosis and molecular biology of diseases", 1991 (28-34)
[0009]
[Problems to be solved by the invention]
Accordingly, an object of the present invention is to provide a small apparatus for amplifying a DNA fragment by chemical denaturation.
[0010]
[Means for Solving the Problems]
The object is an apparatus for amplifying a DNA fragment by chemical denaturation, the first flow path extending in a predetermined direction for causing an amplification reaction of the DNA fragment by chemical denaturation, and the first A second flow channel arranged in parallel on one side of the flow channel and extending in a predetermined direction for supplying the alkaline denaturing reagent solution; and in parallel with the other side of the first flow channel A predetermined amount of alkali denaturation having a third flow path arranged in a predetermined direction for supplying the neutralization reaction reagent solution, the second flow path opening in the flow path wall; A plurality of fourth channels for weighing the reagent solution, the third channel opening to the channel wall, and a plurality of units for weighing a predetermined amount of the neutralization reaction reagent solution A fourth flow path, and the fourth flow path is more than these flow paths communicating the flow path and the first flow path. A sixth flow path, and the fifth flow path has a seventh flow path that is narrower than these flow paths that connect the flow path and the first flow path, The sixth flow path and the seventh flow path are solved by a chemically denatured DNA fragment amplification device, wherein the sixth flow path and the seventh flow path are spaced apart from each other so as not to be opposed to both sides of the first flow path. The
[0011]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, a chemically modified DNA fragment amplification apparatus according to the present invention will be described in detail with reference to the accompanying drawings.
[0012]
FIG. 1 is a partial schematic plan view of an example of a chemically modified DNA fragment amplification apparatus according to the present invention. As shown in FIG. 1, the chemically modified DNA fragment amplification apparatus 1 of the present invention basically includes a first flow path 3 extended in a predetermined direction for causing a chemical denaturation reaction, A second flow path 5 arranged in parallel on one side of the flow path 3 and extending in a predetermined direction for supplying an alkali denaturing reagent, and parallel to the other side of the first flow path And a third flow path 7 extending in a predetermined direction for supplying the neutralization reaction reagent. The second flow path 5 has a plurality of fourth flow paths 9 that open to the flow path wall and for weighing a predetermined amount of the alkali-denaturing reagent. For example, 30 fourth channels 9 can be provided. This number is preferably matched with the number of PCR amplification reactions. The third flow path 7 also has a plurality of fifth flow paths 11 that weigh out a predetermined amount of the neutralization reaction reagent that opens to the flow path wall. The number of the fifth flow paths 11 is preferably the same as the number of the fourth flow paths 9. The fourth flow path 9 for measuring the alkali denaturing reagent is communicated with the first flow path 3 by a sixth flow path 13. The sixth channel 13 is thinner than any of the first channel 3, the second channel 5, and the fourth channel 9, and may have a property that it is difficult to wet or a capillary attraction force is relatively difficult to work. preferable. Similarly, the fifth flow path 11 for measuring the neutralization reaction reagent is connected to the first flow path 3 by the seventh flow path 15. The seventh flow path 15 is thinner than any of the first flow path 3, the third flow path 7 and the fifth flow path 11, and has a property that it is difficult to wet or a capillary attraction force is relatively difficult to work. preferable.
[0013]
The first flow path 3, the second flow path 5 and the third flow path 7 are respectively connected to ports 17a and 17b opened to the atmosphere for taking in and out the liquid in the flow path at both ends thereof. 19a and 19b and 21a and 21b. For example, a pressurizing means (not shown) for pumping the liquid in the flow path can be connected to the ports 17a, 19a and 21a.
[0014]
It is preferable that the sixth flow path 13 and the seventh flow path 15 are spaced apart from each other so as not to fold relative to both sides of the first flow path. This is because the alkali-denaturing reagent in the fourth flow path 9 is pushed into the first flow path 3 through the sixth flow path 13, and in the fifth flow path 11. In order to prevent the neutralization reaction reagent from being pushed into the first flow path 3 via the seventh flow path 15 and inadvertently reacting with the liquid droplets formed. is there. Accordingly, the distance between the sixth channel 13 and the seventh channel 15 gradually increases as the size of the droplets formed by extrusion from these channels and the progress of the chemically denatured DNA fragment amplification reaction proceeds. It can be determined in consideration of the volume of the reaction solution.
[0015]
FIG. 2 is a cross-sectional view taken along the line II-II in FIG. As shown in the figure, the chemically denatured DNA fragment amplification apparatus 1 of the present invention basically includes a polymer material (for example, polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA), silicon resin), glass, and the like. It can be configured by stacking and joining two flat substrates of an upper substrate 23 and a lower substrate 25 formed of a material. In FIG. 2A, the first flow path 3, the second flow path 5, the third flow path 7, the fourth flow path 9, the fifth flow path 11, and the ports 17a to 21b are connected to the upper substrate. The sixth channel 13 and the seventh channel 15 are formed in the lower substrate 25. Thereby, each flow path is sealed and only the port is opened to the atmosphere. Alternatively, as shown in FIG. 2B, all the channels and ports can be formed in the upper substrate 23 and these channels can be sealed with the lower substrate 25. The substrates 23 and 25 may be transparent or opaque.
[0016]
The depth (or height) of the first flow path 3, the second flow path 5, the third flow path 7, the fourth flow path 9, and the fifth flow path 11 is not particularly limited. Generally, it can select suitably within the range of 1-300 micrometers. Moreover, the width | variety can also be suitably selected within the range of 100-300 micrometers. The first channel 3 can also be wider than the second channel 5 and the third channel 7. The height and width of the fourth flow path 9 and the fifth flow path 11 can be determined in consideration of the amount of alkali-denaturing reagent and the amount of neutralizing solution to be weighed in this flow path. The widths of the sixth channel 13 and the seventh channel 15 are preferably in the range of several μm to several tens of μm.
[0017]
As a technique for forming the flow path and the port in the substrate, for example, a conventional technique known to those skilled in the art can be used, which comprises producing a template by photolithography and producing a replica from the mold. In addition, it can also be produced using techniques such as mold casting.
[0018]
FIG. 3 is a diagram for explaining the principle by which a predetermined amount of alkali-denaturing reagent or neutralizing solution is weighed by the fourth flow path 9 and the fifth flow path 11. Here, for convenience of explanation, a mode in which the neutralizing liquid is weighed in the third flow path 7 and the fifth flow path will be described. Neutralizing liquid is injected from the port 21a (see FIG. 1), and the entire inside of the third flow path 7 is filled with the neutralizing liquid until the port 21b (see FIG. 1) is reached. When the neutralizing liquid 100 is introduced into the third flow path 7, the neutralizing liquid 100 is supplied through the opening c <b> 1 of the fifth flow path 11 that opens at the flow path wall aa of the third flow path 7. It can be introduced into the five flow paths 11. If the third flow path 7 and the fifth flow path 11 have easy-to-wet flow path walls, if the fifth flow path 11 is made thinner than the third flow path 7, the neutralizing liquid 100 is more It is spontaneously drawn into the fifth channel 11 through the opening c1 of the fifth channel 11 that opens to the channel wall aa of the third channel 7 by a strong capillary attraction. When the third flow path 7 and the fifth flow path 11 have flow path walls that are difficult to wet, the neutralization liquid is applied to the fifth flow path 11 by applying an appropriate pressure to the neutralization liquid 100 from the port 21a. 100 can be introduced (see FIG. 3A).
[0019]
At this time, the neutralization liquid 100 that has reached the end face c2 on the fifth flow path 11 side of the seventh flow path 15 (the flow path section between c2 and d1) has a flow path wall that is difficult to wet. It is blocked by the capillary repulsion of the flow path 15 and does not enter the seventh flow path 15. Even in the case where the fifth channel 11 is a channel wall that is difficult to get wet, the capillary repulsion of the seventh channel 15 is stronger than the capillary repulsion of the fifth channel 11. The neutralization liquid 100 does not enter (see FIG. 3A).
[0020]
Subsequently, the neutralizing liquid 100 remaining in the third flow path 7 is moved to a lower pressure side by causing an appropriate pressure difference at both ends of the ports 21a and 21b (see FIG. 1), for example. , Removed from the third flow path 7. At this time, the neutralizing liquid 100 in the fifth flow path 11 usually does not return into the third flow path 7 and enter. As a result, the end face 100a and the end face 100b which are both end faces of the neutralizing solution 100 in the fifth flow path 11 are connected to the opening c1 of the fifth flow path 11 and the third flow path 7 and the end face c2 on the side. The neutralization liquid 100 having a volume corresponding to the volume of the section between c1 and c2 of the fifth flow path 11 can be weighed (see FIG. 3B).
[0021]
Further, the neutralizing solution 100 weighed in the fifth flow path 11 closes the port 21b so that the pressure of the third flow path 7 is slightly higher than the pressure of the first flow path 3. It can be introduced into the first flow path 3 through the seventh flow path 15 and its opening d1 by applying pressure from the port 21a. At this time, if the neutralization liquid 100 is not completely pushed out into the first flow path 3 and is partially left in the seventh flow path 15, the neutralization liquid 100 remains at the position of the seventh flow path 15. (See FIG. 3C).
[0022]
FIG. 4 is a schematic diagram illustrating the principle of amplification of a DNA fragment by the chemically modified DNA fragment amplification apparatus 1 of the present invention. The reaction solution 200 is injected into the first channel 3 from the port 19a (see FIG. 1), and the inside of the channel is moved in the direction of the arrow. For example, the reaction solution 200 is mixed with PCR reagents, DNA samples, primers, enzymes, and the like. The reaction solution 200 is weighed in the fourth channel 9 and comes into contact with the alkali-denaturing reagent solution 300 pushed into the first channel 3 through the sixth channel 13, resulting in a pH of 10 or more and two DNA samples. The chains dissociate. The reaction solution 200 denatured to a pH of 10 or more is further moved, brought into contact with the neutralization solution 100, returned to the vicinity of pH 7, and annealed (re-paired) to amplify the DNA sample. By repeating this alkali denaturation and neutralization a predetermined number of times (for example, 30 times) by the chemically denatured DNA fragment amplification apparatus 1 of the present invention, DNA samples are successively amplified. The amplification reaction product can be taken out from the port 19b (see FIG. 1) at the other end of the first flow path 3.
[0023]
In order to efficiently amplify a DNA sample by the chemically denatured DNA fragment amplification apparatus 1 of the present invention, it is preferable to keep the chemically denatured DNA fragment amplification apparatus 1 of the present invention at around 54 ° C. suitable for annealing reaction. For such a purpose, for example, a heating means (not shown) is arranged on the lower substrate 25 side, and the temperature is controlled around 54 ° C., or the chemically denatured DNA fragment amplification apparatus 1 of the present invention is used. The amplification reaction is carried out by placing in a thermostat maintained at a temperature around 54 ° C.
[0024]
As the alkali denaturation reaction and the neutralization reaction proceed alternately, the volume of the reaction solution 200 gradually increases. Therefore, as shown in FIG. 5, the width of the first flow path 3 can be configured to increase in the direction of travel of the reaction solution 200.
[0025]
Although the chemically denatured DNA fragment amplification apparatus 1 of the present invention can be shaped into a rectangular shape (for example, a rectangular shape) as shown in FIG. 1, a spiral shape as shown in FIG. It can also be shaped on a disk in the form of concentric circles as shown in b).
[0026]
As described above, the chemically modified DNA fragment amplifying apparatus 1 of the present invention has been specifically described with reference to preferred embodiments. However, the present invention is not limited to the above embodiments. For example, the reaction liquid injection port 19a of the first flow path 3 can be changed as shown in FIG. In this embodiment, there is an eighth channel 25 connecting the port 23a and the port 23b, and this eighth channel 25 opens to the channel wall and is used for weighing a predetermined amount of reaction liquid. A ninth flow path 27 is provided. The ninth flow path 27 communicates with the first flow path 3 through a thin tenth flow path 29. With such a configuration, a constant amount of reaction solution can always be easily sent into the first flow path 3.
[0027]
【Example】
Hereinafter, a DNA amplification reaction by the chemically denatured DNA fragment amplification apparatus 1 of the present invention is specifically illustrated by Examples.
[0028]
Example 1
A DNA amplification reaction was carried out using a chemically modified DNA fragment amplification apparatus 1 as shown in FIG. The apparatus of FIG. 1 had 30th 4th flow paths each weighing the 4th flow path which measures an alkali denaturation reagent, and the neutralization liquid. The capacity of each fourth channel 9 and the fifth channel was 40 pl (picoliter). A heating means was disposed at the lower part of the chemically denatured DNA fragment amplification apparatus 1, and the apparatus 1 was maintained at a temperature of 54 ° C.
First, a 0.1 M sodium hydroxide (NaOH) solution was injected as an alkali denaturing reagent from the port 17a into the second channel 5, and each fourth channel 9 was filled with an alkali denaturing reagent solution. The excess alkali-denaturing reagent in the second flow path 5 was discharged from the port 17b. Thereafter, the port 17b was closed, and the alkali-denaturing reagent solution in each fourth flow path 9 was pushed out from the port 17a into the first flow path 3 via the sixth flow path 13. At this time, a part of the alkaline denaturing reagent solution remained in the sixth flow path 13. Thereby, the alkali-denaturing reagent solution can be placed at the position of the sixth flow path 13.
Similarly, a 0.1M hydrochloric acid solution as a neutralizing solution was injected from the port 21a into the third flow path 7, and each of the fifth flow paths 11 was filled with the neutralizing liquid. The remaining neutralized solution in the third flow path 7 was discharged from the port 21b. Thereafter, the port 21 b was closed, and the neutralized liquid in each fifth flow path 11 was pushed out from the port 21 a into the first flow path 3 via the seventh flow path 15 using air pressure. At this time, a part of the alkali-denatured reagent solution remained in the seventh flow path 15. Thereby, the neutralization liquid can be placed at the position of the seventh flow path 15.
Then, 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl _2, 0.1% Triton X-100, 4 kinds of dNTPs (deoxyribonucleotide triphosphates) each 200 micromolar , 2nl (nanoliter) of a reaction solution having a composition consisting of 50 to 10 ng of genomic DNA, 0.2 units of Taq DNA polymerase and 10 pmoles of each of two kinds of primers, and this reaction solution is connected to the first channel from the port 19a. 3 was injected at the start of movement. Thereafter, the reaction solution is moved forward using the air pressure from the port 19a, and the DNA is denatured by contacting with the alkali denaturing reagent solution placed at the position of the first sixth flow path 13, and the complementary DNA strand is separated. And changed to a random structure. Next, the denatured reaction solution is further advanced and brought into contact with the neutralization solution placed at the position of the first seventh flow path 15, thereby recombining the separated complementary strands (annealing). The DNA was amplified by returning to the original double strand. The amplified reaction is contacted with the next alkaline denaturing reagent solution, denatured again, then neutralized and re-paired, and the denaturation-neutralization reaction is subsequently referred to as the last 30th sixth stream. It repeated to the path | route 13 and the 7th flow path 15. The amplification reaction product was removed from port 19b. When this product was subjected to electrophoretic analysis, it was confirmed that the product was sufficiently amplified to an analyzable amount.
[0029]
【The invention's effect】
As described above, the conventional heat-denaturing PCR method requires three kinds of temperatures of 94 ° C., 54 ° C., and 72 ° C. repeatedly, and the temperature control is very complicated and difficult. According to the chemically denatured DNA fragment amplification apparatus, a DNA sample can be amplified only at a temperature necessary for annealing. As a result, the chemically denatured DNA fragment amplification apparatus of the present invention can be remarkably reduced in size as compared with the conventional heat denatured DNA fragment amplification apparatus, and at the same time, the analysis time can be shortened.
[Brief description of the drawings]
FIG. 1 is a partial plan view of an example of a chemically modified DNA fragment amplification apparatus of the present invention.
2 is a cross-sectional view taken along line II-II in FIG.
FIG. 3 is a conceptual diagram showing the principle of weighing an alkali-denaturing reagent solution or a neutralizing solution with the apparatus shown in FIG.
4 is a conceptual diagram showing a state in which a chemically denatured DNA fragment amplification reaction is carried out with the apparatus shown in FIG.
FIG. 5 is a partial plan view of another example of the chemically modified DNA fragment amplification apparatus of the present invention.
FIG. 6 is a schematic plan view of still another example of the chemically modified DNA fragment amplification apparatus of the present invention.
FIG. 7 is a partial plan view of an example in which the apparatus shown in FIG. 1 is partially changed.
[Explanation of symbols]
1 Chemically modified DNA fragment amplification apparatus 3 of the present invention 1st flow path 5 2nd flow path 7 3rd flow path 9 4th flow path 11 5th flow path 13 6th flow path 15 7th flow path Channels 17a, 17b, 19a, 19b Ports 21a, 21b, 23a, 23b Port 25 Eight channel 27 Ninth channel 29 Tenth channel 100 Neutralization solution 200 Reaction solution 300 Alkaline denaturing reagent solution

Claims

An apparatus for amplifying a DNA fragment by chemical denaturation, comprising: a first channel extending in a predetermined direction for causing a DNA fragment amplification reaction by chemical denaturation; and one of the first channels A second channel extending in a predetermined direction for supplying the alkali-denaturing reagent solution and arranged in parallel on the other side of the first channel, A third flow path extending in a predetermined direction for supplying the sum reaction reagent solution, wherein the second flow path opens to the flow path wall and weighs a predetermined amount of the alkali-denaturing reagent solution A plurality of fourth channels for weighing a predetermined amount of the neutralization reaction reagent solution, the third channel opening to the channel wall. The fourth channel is a sixth channel that is narrower than these channels that connect the channel and the first channel. And the fifth channel has a seventh channel that is narrower than these channels communicating the channel and the first channel, and the sixth channel and the seventh channel. The chemically denatured DNA fragment amplification device is characterized in that it is alternately arranged so as to be spaced apart from both sides of the first channel.

2. The chemically denatured DNA fragment amplification according to claim 1, wherein each of the first flow path, the second flow path, and the third flow path further has ports communicating with the atmosphere at both ends of the flow path. apparatus.

2. The chemically denatured DNA fragment according to claim 1, wherein the first channel is configured such that the width of the channel gradually increases in a direction in which the amplification reaction is performed stepwise. Amplification equipment.

The chemically denatured DNA fragment amplification apparatus according to claim 1, wherein the first flow path, the second flow path, and the third flow path are arranged in a spiral shape in parallel.

The chemically denatured DNA fragment amplification apparatus according to claim 1, wherein the first flow path, the second flow path, and the third flow path are arranged concentrically in parallel.

A reaction liquid weighing mechanism for weighing a predetermined amount of reaction liquid is disposed at one end of the first flow path, and the reaction liquid weighing mechanism has a port at both ends. An eighth flow path, a ninth flow path for weighing a predetermined amount of reaction liquid that opens in the flow path wall of the eighth flow path, and the ninth flow path and the first flow path. 2. The chemically denatured DNA fragment amplification device according to claim 1, comprising a tenth channel narrower than these channels communicating with the channel.

7. The chemistry according to claim 1, wherein the sixth sixth channel, the seventh channel, and the tenth channel have a property of being difficult to wet (or relatively difficult to perform capillary attraction). Denatured DNA fragment amplification device.

8. The chemically denatured DNA fragment amplification apparatus according to claim 1, further comprising means for maintaining the apparatus at a temperature of about 54 ° C.

Using the chemically denatured DNA fragment amplification device according to claim 1, the DNA fragment is dissociated into single strands by mixing the DNA fragment-containing reaction solution with an alkali denaturing reagent, and then the mixture solution is used as a neutralizing solution. A chemically denatured DNA fragment amplification method comprising amplifying a DNA fragment by repeating a step consisting of annealing a DNA fragment dissociated into a single strand by neutralization in step 1 and returning it to a double strand.

10. The method of claim 9, wherein the number of iterations of the step is a number in the range of 15 to 30 times.