JP3616107B2 - Polynucleotide detection method - Google Patents

Description

【００３６】
なお、 （式１） のアミノ化チミジンは、「Ｇｅｏｆｆｒｅｙ Ｂ． ｅｔ ａｌ．， Ｐｒｏｃ． Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ． ＵＳＡ．， ｖｏｌ．８２， ｐｐ．９６８−９７２，（１９８５）」の方法に従い調製した。
そして、それぞれ合成したアミノ基を有するポリヌクレオチドを、配列番号２に示す塩基配列に従い、Ｔ４ リガーゼを用いそれぞれを連結して、６個のアミノ基を有するポリヌクレオチドを得た。次いで遊離のアミノ基をトリフルオロアセチル化し、３’の水酸基をクロロ−Ｎ， Ｎ−ジイソプロピルアミノメトキシホスフインで活性化し、これにスルホスクシンイミジル６− （ビオチンアミド） ヘキサノエート （ＰＩＥＲＣＥ（イリノイ州 ＵＳＡ）製） を公知の条件で反応させて、各々のアミノ基をビオチン化して、所望のビオチン化プローブを得た。
【００３７】
（３） 標的ポリヌクレオチドの捕捉用のプローブである５’末端をアミノ化した配列番号１に示す塩基配列を有する第一のプローブの反応担体への固定は窒素気流中で以下の様に行なった。
まず、ガラス製の実施例１に示す反応担体の表面を十分に洗浄し、３− （２−アミノエチルアミノプロピル） トリメトキシシラン３２ｍｍｏｌ／Ｌを反応部９１に作用させて、当該反応部にアミノ基を導入した。このアミノシラン化した反応担体を、２．５％グルタルアルデヒド水溶液で活性化して、反応部９１以外の部分をテープでマスクした後、当該反応部に第一のプローブを１０ｐｍｏｌ添加し、室温で３時間反応させた。次にテープをはがして未反応部分を５０ｍＭのエタノールアミンでマスクした。この反応担体を、０．５％ＳＤＳ、 １００μｇ／ｍｌキャリヤーＤＮＡ （変性サケスペルマＤＮＡ） ５ｍｇ／ｍｌ牛血清アルブミン、５０％ホルムアミド、０．５ ％ＮａＣｌを含むｐＨ７の５０ｍＭ Ｔｒｉｓ・ＨＣｌ からなるハイブリダイゼーションバッファー中で保存した。
【００３８】
（４） ハイブリダイゼーション反応は、実施例２に示す装置を用いて、以下の手順に従い行なった。
試料となるバクテリオファージλのＤＮＡの濃度は、モル分子吸光係数＝５．１８×１０^８ Ｍ^−１・ｃｍ^−１として求めた。キャリアーＤＮＡとして加えた １００μｇ／ｍｌの変性サケスペルマＤＮＡを含む各種濃度のバクテリオファージλＤＮＡを容器９１にいれ、容器保持ステージ９２にセットした。当該試料は、加熱器４により９５℃に加熱され、バクテリオファージλＤＮＡを変性して一本鎖状態とした。次に容器１中の試料を１０μｌ 吸い上げ反応担体ステージ９にセットした前記 （３） において調製した反応担体１０の試料添加部 （図３の９２記載） に添加し、当該反応担体１０をステッピングモーター１１で約３０°傾斜させて、反応部 （図３の９１記載） に試料溶液を導入した。そして３０秒毎に反応担体１０を傾け試料溶液を反応部に出し入れすることで、当該試料溶液をゆるやかに攪拌した。なお、反応担体ステージ９は、内部に加熱器を有し、本実施例においては、６５℃に設定した。４時間後に、反応残液を反応担体９の反応残液排出部 （図３の９３記載） にロール状のろ紙１２を用いて排出させた。
【００３９】
ろ液を排出させた後、前記 （２） で調製したビオチン化プローブ１×１０^−１０ｍｏｌ／Ｌ〜１×１０^−９ｍｏｌ／Ｌを１０μｌ を反応担体反応部に添加した。操作は上記試料添加方法と同様である。試料反応と同様な方法で担体ステージを傾斜させることで反応液をゆるやかに攪拌した。４時間後、未反応ビオチン化プローブ残液を反応担体の反応残液排出部 （図３の９３記載） にろ紙１２をあてがい排出した。ろ紙をあてがったままの状態で、Ｚ軸ステージ１５に装着した洗浄液ノズル１６を反応担体の添加部に移動しポンプ１７を用いて洗浄液を添加した。
【００４０】
すなわち、０．１％ドデシル硫酸ナトリウム５ｍｇ／ｍｌ、牛血清アルブミン、０．５Ｍ ＮａＣｌ を含む０．３Ｍクエン酸緩衝液（ｐＨ７．０）で２５分間洗浄した後、さらに、０．１％ドデシル硫酸ナトリウム、２ｍＭ ＥＤＴＡ、３Ｍ テトラメチルアンモニウム、トリス塩酸 （ｐＨ８．０） 溶液で５８℃で３０分間洗浄した。次に、０．０５％ Ｔｗｅｅｎ２０、５ｍｇ／ｍｌ（ミリリットル） 牛血清アルブミン、０．１５ｍｏｌ／Ｌ（リットル） のＮａＣｌ、０．０５ｍｏｌ／Ｌ（リットル） のリン酸緩衝液 （ｐＨ７．４） で洗浄した。
【００４１】
次に、ストレプトアビジン化Ｂ−フィコエリスリン（Ｍｏｌｅｃｕｌａｒ Ｐｒｏｂｅｓ， Ｉｎｃ．製） （濃度約１×１０^９ ｍｏｌ／Ｌで０．０５％ Ｔｗｅｅｎ２０、５ｍｇ／ｍｌ牛血清アルブミン、０．１５ＭＮａＣｌを含む０．０５ｍｏｌ／Ｌリン酸緩衝液（ｐＨ７．４）で希釈） を添加した。操作方法は上記試料添加法と同じである。３７℃で１時間反応させた後、０．０５％ Ｔｗｅｅｎ２０、５ｍｇ／ｍｌ 牛血清アルブミン、０．１５ｍｏｌ／Ｌ のＮａＣｌ、０．０５ｍｏｌ／Ｌ のリン酸緩衝液 （ｐＨ７．４） で洗浄し、反応担体上に捕捉されたビオチン化プローブをＢ−フィコエリスリンで標識した。この操作により反応担体上に捕捉した標的ポリヌクレオチドであるバクテリオファージλＤＮＡを、蛍光体であるＢ−フィコエリスリンで標識した。
【００４２】
（５） 反応の終了した反応担体上の蛍光体結合部を検出するには、ＹＡＧレーザー （５３２ｎｍ）とホトンカウンティング可能な光電子増倍管を用いた実施例３の検出装置で測定した。レーザー光源１０１ の光は、ビームエキスパンダー１０２ でビーム径を拡張し、ダイクロイックミラー１０３ で反射される。当該検出装置は本実施例においては、ダイクロイックミラー１０３ として５３２ｎｍ の光を反射し、５７５ｎｍ 以上の光を８０％以上透過する物を使用した。
【００４３】
反応担体上の標識試料により生じた蛍光を前記ダイクロイックミラー１０３ を通じて透過させ、光電子増倍管１１１ で信号として検出して、これをマイクロプロセッサー１１２ で処理して、サーボモーター１０５ ・ １０７ からの角度情報と同期させて処理することで、反応担体表面の各位置における蛍光強度を算出した。そして、かかるデータをもとに検量線を作成した。
【００４４】
結果を図６に示す。
なお、同様な操作により反応担体上に標的ポリヌクレオチドであるバクテリオファージλを捕捉し、蛍光体であるＢ−フィコエリスリンで標識し、従来の検出法として蛍光光度計を用いホトンカウンティングモードで蛍光強度を測定した結果も○で示す。その結果本発明によれば標的ポリヌクレオチドであるバクテリオファージλを従来法に比べ一桁以上高感度で測定できることが判明した。すなわち、蛍光測定領域を微小な領域に区切って測定することによる背景光除去による高感度変化が確認された。
【００４５】
【実施例５】
ポリヌクレオチドの検出 （２）
（１） 本実施例においては、実施例４と同様に、検出の対象となる標的ポリヌクレオチド試料として、パクテリオファージλのＤＮＡを用いた。
そして、当該バクテリオファージλのＤＮＡに対して３種類のプローブを用いて、かかる標的ポリヌクレオチド試料を検出した。
【００４６】
標的ポリヌクレオチドの捕捉用の第一のプローブとしては、実施例４と同様に配列番号１に示す塩基配列を有するポリヌクレオチド鎖を用いた。また、第一の標識用プローブとして用いられるポリヌクレオチドとして配列番号３で示される塩基配列を有するポリヌクレオチドを、第二の標識用プローブとして用いられるポリヌクレオチドとして配列番号４で示される塩基配列を有するポリヌクレオチドを用いた。標的ポリヌクレオチドであるバクテリオファージλのＤＮＡ上で第三の標識プローブとして用いられるポリヌクレオチドは第二の標識プローブとして用いられるポリヌクレオチドの３’末端側に隣接する配列である。
【００４７】
第二の標識用プローブとして用いられるポリヌクレオチドの５’末端には、ポリＴスペーサーを介してスルホローダミン１０１ 様蛍光団が結合している。本実施例においては、かかる蛍光団として、スルホローダン１０１ 様色素と思われる蛍光色素を直径が０．０２μｍ で表面にカルボキシル基を有する粒子ポリマーを用いた。かかる粒子ポリマーとしてＭｏｌｅｃｕｌａｒ Ｐｒｏｂｅｓ， ＩＮＣ．製のＣａｒｂｏｘｙｌａｔｅ−ｍｏｄｉｆｉｅｄ Ｌａｆｅｘ ｒｅｄ （５８０／６０５）を用いた。そして、当該スルホローダミン１０１様蛍光団を含む粒子による第２のプローブの標識は以下の方法で行った。
すなわち、ポリヌクレオチドの合成最終段階に、Ｎ−モノメトキシトリチルアミノヘキサ−６−オキシ−β−シアノエチル−Ｎ， Ｎ−ジイソプロピルアミノホスホアミダイトを反応させて５’末端にアミノ基を導入し、これに水溶性カルボジイミドで当該粒子のカルボキシル基を活性化して結合させた。そして、結合後、弱アルカリ条件下で未反応のカルボキシル基を再生した。
【００４８】
第三の標識用プローブとして用いられるポリヌクレオチドの３’末端にポリＴスペーサーを介してフルオレッセイン様蛍光団が結合している。本実施例においては、当該蛍光色素を含む直径０．０２μｍ の表面にカルボキシル基を有する粒子ポリマーを用いた。かかる粒子ポリマーとして、Ｍｏｌｅｃｕｌａｒ Ｐｒｏｂｅｓ， ＩＮＣ．製のＣａｒｂｏｘｙｌａｔｅ−ｍｏｄｉｆｉｅｄ Ｌａｔｅｘ Ｙｅｌｌｏｗ−ｇｒｅｅｎ （４９０／５１５）を用いた。そして当該フルオレッセイン蛍光団を含む粒子による第３のプローブの標識は以下の方法で行った。すなわち、ポリヌクレオチドの合成後に、アナリティカルバイオケミストリー記載のラリー イー． モリソン等の方法 （Ｌａｒｒｙ Ｅ． Ｍｏｒｒｉｓｏｎ ｅｔ ａｌ．， Ａｎａｌｙｔｉｃａｌ Ｂｉｏｃｈｅｍｉｓｔｒｙ， １８３， ｐｐ．２３１−２４４（１９８９）） に従い、ターミナル デオキシヌクレオチジル トランスフェラーゼを用いて８− （６−アミノヘキシル） アミノアデノシンを結合して、ポリヌクレオチドの３’末端にアミノ基を導入し、これと水溶性カルボジイミドで当該カルボキシル基を活性化した粒子ポリマーを結合させ当該蛍光団を導入した。そして、結合後、弱アルカリ条件下で未反応のカルボキシル基を再生した。
【００４９】
調製したこれら第二第三の標識プローブは、０．０５％ＳＤＳ、 １００μｇ／ｍｌのキャリヤーＤＮＡ （変性サケスペルマＤＮＡ） を含む実施例４と同様のｐＨ７のハイブリダイゼーションバッファーに懸濁して保存した。
（２） ハイブリダイゼーション反応は、実施例２に示す装置を用いて、実施例４に記載した方法に従い行なった。なお前記、第二、第三の標識プローブは予め混合して、一回のピペット操作で同時に添加した。
【００５０】
（３） 反応担体表面に捕捉された蛍光プローブの位置を、実施例３ （２） 記載の検出装置で測定した。
レーザー発振器１４１ 及び１４２ から出たレーザー光は４８８ｎｍ の光を反射し５９０ｎｍ の光を透過するダイクロイックミラー１５１ で合成された後、レンズ１５２ 及び１５３ からなるビームエキスパンダーでビーム径を拡張させ、リング状のミラー１５５ で反射され集光レンズ１５４ で集光されて、反応担体の反応部に照射される。本発明においてはレーザーとしてＡｒ レーザーで励起した可変波長色素レーザー （波長５９０ｎｍ）と、Ａｒ レーザー （波長４８８ｎｍ）を用いた。レーザーで励起されることにより標識プローブより発した蛍光は、集光レンズ１５４ で集められ、リング状ミラー１５５ の中心部の素通り部分を通過し、さらに５１０ｎｍ の光を反射し６１０ｎｍ の光を透過するダイクロイックミラー１５６ でフルオレッセイン様蛍光団由来の蛍光とスルホローダミン１０１ 様蛍光団由来の蛍光を分離した。これらの蛍光はバンドパスフィルター１５７、 １５８によりそれぞれの蛍光成分のみを取りだし、結像レンズ１５９、 １６０により高感度ＣＣＤカメラ１６１、 １６２で撮像した。高感度ＣＣＤカメラでとらえた信号は画素ごとに画像処理用マイクロプロセッサー１６４ で処理し反応担体上の蛍光発光位置を特定した。各画素は反応担体表面の約３５０ｎｍ 四方に相当する。フルオレッセイン様蛍光団由来の蛍光発光位置とスルホローダミン１０１ 様蛍光団由来の蛍光発光位置の一致する場合は標的ＤＮＡに標識プローブがハイブリダイゼーションしていると判断し、この位置を計数し標的ＤＮＡの分子数とした。いずれか一方の蛍光団由来の蛍光しか検出されない部位には標識プローブが非特異的に吸着しているとみなし計数の対象から除外した。
【００５１】
上記方法に基づいて各種濃度のバクテリオファージλの量を測定した結果を図８に示す。ここで、●は２種の蛍光プローブが同一位置に検出される場合のみを示している。本発明によればバクテリオファージλが高感度に検出できることがわかる。なお、○は同様な操作により反応担体上に標的ポリヌクレオチドであるバクテリオファージλを捕捉し、スルホローダミン１０１ 様蛍光団及びフルオロレッセイン様蛍光団で標識し、従来の検出量である蛍光光度計にセットしホトンカウンティングモードで蛍光強度を測定した結果である。
【００５２】
【発明の効果】
本発明により、放射性同位元素の標識を必要とせずに、高感度で精度が高く、定量的な測定が可能で、かつ自動化により単位時間あたりの測定サンプル数を多くすることで臨床フィールドで多量の検体を検査することが可能なポリヌクレオチド検出法、及び当該方法を用いた検出装置が提供される。
【配列表】
配列番号 ：１
配列の長さ：２７５
配列の型 ：核酸
鎖の数 ：１本鎖
トポロジー：直鎖状
配列の種類：他の配列 他の構造体を含む合成ポリヌクレオチド
配列の特徴：１ ＮＨ_２（ＣＨ_２）_６ 付加

配列番号 ：２
配列の長さ：２００
配列の型 ：核酸
鎖の数 ：１本鎖
トポロジー：直鎖状
配列の種類：他の配列 他の構造体を含む合成ポリヌクレオチド
配列の特徴：２７， ５７， ８９， １３０， １６２ ビオチン付加

配列番号 ：３
配列の長さ：６５
配列の型 ：核酸
鎖の数 ：１本鎖
トポロジー：直鎖状
配列の種類：他の配列 他の構造体を含む合成ポリヌクレオチド
配列の特徴：１ スルホローダミン１０１ 様蛍光団を含む粒子

配列番号 ：４
配列の長さ：６５
配列の型 ：核酸
鎖の数 ：１本鎖
トポロジー：直鎖状
配列の種類：他の配列 他の構造体を含む合成ポリヌクレオチド
配列の特徴：１ フルオレッセイン様蛍光団を含む粒子

【図面の簡単な説明】
【図１】従来の蛍光測定法と本発明に基づく蛍光測定法の概念図。
【図２】本発明による方法の反応の概念図。
【図３】本発明反応担体の模式図。
【図４】本発明反応装置の構成図。
【図５】本発明測定装置の概念図。
【図６】実施例１による測定結果。
【図７】２種類の蛍光色素を用いた本発明測定装置の概念図。
【図８】実施例２による測定結果。[0001]
[Industrial application fields]
The present invention is a polynucleotide detection method that can easily detect the presence or absence of a foreign polynucleotide such as a virus, rickettsia, or bacteria that causes disease, or the presence of a polynucleotide carrying specific genetic information in an organism. And a reaction carrier, a reaction apparatus, and a detection apparatus used in the method.
[0002]
[Prior art]
In “Proc. Natl. Acad. Sci. USA, vol. 80, p278-282 (1983)”, a target polynucleotide (DNA or RNA) sample is directly captured on a solid phase or complemented with a target polynucleotide. A target polynucleotide is captured on a solid phase using a hybridization reaction in which another polynucleotide probe containing a typical nucleotide sequence is immobilized on the solid phase, and then a labeled polynucleotide to which a label is bound is associated. A polynucleotide detection method is disclosed.
[0003]
In this method, 1) a polynucleotide fragment sample separated by molecular weight by electrophoresis is transferred to and fixed on a nitrocellulose membrane, and then the nitrocellulose membrane is immersed in a solution containing a polynucleotide probe labeled with a radioisotope to perform an association reaction. 2) After reaction, after washing away unreacted labeled probe from the membrane, the amount of residual labeled probe bound to the membrane is detected by autoradiography to determine the presence or absence of the target polynucleotide.
[0004]
Japanese Patent Laid-Open No. 58-31998 discloses a method for detecting foreign target polynucleotides such as bacteria and viruses in samples such as blood and excreta.
In this method, 1) a polynucleotide in a purified sample is denatured by heating or the like to form a single strand, which is then fixed to a nitrocellulose membrane, and then 2) a bacterial or viral polynucleotide to be examined. After reacting with a radioisotope-labeled polynucleotide probe having a base sequence complementary to the membrane, the membrane is washed, and the amount of residual labeled probe bound to the membrane is detected by autoradiography. This is a method for determining the presence or absence of nucleotides.
[0005]
[Problems to be solved by the invention]
However, the radioisotopes used for labeling in both of the above detection methods must minimize the radiation exposure of workers and can only use tracer amounts, and the specific radioactivity per probe. In addition, it is difficult to increase the exposure time, and a background of radiation is likely to be generated from the labeled probe adsorbed on the nitrocellulose membrane, so that detection takes a long time and detection sensitivity is often insufficient. For this reason, quantitative measurement is difficult, and most of them are used to determine the presence or absence of a target polynucleotide. At most, only semi-quantitative measurement is performed to determine the degree of blackening of an autoradiogram film. There was an unsatisfactory aspect. Furthermore, since autoradiography is used, it is difficult to automate detection. In addition, it is necessary to consider the radiation exposure of workers as described above, and it is pointed out that it is inconvenient for measuring a large amount of specimens in the clinical field. It was.
[0006]
Therefore, the present invention is capable of high-sensitivity, high-accuracy and quantitative measurement without the need for radioisotope labeling, and increases the number of measurement samples per unit time by automation, resulting in a large amount in the clinical field. It is an object of the present invention to provide a polynucleotide detection method and a detection apparatus using the method that can inspect the specimen.
[0007]
[Means for Solving the Problems]
As a result of intensive studies on the above problems, the present inventors have found that the problems can be solved by the inventions shown in the following (1) to (7). That is, the present invention
(1) A polynucleotide that detects a quantity of associated labeled probe by associating a polynucleotide probe that is immobilized on a reaction carrier with a labeled probe that binds a labeled substance to a polynucleotide chain that is complementary to the polynucleotide chain. In the nucleotide detection method, all or part of the surface of the reaction carrier on which the association reaction has been performed is divided into minute regions, and the intensity of the signal derived from the label for each minute region is detected, and the minute region showing a strong signal is detected. A polynucleotide detection method characterized by measuring the number,
(2) As a labeled probe in which a label is bound to a polynucleotide chain complementary to the polynucleotide strand that is the object to be measured, a plurality of polynucleotide chains that can associate with different sites in the polynucleotide chain that is the object to be measured (1) the polynucleotide detection method according to (1), wherein a labeled body is used;
(3) The polynucleotide detection method according to (2), wherein the labeled products of the plurality of polynucleotide chains used as the labeled probe are different phosphors,
(4) The distance between the labeled products in the polynucleotide chain when a plurality of labeled probes are associated with the polynucleotide chain to be measured is within 2000 base lengths. (2) or (3 ) The polynucleotide detection method described,
(5) On the flat plate, a sample addition part for adding a sample containing a polynucleotide chain to be measured and a polynucleotide chain complementary to the polynucleotide chain to be measured for performing an association reaction are fixed. The reaction part and the reaction residue discharge part for discharging the reaction residual liquid are provided with appropriately shaped recesses, respectively, and the sample addition part, the reaction part, the reaction part, and the reaction residue discharge part are respectively A reaction carrier characterized by being connected by a groove having the same depth as a line connecting the deepest part of each part, the sample addition part, the reaction part, and the reaction residual liquid discharge part,
(6) The reaction carrier described in (5), the sample added to the sample addition part in the reaction carrier, or the cleaning solution can be taken in and out from the sample addition part to the reaction part and the reaction residual liquid discharge part, or in the opposite direction. A mechanism for shaking the reaction carrier, a mechanism for adding a sample or washing liquid to the reaction liquid addition part of the reaction carrier, and a mechanism for discharging the reaction residual liquid or washing liquid from the reaction liquid discharge part of the reaction carrier. A reactor used for the polynucleotide detection method according to any one of (1) to (4),
(7) A light source that emits at least one type of excitation light, a beam expander that expands the beam diameter of the excitation light emitted from the light source, and a sample that bends the excitation light with the expanded beam diameter in a certain direction. A mirror that can transmit the emitted fluorescence, the excitation light bent by the mirror is synchronized with the mirror and the condenser lens to adjust the irradiation position of the excitation light to the sample, and the beam diameter of the fluorescence emitted by the sample is expanded A mechanism for irradiating a mirror that can bend the excited light in a certain direction and transmit the fluorescence emitted by the sample, and the fluorescence detection mechanism, and the signal from the fluorescence detection mechanism is identified as the location of the labeled probe And a fluorescence detection device used for the polynucleotide detection method according to any one of (1) to (4).
[0008]
First, the polynucleotide detection method of the present invention will be described.
The type of polynucleotide chain used in the method of the present invention as an object to be measured is not particularly limited. For example, specific genetic information in an organism, as well as foreign polynucleotides such as viruses, rickets, and bacteria that cause disease. In the case of examining the presence or absence of a polynucleotide that bears, an endogenous polynucleotide can be used as the analyte.
[0009]
In addition, regardless of whether the polynucleotide is DNA or not, any known method can be used as a preparation method according to the type of the specific object to be measured.
In a labeled probe in which a label is bound to a polynucleotide strand complementary to the polynucleotide strand to be measured, “complementary” means that hydrogen bonds are mutually connected to such an extent that the labeled probe can bind at least to the polynucleotide strand to be measured. In other words, all bases do not necessarily have to be hydrogen bonds with each other as long as such a condition is satisfied. The length of the polynucleotide used as the labeled probe is what is used as the polynucleotide to be measured, that is, how long the polynucleotide can be detected as the labeled probe. And is not particularly limited as long as it is determined. The polynucleotide used as the labeled probe is a phosphite triester method (Nature, 310 , 105 (1984)), and the phosphoramidide method (Mc Bride, L. J. and Caruthers, MH Tetrahedron Letters, vol. 24, pp. 245-248 (1983)). It is also possible to obtain a specific gene portion of an organism by amplifying it by a generally known method such as a PCR method (Saiki, RK, et al., Science, vol. 37, p170 (1985)). it can.
[0010]
The labeling substance used here is not particularly limited as long as it can detect the polynucleotide to be measured by performing minute division described later. Particularly preferred from the viewpoint of detection sensitivity and safety, fluorescent dyes for fluorescence analysis can be mentioned. The kind of fluorescent dye used is not particularly limited, and for example, phycobiliproteins such as B-phycoerythrin and R-phycoerythrin; rhodamine, fluorescein, 4-nitrobenzo-2-oxa-1, 3-diazole, It is possible to use phthalocyanine and the like and derivatives thereof; and polymers containing these phosphors. However, since phycobiliprotein has problems with thermal stability and stability against denaturing agents, it is necessary to label the probe associated with the target polynucleotide in advance using biotin-avidin or hapten-anti-hapten antibody. . The polymer containing the phosphor can be obtained by sensitizing a specific dye to the polymer by a known method, but a commercially available product can also be used directly.
[0011]
The method for binding these fluorescent dyes to the polynucleotide as a labeled probe is not particularly limited. For example, a method of directly labeling a nucleobase with a fluorophore using an amino group introduction reagent or a method disclosed in Japanese Patent Application Laid-Open No. 61-44353, that is, a phosphone having a functional group with a phosphoric acid bond at a phosphor labeling site of a polynucleotide. A method of introducing a labeled substance at an arbitrary position of a polynucleotide by replacing with an acid bond and realizing a fluorescent label by binding the functional group to a fluorescent substance can be employed.
[0012]
The polynucleotide chain can be bound to the reaction carrier by a known method, for example, by binding a functional group to the surface of the carrier, then activating the functional group and binding to the activated functional group. Is carried out by a method of bringing a polynucleotide chain into which is previously introduced into contact with each other. For example, the functional group and the polynucleotide chain on the reaction carrier can be bonded to the carboxyl group on the surface of the carrier via an amide bond.
[0013]
In such a case, the polynucleotide to be measured can be directly immobilized on the reaction carrier, but it is possible to selectively immobilize the polynucleotide to be measured by preparing a capture probe in advance and immobilizing it. Is preferable.
In this way, a polynucleotide probe, which is an object to be measured immobilized on a reaction carrier, and a labeled probe in which a label is bound to a polynucleotide chain complementary to the polynucleotide chain, are associated under normal conditions. As a labeled probe in which a label is bound to a polynucleotide strand complementary to the polynucleotide strand that is the analyte, it can associate with different sites in the polynucleotide strand that is the analyte. Using a label of multiple polynucleotide strands distinguishes the signal generated by nonspecific binding of the labeled probe to the nucleotide strand to be measured from the signal generated by nonspecific adsorption of the multiple labeled probes. It is preferable in terms of obtaining. Furthermore, in consideration of detection accuracy, when a plurality of labeled probes are associated with a polynucleotide chain that is an object to be measured, the distance between the labeled substances in the polynucleotide chain is at least 2000 base lengths or less, and 600 to It is preferably within 700 base length.
[0014]
Further, in the case of using the above-described labeled probe, it is preferable to use a fluorescent substance having a different label for further easy detection. In such a case, the combination of the phosphors used is preferably a fluorescein system and a rhodamine system, or a phycobiliprotein and a rhodamine system, more specifically, fluorescein and sulforhodamine 101, or phycoerythrin and sulforhodamine. A combination such as 101 can be cited as a suitable one.
[0015]
The shape of the reaction carrier is not particularly limited as long as it is suitable for carrying out the method of the present invention. However, a sample addition unit for adding a sample containing a polynucleotide chain as a measurement object on a flat plate. , A reaction part in which a polynucleotide chain complementary to the polynucleotide chain, which is an object to be measured for conducting the association reaction, is fixed, and a concave part of an appropriate shape are provided as a reaction liquid discharge part for discharging the reaction liquid. And the sample addition part, the reaction part, and the reaction part and the reaction residual liquid discharge part are the same as the lines connecting the deepest parts of the sample addition part, the reaction part, and the reaction residual liquid discharge part, respectively, at the maximum. A preferable example is a reaction carrier characterized by being connected by a groove having a depth. The exemplary reaction carrier will be described in detail in the examples below.
[0016]
A major feature of the method of the present invention is that, after the association reaction, the surface or part of the reaction carrier on which the reaction has been performed is divided into minute regions, and the intensity of the signal derived from the label for each minute region is detected, The purpose is to measure the number of minute regions showing a strong signal.
That is, by dividing the measurement target into minute regions, it is possible to minimize the influence of background light generated from the reaction carrier, and as a result, it is possible to relatively increase the detection sensitivity. In particular, when a plurality of labeled probes are used, if the distance in the polynucleotide to be measured in which the plurality of labels are associated is sufficiently close, that is, within 2000 base lengths as described above, the signals derived from the plurality of labels are generated. At the same time, it is detected in the microregion, and the labeled probe is adsorbed non-specifically, so that it can be clearly distinguished from the microregion in which only a signal derived from a single labeled probe is detected. It is possible to prevent a decrease in sensitivity due to a dependent ghost.
[0017]
The above-mentioned minute area division is a means for dividing and measuring two-dimensionally with a one-dimensional or two-dimensional detector, that is, a diode array detector or a CCD two-dimensional detector when detecting a polynucleotide. However, it can be performed by means for fetching data at regular intervals.
In addition, although it is preferable in terms of increasing detection sensitivity that the minute region is as small as possible, there is a limit depending on the nature of the substance used as the label. For example, when a fluorescent substance is used as a label, it cannot be divided into regions smaller than the wavelength of light.
[0018]
A conceptual diagram of the measurement method of the present invention described above is shown in FIGS.
The means for detecting the strength of the signal derived from the label for each minute region is selected according to the type of substance used as the label. For example, when a fluorescent dye is used as the label, the reaction carrier, the sample added to the sample addition part of the reaction carrier, or the cleaning solution is moved from the sample addition part to the reaction part and the reaction residual liquid discharge part. Or a mechanism for rocking the reaction carrier so that it can be taken in and out in the opposite direction, a mechanism for adding a sample or washing liquid to the reaction liquid addition part of the reaction carrier, and a reaction residual liquid or washing liquid from the reaction liquid discharge part of the reaction carrier A reaction apparatus characterized by having a discharge mechanism; a light source that emits at least one type of excitation light, a beam expander that expands the beam diameter of the excitation light emitted from the light source, and excitation with an expanded beam diameter A mirror that can bend light in a certain direction and transmit the fluorescence emitted from the sample, and collect the excitation light bent by the mirror with the mirror. A mirror that adjusts the irradiation position of the excitation light to the sample by synchronizing the lens and bends the fluorescence emitted by the sample in a certain direction and transmits the fluorescence emitted by the sample. Detection by a fluorescence detection device characterized by comprising an irradiation mechanism, a fluorescence detection mechanism, and a device that identifies and counts the signal from the fluorescence detection mechanism as the presence position of the labeled probe is adopted as a preferred detection means. Can do. Such a reaction apparatus and fluorescence detection apparatus will be described in detail in Examples below.
[0019]
【Example】
Hereinafter, the present invention will be described more specifically with reference to examples.
[0020]
[Example 1]
Reaction carrier of the present invention
FIG. 3 shows one embodiment of the reaction carrier of the present invention.
A reaction unit 91, a sample addition unit 92, and a reaction residue discharge unit 93 are provided on a flat plate, and the reaction unit 91, the sample addition unit 92, the reaction unit 91, and the reaction residue discharge unit 93 are connected by a groove portion. Yes.
[0021]
The material of the reaction carrier is not particularly limited as long as the label signal in the reaction part can be detected. For example, when a fluorescent dye is used as the label, it is preferable to use an optically flat material such as glass, a silicon wafer, or a plastic having an optically flat structure. The upper surface portion 94 of these reaction carriers is coated with a fluorine resin, for example, polytetrafluoroethylene, for the purpose of holding the reaction solution in the sample addition portion, reaction portion, and reaction residual solution discharge portion.
[0022]
The groove connecting each part usually connects each part in a straight line, and is the same depth as the deepest part of each part, but in order to adjust the outflow speed etc. of the reaction solution etc., it is deeper than the deepest part, It is possible to make it shallow, and to meander further.
In the use of the reaction unit 91 or in advance, the functional group is bonded to the surface of the reaction unit 91, and then the functional group is activated and the functional group bonded to the activated functional group is previously introduced. The polynucleotide chain to be measured is immobilized by a known method typified by the method of bringing the measured polynucleotide chain into contact. In carrying out such immobilization, it is preferable to mask other portions in the carrier with tape or the like.
[0023]
A sample containing a labeled probe is added to the sample addition unit 92 by, for example, the reaction apparatus in Example 2 below, and the reaction carrier of the present invention is shaken to transfer the sample to the reaction unit 91 through the groove. The resulting reaction residual liquid is transferred to the reaction residual liquid discharge section 93 through the groove as described above, and the reaction residual liquid in the reaction residual liquid discharge section 93 can be discharged and removed by, for example, a roll-up filter paper.
[0024]
By the way, the shape of the reaction part and the groove in the reaction carrier of the present invention is not particularly limited as long as the sample can be subjected to the reaction / discharge process described above after the sample is added. For example, a shape in which the reaction part and the entire groove are taken in one groove is also acceptable.
[0025]
[Example 2]
The reactor of the present invention
FIG. 4 shows one embodiment of the reaction apparatus of the present invention. The association reaction between polynucleotides is preferably carried out by the following procedure using the apparatus.
First, the sample is put in the container 1 and set on the container holding stage 2. A labeled probe or the like is placed in a similar container and set on the container holding stage 2. The container stage is equipped with a dedicated disposable pipette tip 3 for each reaction container. The sample or the like in the container 1 is heated by the heater 4 as necessary, and the sample or the like can be denatured into a single-stranded oligonucleotide. The container holding stage 2 has a disk shape and can be rotated by a stepping motor 5, and an arbitrary container can be set at the position of the automatic pipette 6. The automatic pipette 6 can be moved in the X-axis direction and the vertical-axis (Z-axis) direction by the stage 8 and the Z-axis (vertical axis) stage 8. First, the automatic pipette 6 is moved in the X-axis direction, and the pipette tip 3 is set.
[0026]
Next, the sample in the container 1 is sucked up and added to the sample addition part of the reaction carrier 10 described in Example 1 in which the first probe set on the reaction carrier stage 9 is fixed, and the sample solution is introduced into the reaction part. . Used pipette tips are discarded in the tip discard box 50. The reaction carrier stage 9 has a structure that can move in the Y-axis direction (the front side of the paper), and the sample solution can be sequentially added to a plurality of reaction carrier addition portions. Further, the reaction carrier stage 9 has a structure having a heater inside, and can keep the reaction carrier at a constant temperature. The temperature can be appropriately set according to the combination of the sample to be used and the labeled probe. The reaction carrier stage 9 has a structure that can be tilted up and down by a stepping motor 11. Such an inclination angle is appropriately determined according to the intended reaction time, but is usually preferably about 15 to 30 °. Using this mechanism, the reaction solution is tilted at regular intervals, and the reaction solution is gently stirred by taking the sample solution into and out of the reaction section. After the reaction, the reaction residual liquid is discharged by applying the filter paper 12 to the reaction liquid discharge part (described in 93 of FIG. 3) of the reaction carrier described in Example 1. The form of the filter paper 12 is not particularly limited, but a roll-like one is preferable in that it can be continuously used and can be easily replaced. And the filter paper 12 in a present Example can be easily supplied with the roller 13. FIG. During the reaction of the sample or the like, the filter paper is separated from the reaction carrier, but when the reaction residual liquid is discharged, the roller 14 moves to the reaction residual liquid discharge part on the reaction carrier.
[0027]
After retracting the filter paper, the labeled probe is added to the reaction carrier reaction part described in Example 1. The operation is the same as the above sample addition method. The reaction solution is gently stirred by tilting the carrier stage in the same manner as the sample reaction. The unreacted labeled probe residual liquid is discharged by applying the filter paper 12 to the reaction carrier liquid discharge part (denoted by 93 in FIG. 3) described in Example 1. With the filter paper still applied, the cleaning liquid nozzle 16 mounted on the Z-axis stage 15 is moved to the addition portion of the reaction carrier, and the cleaning liquid placed in the cleaning liquid holding container 18 connected through the valve 19 using the pump 17 is supplied. Added. The cleaning liquid is preheated to 42 to 65 ° C. The cleaning liquid passes through the reaction section and is discharged by filter paper. Then, the used filter paper is discarded in the filter paper discarding box 51. The cleaning liquid holding container 18 may be single, or a plurality of cleaning liquid holding containers may be connected via a valve 19.
[0028]
[Example 3]
The detection device of the present invention
When a fluorescent dye is used as the label, it is preferable to detect the phosphor-binding portion on the reaction carrier using the detection apparatus shown in this example.
(1) FIG. 5 shows an embodiment of the detection apparatus of the present invention.
The light from the laser light source 101 is expanded by the beam expander 102 and reflected by the dichroic mirror 103. The dichroic mirror 103 uses an object that reflects light of the wavelength of the laser light source 101 and transmits 80% or more of light of other wavelengths. The light reflected by the dichroic mirror 103 is reflected by the mirror 104 whose angle can be changed by the servo motor 105, and further reflected by the mirror 106 whose angle can be changed in a direction orthogonal to the mirror 104, and then by the condenser lens 108. The light is collected and irradiated on a specific area on the reaction carrier 109. Here, since the angles of the mirror 104 and the mirror 106 can be changed in directions orthogonal to each other, the reaction carrier can be scanned in the XY direction. When a laser spot hits a site where the phosphor on the surface of the reaction carrier exists, fluorescence is generated from that site. The fluorescence proceeds in the order of the condenser lens 108, the mirror 106, and the mirror 104, and passes through the dichroic mirror 103. After passing through the band pass filter 110, it is detected by the photomultiplier tube 111. The detected signal is taken into the microprocessor 112 and processed in synchronization with the angle information from the servo motors 105 and 107 to calculate the fluorescence intensity at each position on the surface of the reaction carrier. By this method, a site emitting weak fluorescence derived from background light and a site emitting strong fluorescence derived from a fluorescent probe can be distinguished. Since it is considered that there is one fluorescently labeled probe at one position having a constant fluorescence, the amount of target polynucleotide can be quantified by counting the sites emitting fluorescence.
[0029]
In this fluorescence measuring apparatus, if a condensing lens and a pinhole are provided in front of the photomultiplier tube and the pinhole position is a confocal position, that is, a confocal microscope system, the position resolution and sensitivity are improved. The target polynucleotide can be detected with higher sensitivity.
(2) FIG. 7 shows one embodiment of the detection apparatus of the present invention using two types of fluorescent dyes.
[0030]
The reaction carrier is irradiated with laser light. Laser light emitted from the laser oscillators 141 and 142 is synthesized by a dichroic mirror 151 that reflects light having the wavelength of the laser light 142 and transmits light having the wavelength of the laser light source 141, and then is beamed by a beam expander including lenses 152 and 153. The diameter is expanded, the light is reflected by a ring-shaped mirror 155, condensed by a condenser lens 154, and irradiated to the reaction part of the reaction carrier. The fluorescence emitted from the labeling probe by being excited by the laser is collected by the condenser lens 154, passes through the center portion of the ring-shaped mirror 155, and further reflects the fluorescence derived from one laser beam, and The fluorescence is separated by a dichroic mirror 156 that transmits the light derived from the other laser beam. These fluorescences are picked up by the band-pass filters 157 and 158, and are picked up by the high-sensitivity CCD cameras 161 and 162 by the imaging lenses 159 and 160, respectively. The signal taken by the high-sensitivity CCD camera is processed by the image processing microprocessor 164 for each pixel to identify the fluorescence emission position on the reaction carrier. When the fluorescence emission positions of the two types of fluorescent dyes coincide with each other, it is determined that the labeled probe is hybridized with the target DNA, and this position is counted to obtain the number of molecules of the target DNA. It is considered that the labeled probe is adsorbed nonspecifically at a site where only the fluorescence derived from either one of the fluorophores is detected, and is excluded from the object of counting. In FIG. 7, reference numeral 163 denotes a camera controller, and reference numeral 165 denotes an output device.
[0031]
Note that the fluorescence detection apparatus can be a measurement apparatus having a confocal optical system, similarly to the fluorescence detection apparatus described in (2).
[0032]
[Example 4]
Detection of polynucleotide (1)
(1) In this example, DNA of bacteriophage λ was used as a target polynucleotide sample to be detected.
Then, the target polynucleotide sample was detected using two kinds of probes for the DNA of the bacteriophage λ.
[0033]
The polynucleotide serving as the first probe is a polynucleotide chain having the base sequence shown in SEQ ID NO: 1, and for each base of the base sequence 6601 to 6875 of the DNA sequence of the target bacteriophage λ, A complementary strand of polynucleotide. The polynucleotide that serves as the second probe is a polynucleotide chain that has the base sequence shown in SEQ ID NO: 2 and can bind to different sites on the DNA of the target bacteriophage λ. An amino group for immobilization on the reaction carrier is introduced at the 5 ′ end of the first probe. Biotin is bound to the 5 ′ end of the second probe and the thymine residues of Nos. 27, 57, 89, 130, and 162 for introduction of the phosphor B-phycoerythrin.
(2) {circle over (1)} The bacteriophage λ DNA, which is the target polynucleotide sample, was λDNA (derived from bacteriophage λ cI857 Sam 7) manufactured by Takara Shuzo.
(2) The polynucleotide chain used as the first probe was synthesized according to the phosphoramidide method. In the final stage of the synthesis, N-monomethoxytritylaminohex-6-oxy-β-cyanothiel-N, N-diisopropylaminophosphoamidide was reacted to introduce an amino group at the 5 ′ end.
(3) The second probe was synthesized as follows.
[0034]
The polynucleotide having the base sequence shown in SEQ ID NO: 2 was divided into oligonucleotides having a length of 40 to 60 bases, and each was chemically synthesized by the phosphoramidide method. At that time, in the final stage of the synthesis of the 1st to 20th polynucleotides, N-monomethoxytritylaminohexa-6-oxy-β-cyanoethyl-N, N-diisopropylaminophosphoamidide is reacted to convert the amino group to 1 Introduced at the 5 'end of the second nucleotide. In addition, in other polynucleotides, amino groups were introduced at the 5-position of uridine instead of thymidine at sites 27, 57, 89, 130 and 162 where thymidine originally remained (Formula 1) Synthesis of each polynucleotide was completed by introducing nucleotides in which the 4′-dimethoxytrityl group of the aminated thymidine was protected at the 4′-hydroxy group.
[0035]
[Chemical 1]

[0036]
The aminated thymidine of (Formula 1) was prepared according to the method of “Geoffrey B. et al., Proc. Natl. Acad. Sci. USA., Vol. 82, pp. 968-972, (1985)”. .
Then, each synthesized polynucleotide having an amino group was ligated using T4 ligase according to the base sequence shown in SEQ ID NO: 2 to obtain a polynucleotide having 6 amino groups. The free amino group is then trifluoroacetylated and the 3 'hydroxyl group activated with chloro-N, N-diisopropylaminomethoxyphosphine, to which sulfosuccinimidyl 6- (biotinamide) hexanoate (PIERCE (IL) USA)) under the known conditions, and each amino group was biotinylated to obtain the desired biotinylated probe.
[0037]
(3) Immobilization of the first probe having the base sequence shown in SEQ ID NO: 1, which was aminated at the 5 'end, which was a probe for capturing the target polynucleotide, to the reaction carrier was carried out in a nitrogen stream as follows. .
First, the surface of the reaction carrier shown in Example 1 made of glass was sufficiently washed, and 3- (2-aminoethylaminopropyl) trimethoxysilane (32 mmol / L) was allowed to act on the reaction part 91, and the reaction part was subjected to amino A group was introduced. The aminosilanized reaction carrier was activated with 2.5% glutaraldehyde aqueous solution, and the parts other than the reaction part 91 were masked with a tape. Then, 10 pmol of the first probe was added to the reaction part, and the reaction was performed at room temperature for 3 hours. Reacted. Next, the tape was removed and the unreacted part was masked with 50 mM ethanolamine. This reaction carrier is a hybridization buffer comprising 0.5% SDS, 100 μg / ml carrier DNA (denatured salmon sperm DNA) 5 mg / ml bovine serum albumin, 50% formamide, 50 mM Tris · HCl containing 0.5% NaCl. Saved in.
[0038]
(4) The hybridization reaction was performed according to the following procedure using the apparatus shown in Example 2.
The concentration of bacteriophage λ DNA serving as a sample is the molar extinction coefficient = 5.18 × 10 6. ⁸ M ^-1 ・ Cm ^-1 As sought. Various concentrations of bacteriophage λDNA containing 100 μg / ml denatured salmon sperm DNA added as carrier DNA were placed in a container 91 and set on a container holding stage 92. The sample was heated to 95 ° C. by the heater 4 to denature the bacteriophage λDNA into a single-stranded state. Next, 10 μl of the sample in the container 1 is sucked up and set on the reaction carrier stage 9 and added to the sample addition part (described in 92 of FIG. 3) of the reaction carrier 10 prepared in (3), and the reaction carrier 10 is added to the stepping motor 11. The sample solution was introduced into the reaction part (described in 91 in FIG. 3) at an inclination of about 30 °. The sample solution was gently stirred by tilting the reaction carrier 10 every 30 seconds and putting the sample solution into and out of the reaction section. The reaction carrier stage 9 has a heater inside, and was set to 65 ° C. in this example. After 4 hours, the reaction residue was discharged using a roll-shaped filter paper 12 into the reaction residue discharge part (described in 93 of FIG. 3) of the reaction carrier 9.
[0039]
After discharging the filtrate, the biotinylated probe 1 × 10 prepared in (2) above is used. ^-10 mol / L to 1 × 10 ^-9 10 μl of mol / L was added to the reaction carrier reaction part. The operation is the same as the above sample addition method. The reaction solution was gently stirred by tilting the carrier stage in the same manner as the sample reaction. After 4 hours, the unreacted biotinylated probe residual solution was discharged by applying the filter paper 12 to the reaction residual solution discharge part (described in 93 in FIG. 3). With the filter paper still applied, the cleaning liquid nozzle 16 mounted on the Z-axis stage 15 was moved to the reaction carrier addition section, and the cleaning liquid was added using the pump 17.
[0040]
Specifically, after washing with 0.3 M citrate buffer (pH 7.0) containing 0.1% sodium dodecyl sulfate 5 mg / ml, bovine serum albumin, 0.5 M NaCl for 25 minutes, 0.1% dodecyl sulfate was further added. The plate was washed with a solution of sodium, 2 mM EDTA, 3M tetramethylammonium, tris-hydrochloric acid (pH 8.0) at 58 ° C. for 30 minutes. Next, it was washed with 0.05% Tween 20, 5 mg / ml (milliliter) bovine serum albumin, 0.15 mol / L (liter) NaCl, 0.05 mol / L (liter) phosphate buffer (pH 7.4). did.
[0041]
Next, streptavidinated B-phycoerythrin (Molecular Probes, Inc.) (concentration of about 1 × 10 ⁹ 0.05% Tween 20, 5 mg / ml bovine serum albumin, diluted with 0.05 mol / L phosphate buffer (pH 7.4) containing 0.15 M NaCl) was added. The operation method is the same as the sample addition method. After reacting at 37 ° C. for 1 hour, it was washed with 0.05% Tween 20, 5 mg / ml bovine serum albumin, 0.15 mol / L NaCl, 0.05 mol / L phosphate buffer (pH 7.4), The biotinylated probe captured on the reaction carrier was labeled with B-phycoerythrin. The bacteriophage λ DNA, which is the target polynucleotide captured on the reaction carrier by this operation, was labeled with B-phycoerythrin, which is a fluorescent substance.
[0042]
(5) In order to detect the phosphor-binding portion on the reaction carrier after the reaction, measurement was performed with the detection apparatus of Example 3 using a YAG laser (532 nm) and a photomultiplier tube capable of photon counting. The light of the laser light source 101 is expanded by the beam expander 102 and reflected by the dichroic mirror 103. In this embodiment, the detection device used is a dichroic mirror 103 that reflects light of 532 nm and transmits light of 575 nm or more by 80% or more.
[0043]
Fluorescence generated by the labeled sample on the reaction carrier is transmitted through the dichroic mirror 103, detected as a signal by the photomultiplier tube 111, processed by the microprocessor 112, and angular information from the servo motors 105 and 107. The fluorescence intensity at each position on the surface of the reaction carrier was calculated by processing in synchronization with. A calibration curve was created based on such data.
[0044]
The results are shown in FIG.
In the same manner, bacteriophage λ, which is a target polynucleotide, is captured on the reaction carrier, labeled with B-phycoerythrin, which is a fluorescent substance, and fluorescent in photon counting mode using a fluorometer as a conventional detection method. The result of measuring the strength is also indicated by ○. As a result, it was found that according to the present invention, the target polynucleotide, bacteriophage λ, can be measured with a sensitivity higher than that of the conventional method by one digit or more. That is, a high-sensitivity change due to background light removal by measuring the fluorescence measurement region divided into minute regions was confirmed.
[0045]
[Example 5]
Detection of polynucleotide (2)
(1) In this example, as in Example 4, pacteriophage λ DNA was used as the target polynucleotide sample to be detected.
Then, the target polynucleotide sample was detected using three types of probes against the DNA of the bacteriophage λ.
[0046]
As the first probe for capturing the target polynucleotide, a polynucleotide chain having the base sequence shown in SEQ ID NO: 1 was used as in Example 4. In addition, the polynucleotide having the base sequence shown by SEQ ID NO: 3 as the polynucleotide used as the first labeling probe has the base sequence shown by SEQ ID NO: 4 as the polynucleotide used as the second labeling probe. Polynucleotide was used. The polynucleotide used as the third labeled probe on the DNA of the target polynucleotide bacteriophage λ is a sequence adjacent to the 3 ′ end of the polynucleotide used as the second labeled probe.
[0047]
A sulforhodamine 101-like fluorophore is bound to the 5 ′ end of the polynucleotide used as the second labeling probe via a poly-T spacer. In this example, a particle polymer having a diameter of 0.02 μm and having a carboxyl group on its surface was used as such a fluorophore. Such particle polymers include Molecular Probes, INC. Carboxylate-modified Lafex red (580/605) manufactured by the same company was used. The labeling of the second probe with particles containing the sulforhodamine 101-like fluorophore was performed by the following method.
That is, in the final synthesis step of the polynucleotide, N-monomethoxytritylaminohex-6-oxy-β-cyanoethyl-N, N-diisopropylaminophosphoamidite is reacted to introduce an amino group at the 5 ′ end. The carboxyl groups of the particles were activated and bound with water-soluble carbodiimide. And after coupling | bonding, the unreacted carboxyl group was reproduce | regenerated under weak alkali conditions.
[0048]
A fluorescein-like fluorophore is bound to the 3 ′ end of the polynucleotide used as the third labeling probe via a poly-T spacer. In this example, a particle polymer having a carboxyl group on the surface having a diameter of 0.02 μm containing the fluorescent dye was used. Examples of such particle polymers include Molecular Probes, INC. Carboxylate-modified Latex Yellow-green (490/515) manufactured by the company was used. And the label | marker of the 3rd probe by the particle | grains containing the said fluorescein fluorophore was performed with the following method. That is, after synthesis of the polynucleotide, Larry E. described in Analytical Biochemistry. According to the method of Morrison et al. (Larry E. Morrison et al., Analytical Biochemistry, 183, pp. 231-244 (1989)), 8- (6-aminohexyl) aminoadenosine was bound using terminal deoxynucleotidyl transferase. Then, an amino group was introduced at the 3 ′ end of the polynucleotide, and this was combined with a particle polymer in which the carboxyl group was activated with water-soluble carbodiimide, to introduce the fluorophore. And after coupling | bonding, the unreacted carboxyl group was reproduce | regenerated under weak alkali conditions.
[0049]
These prepared second and third labeled probes were suspended and stored in the same pH 7 hybridization buffer as in Example 4 containing 0.05% SDS and 100 μg / ml carrier DNA (denatured salmon sperm DNA).
(2) The hybridization reaction was performed according to the method described in Example 4 using the apparatus shown in Example 2. The second and third labeled probes were mixed in advance and added simultaneously by a single pipetting operation.
[0050]
(3) The position of the fluorescent probe captured on the surface of the reaction carrier was measured with the detection device described in Example 3 (2).
The laser light emitted from the laser oscillators 141 and 142 is synthesized by a dichroic mirror 151 that reflects 488 nm light and transmits 590 nm light, and then expands the beam diameter with a beam expander comprising lenses 152 and 153, thereby producing a ring-shaped laser beam. The light is reflected by the mirror 155, condensed by the condenser lens 154, and irradiated to the reaction part of the reaction carrier. In the present invention, a variable wavelength dye laser (wavelength 590 nm) excited by an Ar laser and an Ar laser (wavelength 488 nm) were used as lasers. Fluorescence emitted from the labeled probe by being excited by the laser is collected by the condensing lens 154, passes through the central portion of the ring-shaped mirror 155, reflects 510 nm light, and transmits 610 nm light. A dichroic mirror 156 was used to separate fluorescence derived from a fluorescein-like fluorophore and fluorescence derived from a sulforhodamine 101-like fluorophore. These fluorescences were extracted by the bandpass filters 157 and 158, respectively, and imaged by the high sensitivity CCD cameras 161 and 162 by the imaging lenses 159 and 160, respectively. The signal captured by the high-sensitivity CCD camera was processed for each pixel by the image processing microprocessor 164 to identify the fluorescence emission position on the reaction carrier. Each pixel corresponds to about 350 nm square on the surface of the reaction carrier. If the fluorescence emission position derived from the fluorescein-like fluorophore coincides with the fluorescence emission position derived from the sulforhodamine 101-like fluorophore, it is judged that the labeled probe is hybridized to the target DNA, and this position is counted and the target DNA is counted. The number of molecules. It was considered that the labeled probe was adsorbed nonspecifically at the site where only the fluorescence derived from either one of the fluorophores was detected, and was excluded from the target of counting.
[0051]
The results of measuring the amount of bacteriophage λ at various concentrations based on the above method are shown in FIG. Here, ● indicates only when two types of fluorescent probes are detected at the same position. According to the present invention, it can be seen that bacteriophage λ can be detected with high sensitivity. In addition, (circle) capture | acquires bacteriophage (lambda) which is a target polynucleotide on a reaction support | carrier by the same operation, labels with sulforhodamine 101-like fluorophore and fluororescein-like fluorophore, and is a conventional fluorophore which is a detection amount It is a result of setting the fluorescence intensity in the photon counting mode.
[0052]
【The invention's effect】
According to the present invention, there is no need for radioisotope labeling, high sensitivity, high accuracy, quantitative measurement is possible, and a large amount of measurement samples per unit time can be obtained by automation. A polynucleotide detection method capable of examining a specimen, and a detection apparatus using the method are provided.
[Sequence Listing]
SEQ ID NO: 1
Sequence length: 275
Sequence type: Nucleic acid
Number of chains: 1 strand
Topology: Linear
Sequence type: other sequences Synthetic polynucleotides containing other structures
Sequence features: 1 NH ₂ (CH ₂ ) ₆ Addition

SEQ ID NO: 2
Sequence length: 200
Sequence type: Nucleic acid
Number of chains: 1 strand
Topology: Linear
Sequence type: other sequences Synthetic polynucleotides containing other structures
Sequence characteristics: 27, 57, 89, 130, 162 Biotin addition

SEQ ID NO: 3
Sequence length: 65
Sequence type: Nucleic acid
Number of chains: 1 strand
Topology: Linear
Sequence type: other sequences Synthetic polynucleotides containing other structures
Sequence Features: 1 Particles containing sulforhodamine 101-like fluorophore

SEQ ID NO: 4
Sequence length: 65
Sequence type: Nucleic acid
Number of chains: 1 strand
Topology: Linear
Sequence type: other sequences Synthetic polynucleotides containing other structures
Sequence features: 1 particle containing fluorescein-like fluorophore

[Brief description of the drawings]
FIG. 1 is a conceptual diagram of a conventional fluorescence measurement method and a fluorescence measurement method based on the present invention.
FIG. 2 is a conceptual diagram of the reaction of the method according to the present invention.
FIG. 3 is a schematic view of the reaction carrier of the present invention.
FIG. 4 is a configuration diagram of the reaction apparatus of the present invention.
FIG. 5 is a conceptual diagram of the measurement apparatus of the present invention.
6 is a measurement result according to Example 1. FIG.
FIG. 7 is a conceptual diagram of the measurement apparatus of the present invention using two types of fluorescent dyes.
8 is a measurement result according to Example 2. FIG.

Claims (3)

The labeled probe that is hybridized with a target polynucleotide immobilized on a reaction carrier and a plurality of types of labeled probes that are complementary to the target polynucleotide and labeled with different fluorescent substances, respectively. a polynucleotide detection method of detecting, poly and detecting the fluorescence intensity of the fluorescence which the surface of the reaction carrier by dividing measured minute regions, derived from the different phosphors in each fine small area Nucleotide detection method. The polynucleotide detection method according to claim 1, wherein the distance between the different fluorescent substances in the hybrid of the plurality of types of labeled probes and the target polynucleotide is within 2000 base length. The amount of the target polynucleotide is quantified by detecting the fluorescence intensity of the fluorescence derived from the phosphor and counting the number of minute regions emitting fluorescence with a certain fluorescence intensity. The polynucleotide detection method as described.

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