JP3963422B2 - Nucleic acid measurement method - Google Patents

Description

【０１１６】
実施例２
殺菌したニュウトリエントブロス（ＮＢ）（Difco社製）液体培地５０ｍｌ（組成：ＮＢ、０．０８ｇ／１００ｍｌ）が添加された２００ｍｌ容の（殺菌された）三角フラスコを用いて、大腸菌ＪＭ１０９株を３７℃で一晩振蘯培養した。次に、本培養液に９９．７％エタノールを当量添加した。このエタノール添加培養液２ｍｌを２．０ｍｌ容量のエッペンドルフ遠心チューブで遠心分離し、菌体を得た。３０ｍＭリン酸緩衝液（ソーダ塩）（ｐＨ：７．２）１００μｌで菌体を一回洗浄した。菌体を１３０ｍＭのＮａＣｌ含有の前記リン酸緩衝液１００μｌに懸濁した。当該懸濁液を氷冷中で４０分間超音波処理し（出力：３３ｗ、発振周波数：２０ｋＨｚ、発振法：０．５秒発振、０．５秒休止）、ホモジネートを作製した。
【０１１７】
前記ホモジネートを遠心分離した後、上澄液を採取し蛍光光度計のセルに移した。それを３６℃に温調した。それに３６℃に予め加温した前記核酸プローブの溶液を最終濃度で５ｎＭとなるように添加した。３６℃に温調しながら９０分間大腸菌１６ＳｒＲＮＡと核酸プローブとをハイブリダイズさせた。そして蛍光光度計で蛍光色素の発光量を測定した。
【０１１８】
ハイブリダイゼーション前の蛍光色素の発光量は、上記の上澄液の代りに、１３０ｍＭのＮａＣｌ含有の３０ｍＭのリン酸緩衝液（ソーダ塩）（ｐＨ：７．２）を用いて測定した値を採用した。核酸プローブの量と上澄液の量の比を変えて発光量を測定した（励起光５０３ｎｍ；測定蛍光色５１２ｎｍ）。その結果を図１に示した。図１から分かるように、上澄液の量比が増加することにより蛍光色素の発光が減少した。即ち、本発明においては、核酸プローブにハイブリダイズする標的核酸量に比例して、蛍光色素の発光の減少量の大きさが大きくなることが分かる。
【０１１９】
実施例３
核酸プローブの調製：大腸菌ＪＭ１０９株の２３ＳｒＲＮＡにハイブリダイズする(5')CCCACATCGTTTTGTCTGGG(3')の塩基配列をもつオリゴヌクレオチドの５’末端ヌクレオチドの３’位炭素のＯＨ基に、−（ＣＨ₂)₇−ＮＨ₂を結合したものを、実施例１と同様にメドランド・サーテイファイド・レージント・カンパニー社（Midland Certified Reagent Company、米国）から購入した。更に、実施例１と同様にモレキュラープローブ（Molecular Probes）社からフロオ・リポーターキット（FluoReporter Kit) F-6082 (ボデピーＦＬのプロピオン酸サクシニミジルエステル(BODIPY FL propionic acid succinimidyl ester）の他に、当該化合物をオリゴヌクレオチドのアミン誘導体に結合する試薬を含有するキット）を購入した。当該キットを前記オリゴヌクレオチドに作用させて、ボデピーＦＬで標識した核酸プローブを合成した。得られた合成物を実施例１と同様に精製して、最初のオリゴヌクレオチド原料２ｍＭより２５％の収率でボデピーＦＬで標識した核酸プローブを得た。
【０１２０】
実施例４
実施例２で得られた大腸菌ＪＭ１０９株の菌体に実施例２と同一の培地及び培養条件で調製したシュウドモナス・プウシモビルス ４２１Ｙ株（Pseudomonas paucimobilis）（現在名：スフィンゴモナス・プウシモビルス）（FERM P-5122）の菌体をＯＤ６６０値で大腸菌ＪＭ１０９株と同濃度混合し、複合微生物系を調製した。得られた混合液（大腸菌ＪＭ１０９株の菌体濃度は実施例２と同一）について実施例２と同じ方法によりホモジネートを調製した。実施例３で調製した核酸プローブを用いて、励起光を５４３ｎｍ、また、測定蛍光色を５６９ｎｍにする以外は、実施例２と同様な実験を行った結果、実施例２と同様な結果を得た。
【０１２１】
実施例５
蛍光消光現象における標的核酸の塩基選択性、即ち、本発明の塩基特異性を検討した。下記に示す標的合成デオキシリボオリゴヌクレオチド（３０mer）の１０種類（poly a〜poly j）をＤＮＡ合成機ABI394（Perkin Elmer社製、米国)で調製した。
【０１２２】
更に、上記の合成ＤＮＡに対応するデオキシリボオリゴヌクレオチドの５’末端にボデピーＦＬで標識した下記に示す本発明のプローブを調製した。
上記の合成ＤＮＡに対応するプライマーＤＮＡの５’末端のリン酸基に、−（CH₂)₆-NH₂を結合したものをメドランド・サーテイファイド・レージント・カンパニー社（Midland Certified Reagent Company、米国）から購入した。更に、モレキュラープローブ（Molecular Probes）社からフロオ・リポーターキット（FluoReporter Kit）F-6082（ボデピーFL/C6のプロピオン酸サクシニジルエステル（BODIPY FL propionic acid succinidyl ester)の他に、当該化合物をオリゴヌクレオチドのアミン誘導体に結合させる試薬を含有するキット）を購入した。当該キットを前記購入のプライマーＤＮＡに作用させて下記のボデピーＦＬで標識した本発明のプローブprobe a〜d、及びf〜hのを合成した。そして対応する合成デオキシリボオリゴヌクレオチドとハイブリダイズしたときに、蛍光色素の発光がどの程度減少するかを下記の条件下に調べ、本発明のプローブの特異性を検討した。
【０１２３】

【０１２４】

【０１２５】

【０１２６】

【０１２７】

【０１２８】

【０１２９】
その結果を表１に示した。表１から分かるように、蛍光色素で標識された核酸プローブが標的ＤＮＡ（オリゴデオキシリボヌクレオチド）にハイブリダイズしたときに、当該末端部において、当該プローブと標的ＤＮＡとがハイブリダイズした末端塩基部から１〜３塩基離れて、標的ＤＮＡの塩基配列にＧ（グアニン）が少なくとも１塩基以上存在するように、当該プローブの塩基配列が設計されていることが好適である。また、蛍光色素で標識された核酸プローブが標的ＤＮＡにハイブリダイズしたときに、当該末端部において核酸ハイブリッド複合体の複数の塩基対がＧとＣのペアーを少なくとも一対以上形成するように、当該プローブの塩基配列が設計されていることが好適であることが表１から分かる。
【０１３０】
実施例６
下記のような塩基配列の標的核酸と本発明の核酸プローブを調製した。標的核酸内のＧ及び本発明の核酸プローブ内のＧの数の影響について、前記実施例と同様にして調べた。
【０１３１】

【０１３２】

【０１３３】

【０１３４】

【０１３５】

【０１３６】
表２から分かるように、標的核酸内のＧの数、本発明のプローブ内のＧの数はそれほど蛍光強度の減少に影響しないことが認識される。
【０１３７】
実施例７
前記と同様にして下記のような塩基配列の標的核酸と本発明の核酸プローブを調製した。なお、本実施例における本発明の核酸プローブはオリゴヌクレオチドの５’末端部をボデピーFL/C6で標識したものである。標的核酸内の塩基種及び本発明の核酸プローブ内の塩基種の影響について、前記実施例と同様にして調べた。
【０１３８】

【０１３９】

【０１４０】

【０１４１】
表３及び前記の実施例から分かるように、(i)蛍光色素で標識される本発明のプローブの末端がＣで構成され、標的核酸がハイブリダイズしたとき、ＧＣペアーを形成するとき、(ii)蛍光色素で標識される本発明のプローブの末端がＣ以外の塩基で構成された場合、蛍光色素で標識されている個所の塩基と、標的核酸の塩基との塩基ペアーより、標的核酸の３’末端側にＧが少なくとも１個以上存在する場合に、蛍光強度の減少率が大きい。
【０１４２】
実施例８
本発明の核酸プローブに標識する色素の種類について、前記実施例と同様にして調べた。なお、本発明のプローブは、前記実施例７のプローブｚを、また、標的核酸は前記実施例７のオリゴヌクレオチドｚを用いた。
その結果を、表４に示した。表から分かるように、本発明に用いる蛍光色素として好適なものは、FITC、 BODIPY FL、 BODIPY FL/C3、 6-joe、TMRなどを挙げることができる。
【０１４３】

【０１４４】
実施例９（標的核酸（１６ＳｒＲＮＡ）の加熱処理の効果の実験）
本発明の核酸プローブの調製：大腸菌ＪＭ１０９株の１６ＳｒＲＮＡの１１５６塩基目から１１９０塩基の塩基配列に相当するＫＹＭ−７株の１６ＳｒＲＮＡ塩基配列に特異的にハイブリダイズする(5')CATCCCCACC TTCCT CCGAG TTGACCCCGG CAGTC(3')（３５塩基対）の塩基配列をもち、１〜１６及び２５〜３５塩基目がデオキシリボヌクレオチド、１７〜２４塩基目が２’位炭素のＯＨ基をメチル基で修飾（エーテル結合で修飾）したリボオリゴヌクレオチドからなり、その５’末端のリン酸基のＯＨ基を-(CH₂)₇-NN₂で修飾して結合したオリゴヌクレオチドを、実施例１と同様にメドランド・サーテイファイド・レージント・カンパニー社（Midland Certified Reagent Company、米国）から購入した。また、2-O-Meプローブ（2-O-Meオリゴヌクレオチドからなるプローブを単に2-O-Meプローブという。）に使用する2-O-Meオリゴヌクレオチドは、ＧＥＮＳＥＴ株式会社（フランス）に合成を委託し、得たものである。
更に実施例１と同様にモレキュラープローブ（Molecular Probes）社からフロオ・リポーターキット（FluoReporter Kit) F-6082 （ボデピーFL/C6のプロピオン酸サクシニミジルエステル（BODIPY FL/ C6 propionic acid succinimidyl ester）の他に、当該化合物をオリゴヌクレオチドのアミン誘導体に結合する試薬を含有するキット）を購入した。当該キットを前記オリゴヌクレオチドに作用させて、ボデピーFL/C6で標識した本発明の核酸プローブを合成した。得られた合成物を実施例１と同様に精製して、最初のオリゴヌクレオチド原料２ｍＭより２３％の収率でボデピーFL/C6で標識した本発明の核酸プローブを得た。このプローブを３５塩基鎖2-O-Meプローブと名づけた。
【０１４５】
5')TCCTTTGAGT TCCCGGCCGG A (3')の塩基配列を有するオリゴリボヌクレオチドを前記同様にしてＤＮＡ合成機を用いて合成して、フォワード（forward）型のヘルパープローブとした。一方、(5')CCCTGGTCGT AAGGGCCATG ATGACTTGAC GT(3')の塩基配列を有するオリゴリボヌクレオチドを前記同様にしてＤＮＡ合成機を用いて合成して、バックワード(back ward) 型すなわちリバース型ヘルパープローブとした。
【０１４６】
３５塩基鎖2-O-Meプローブ、フォワード型のヘルパープローブ及びリバース型ヘルパープローブを各々25nMになるように下記に示す組成の緩衝液に溶解させ、７５℃に加温した（プローブ溶液）。
前記の１６ＳｒＲＮＡを９５℃で５分加熱処理した後、それを下記に示す反応条件においた前記のプローブ溶液に添加した後、蛍光測定機器パーキンエルマー（Perkin Elmer) LS-50Bで蛍光強度を測定した。その結果を図２に示した。尚、上記の加熱処理しない１６ＳｒＲＮＡを用いたものをコントロールとした。加熱処理した実験区においては、蛍光強度の減少が大きいことが図２から分かる。この結果は、１６ＳｒＲＮＡを９５℃で加熱処理することで本発明のプローブとより強いハイブリダイゼーションをしていることを示している。

【０１４７】
実施例１０（2'-O-Meオリゴヌクレオチド及びヘルパープローブのハイブリダイゼーション効率向上への寄与の実験）
前記の１６ＳｒＲＮＡにハイブリダイズする下記の各種の本発明の核酸プローブ及び各種のヘルパープローブを前記実施例９と同様にして調製した。また、2-O-Meプローブに使用する2-O-Meオリゴヌクレオチドは、すべてGENSET株式会社(フランス)に合成を委託し、得たものである。そして下記の条件にて、本発明の2-O-Meプローブの効果、当該プローブの塩基鎖の長さの影響、及びヘルパープローブの効果について、下記の図３Ａ、Ｂ、Ｃ、及びＤの実験系で前記実施例９と同様にして検討した。その結果を図３に示した。
図から、本発明の2-O-Meプローブがハイブリダイゼーション効率に寄与していることが分かる。また、2-O-Meプローブの塩基鎖が短い場合にヘルパープローブがハイブリダイゼーション効率を高めるのに役立っている。
【０１４８】
１）３５塩基鎖2-O-Meプローブ： 前記実施例９と同じプローブ、
２）３５塩基鎖ＤＮＡプローブ： 前記１）の３５塩基鎖2-O-Meプローブと同じ塩基配列であるが、オリゴヌクレオチドがデオキシリボースで構成されているプローブ、
３）１７塩基鎖2-O-Meプローブ： 前記１）の３５塩基鎖2-O-Meプローブと同じ塩基配列であるが、５’末端から８塩基分、３’末端から１０塩基分のヌクレオチドを削除したプローブ、
４）１７塩基鎖ＤＮＡプローブ： 前記２）の３３塩基鎖DNAプローブと同じ塩基配列であるが、３’末端から１６塩基分のヌクレオチドを削除したプローブ、
【０１４９】
５）フォワード型2-O-Meヘルパープローブ: 前記実施例９のフォワード型ヘルパープローブの中央８塩基分（５’末端から数えて９塩基〜１６塩基分）のリボースの２’位炭素のＯＨ基を、メチル基で修飾（エーテル結合）したヘルパープローブ、
６）リバース型2-O-Meヘルパープローブ： 前記実施例９のリバース型ヘルパープローブの中央８塩基分（５’末端から数えて９塩基〜１６塩基分）のリボースの２’位炭素のＯＨ基を、メチル基で修飾（エーテル結合）したヘルパープローブ、
７）フォワード型ＤＮＡヘルパープローブ: 前記実施例９のフォワード型ヘルパープローブの塩基配列と同じ塩基配列であるが、オリゴヌクレオチドがデオキシリボヌクレオチドで構成されているヘルパープローブ、
８）リバース型ＤＮＡヘルパープローブ: 前記 前記実施例９のリバース型ヘルパープローブの塩基配列と同じ塩基配列であるが、オリゴヌクレオチドがデオキシリボヌクレオチドで構成されているヘルパープローブ、
９）３５塩基オリゴリボヌクレオチド: (5')CATCCCCACC TTCCTCCGAG TTGA CCCCGG CAGTC(3')なる塩基配列を有するオリゴリボヌクレオチド、
１０）１７塩基鎖オリゴリボヌクレオチド： (5')CCTTCCTCCG AGTTGAC(3')なる塩基配列を有するオリゴリボヌクレオチド。
【０１５０】

【０１５１】

【０１５２】

【０１５３】

【０１５４】

【０１５５】
実施例１１（ｒＲＮＡ測定のための検量線の作成）
前記ｒＲＮＡを、０．１〜１０ｎＭの範囲のさまざまな濃度において、９５℃で５分間加熱後、得られた核酸溶液を予め下記反応条件においた反応液に添加し、１０００秒後、蛍光強度の減少をパーキンエルマーLS-50Bを使用して測定した。その結果を図４に示した。図から検量線は０．１〜１０ｎＭにおいて直線性を示すことが分かる。なお、下記の３５塩基鎖2-O-Meプローブは実施例９と同じプローブである。

【０１５６】
実施例１２(FISH方法)
セルロモナス（Cellulomonas）sp.KYM-7（FERM P-11339）及びアグロバクテリウム（Agrobacterium) sp. KYM-8(FERM P-16806)の各々のｒＲＮＡにハイブリダイズする下記の本発明の35又は36塩基鎖オリゴデオキシリボヌクレオチド 2-O-Meプローブを前記と同様にして調製した。各プローブの塩基配列は下記の通りである。
【０１５７】
セルロモナス sp.KYM-7のｒＲＮＡ測定のための３５塩基鎖オリゴデオキシリボヌクレオチド2-O-Meプローブ：
（5')CATCCCCACC TTCCTCCGAG TTGACCCCGG CAGTC(3')（アンダーライン部分がメチル基で修飾されている。）。
アグロバクテリウム sp. KYM-8のｒＲＮＡ測定のための３６塩基鎖オリゴデオキシリボヌクレオチド2-O-Meプローブ：
(5')CATCCCCACC TTCCTCTCGG CTTATCACCG GCAGTC(3')（アンダーライン部分がメチル基で修飾されている。）。
【０１５８】
セルロモナス sp. KYM-7及びアグロバクテリウム sp. KYM-8を下記の培地組成の培地で下記の培養条件で混合培養し、培養時間毎に培養物を採取した。それらから、ｒＲＮＡをRNeasy Maxikit（QIAGEN社）を用いて調製した。当該ｒＲＮＡを９５℃で５分加熱後、予め反応条件においた反応液に添加し、７０℃、１０００秒間反応させた後、蛍光強度をパーキンエルマーLS-50Bを使用して測定した。その結果を図５に示した。尚、全rＲＮＡはリボグリーン（RiboGreen) total RNA Quantification Kit （会社名：モレキュラープローブ（molecular probes)、所在地名：Eugene,Oregon, USA）を用いて測定した。
図から分かるように、各菌株のｒＲＮＡの動態は全 rＲＮＡの動態と一致した。また、各菌株のｒＲＮＡの合計量は全ｒＲＮＡと一致した。このことは、本発明方法はFISH方法において有効な方法になることを示している。
【０１５９】
・培地組成（g/l）：デンプン，10.0；アスパラギン酸，0.1； K₂HPO₄，5.0;KH₂PO₄,2.0;MgSO₄・7H₂O ,0.2;NaCl，0.1; (NH₄)₂SO₄;0.1.
・培地１００ｍｌを５００ｍｌ容のコニカルフラスコに分注し、該フラスコを１２０℃で１０分間、オートクレーブ釜を用いて殺菌した。
・培養条件：前記の菌株を斜面培地で予め培養した。該斜面培地より１白金耳の菌体をとり、前記の殺菌したコニカルフラスコの培地に接種した。３０℃、１５０ｒｐｍで撹拌培養した。
【０１６０】

【０１６１】
以下実施例１３に、標的核酸の多型及び変異を解析若しくは測定する方法を記す。
実施例１３
下記に示した塩基配列をもつ４種類のオリゴヌクレオチドを前記実施例５のＤＮＡ合成機を用いて合成した。また、前記実施例５と同様にして、下記の塩基配列の本発明の核酸プローブを合成した。該プローブと各々のオリゴヌクレオチドを溶液中でハイブリダイズさせた後、蛍光強度の変化から１塩基置換の評価ができるかどうか検討した。本発明の核酸プローブの塩基配列は、標的オリゴヌクレオチドのうちのいずれかの３’末端にＧが存在する場合に、そのオリゴヌクレオチドの塩基配列に１００％マッチするように設計されている。ハイブリダイゼーション温度は、プローブと標的オリゴヌクレオチドとの間の全塩基対（base-pairs）が１００％ハイブリダイズできる４０℃に設定した。プローブ及び標的オリゴヌクレオチドの濃度、緩衝液の濃度，蛍光測定装置、蛍光測定条件、実験操作などは、前記実施例５と同様である。
【０１６２】

【０１６３】
その結果を表５に示した。表から、標的オリゴヌクレオチドＮｏ．１〜３においては、蛍光強度に変化は観察されなかったが、標的オリゴヌクレオチドＮｏ．４においては８４％の減少が観察された。
【０１６４】

【０１６５】
本発明において、標的核酸（例えば上記標的オリゴヌクレオチドＮｏ．１〜４）の多型及び／又は変異を解析若しくは測定する方法により得られるデータ（例えば表５のカラムＡ及びＢのデータ）を解析する方法において、標的核酸が本発明の核酸プローブ（上記の核酸プローブ）とハイブリダイズしたときの反応系の蛍光強度値を、前記のハイブリダイズしていないときの反応系の蛍光強度値により補正演算処理するとは、表３の（Ａ−Ｂ）／Ｂの計算をいう。
以上の結果より、標的核酸が２本鎖の場合、Ｇ→Ａ、Ｇ←Ａ、Ｃ→Ｔ、Ｃ←Ｔ、Ｇ→Ｃ、Ｇ←Ｃの置換を検出できることが明らかになった。
【０１６６】
実施例１４
図６に本発明のＤＮＡチップのモデルの一例を図示した。先ず、実施例１３で調製した本発明のプローブである3'TTTTTTTTGGGGGGGGC5'BODIPY FL/C6のの３’末端のリボースの３’位炭素のＯＨ基にアミノ基を導入して調製した修飾プローブ、また、スライドガラスを反応基としてエポキシ基を有するシランカップリン剤でスライドガラスの表面を処理した表面処理済スライドガラスを用意した。上記の修飾プローブを含む溶液をＤＮＡチップ作成装置GMS^TM417ARRAYER(TAKARA)で該表面処理済スライドガラス上にスポットした。そうすると、３’末端で上記修飾プローブがガラス面に結合した。該スライドガラスを密閉容器内に４時間位おき反応を完結させた。そして該スライドガラスを０．２％ＳＤＳ溶液、水に１分程度交互に２回づつ漬けた。更にホウ素溶液（水３００ｍｌにNaBH₄１．０ｇを溶かしたもの。）に５分位つけた。９５℃の水に２分つけてから、素早く０．２％ＳＤＳ溶液、水に１分程度交互に２回づつ漬けて試薬を洗い流した。室温で乾燥した。このようにして本発明のＤＮＡチップを調製した。
【０１６７】
更に、ガラスの下面の修飾プローブの各スポットに対応する位置に図のような微小な温度センサーとヒータを設けることにより、本発明のＤＮＡチップに高性能を付与することができた。
このＤＮＡチップを用いて標的核酸を測定する場合を説明する。該プローブに標的核酸がハイブリダイズしていないとき、又はハイブリダイズしても蛍光色素標識末端でＧＣペアーを形成しないとき、若しくは当該プローブと標的核酸とがハイブリダイズした末端塩基部分から１ないし３塩基離れて、標的核酸の塩基配列にＧ（グアニン）又はシトシン（Ｃ）が少なくとも１つ存在しないときは、蛍光強度に変化ない。しかし、その反対に、該プローブに標的核酸がハイブリダイズしているとき、又はハイブリダイズしても蛍光色素標識末端でＧＣペアーを形成しているとき、若しくは当該プローブと標的核酸とがハイブリダイズした末端塩基部分から１ないし３塩基離れて、標的核酸の塩基配列にＧ（グアニン）又はシトシン（Ｃ）が少なくとも１つ存在するときは蛍光強度が減少する。この蛍光強度はＤＮＡチップ解析装置ＧＭＳ^TM４１８アレースキャナー（Array Scanner）（TAKARA）を使用して測定できる。
【０１６８】
実施例１５：本発明のＤＮＡチップを用いた一塩基多型（ＳＮＰｓ）の検出実験
Ｉ）標的核酸の調製：(5')AAACGATGTG GGAAGGCCCA GACAGCCAGG ATGTTGGCTT AGAAGCAGCC(3')の塩基配列をもつオリゴデオキシリボヌクレオチドを、ＤＮＡ合成機ABI394(Perkin Elmer社製、米国）を用いて合成し、標的核酸とした。
II）核酸プローブの調製：標的核酸の５’末端から１５塩基の配列（アンダーライン部）にハイブリダイズする塩基配列をもつ、下記の６個のオリゴデオキシリボヌクレオチドを、ＤＮＡ合成機ABI394(Perkin Elmer社製、米国）を用いて合成した。そして、3'-Amino-Modifier C7 CPG（グレンリサーチ、カタログ番号20ー2957）を用いて、３’末端のデオキシリボースの３’位のＯＨ基をアミノ化した。更に５’末端のリン酸基を実施例５と同様な方法でBODIPY FLで標識した。
【０１６９】
１）プローブ100（１００％マッチ）：（5')CCTTCCCACA TCGTTT(3')、
２）プローブ−T（１塩基ミスマッチ）：(5')CCTTCCCATA TCGTTT(3')、
３）プローブ−A（１塩基ミスマッチ）: (5')CCTTCCCAAA TCGTTT(3')、
４）プローブ−G（１塩基ミスマッチ）: (5')CCTTCCCAGA TCGTTT(3')、
５）プローブ−TG（２塩基ミスマッチ）:(5')CCTTCCCTGA TCGTTT(3')、
６）プローブ−TGT（３塩基ミスマッチ）:(5')CCTTCCCTGT TCGTTT(3')、
【０１７０】
III）ＤＮＡチップの調製
全てのＤＮＡプローブを、滅菌蒸留水に溶解して、１μＭ濃度の溶液にした。ＤＮＡマイクロアレイヤー装置（ＤＮＡマイクロアレイヤーNo.439702、３２ピン型、およびＤＮＡスライドインデックスNo.439701からなる手動式のチップアレイヤーである。greiner社）を用いて、ＤＮＡチップ用スライドグラス（Black silylated slides、greiner社）の上に、前記プローブ溶液をスポテイング（spotting）した。スポット終了後１０分間反応させ、プローブをスライドグラス上に固定化した。その後、５０ｍＭのＴＥ緩衝液（ｐＨ：７．２）にて洗浄した。なお、各プローブ溶液につき、４スポットづつスポッテングした。
本発明のＤＮＡチップの模式図を図６に示した。スライドグラス上に固定化された本発明のプローブは、標的核酸にハイブリダイズしないときはBODIPY FLは発色しているが、ハイブリダイズしているときは発色が、ハイブリダイズしないときのものよりも少ない。すなわち減少する。スライドグラスはマイクロヒーターで加熱されるようになっている（本発明では、下記に示すように顕微鏡用透明加熱板（MP-10MH-PG、（株式会社）北里サプライ）で行われた。）。
【０１７１】
IV）ＳＮＰｓの検出測定
１００μＭ濃度の標的核酸溶液｛５０ｍＭのＴＥ緩衝液（ｐＨ：７．２）使用｝を上記のごとくに調製したＤＮＡチップの上にのせた。カバーグラスで覆い、標的核酸が漏れないようにマニキュアにてカバーグラスをシールした。
検出測定のための装置類の概略は図７に示した。先ず、オリンパス正立顕微鏡（AX80型）の試料台に顕微鏡用透明加熱板（MP-10MH-PG、（株式会社）北里サプライ）をのせた。当該板上に前記に調製した本発明のＤＮＡチップを置き、９５℃から３３℃まで３℃刻みで変化させ、３０分かけて反応させた。各スポットの反応過程の蛍光強度変化を、冷却ＣＣＤカメラ（C5810型、浜松フォトニクス社）にて画像取り込み形式で測定した。
取り込み画像を画像解析装置（画像解析ソフト（TPlab spectrum； Signal Analytics社，Verginia）がインストールされたパソコン（NEC））にて解析し、各スポットの輝度を算出し、温度と輝度の関係を求めた。
【０１７２】
実験結果を図８に示した。図から全てのプローブで蛍光強度が減少していることが分かる。従って、本発明の方法により、本発明のプローブと標的核酸との解離曲線を簡便にモニタリングすることができる。また、標的核酸と１００％マッチするプローブ100と一塩基ミスマッチするプローブとのＴｍ値の差は１０℃以上あるので、解離曲線から両者を容易に識別することができる。すなわち本発明のＤＮＡチップを使用することによりＳＮＰｓの解析が容易にできることがわかる。
【０１７３】
以下、実施例１６〜１９に本発明のＰＣＲ方法を記す。
実施例１６
大腸菌のゲノムＤＮＡにおける１６ＳｒＲＮＡ遺伝子を標的核酸として、当該核酸の増幅のための（BODIPY FL/C6で標識した）プライマー（本発明の核酸プローブ）を調製した。
【０１７４】
プライマー１（Eu800R：リバース型）の調製：(5')CATCGTTTAC GGCGTGGAC(3')の塩基配列をもつオリゴデオキシリボヌクレオチドを、ＤＮＡ合成機ABI394(Perkin Elmer社製、米国）を用いて合成し、更に当該オリゴデオキシリボヌクレオチドの５’末端のリン酸基をホスファターゼ処理してシトシンとし、そのシトシンの５’位の炭素OH基に、-(CH₂)₉-NH₂を結合したオリゴヌクレオチドを、ミドランド・サーテイファイド・レージンド・カンパニー社（米国）から購入した。更に、モレキュラープローブ社からフロオ・リポーターキット（FluoReporter Kits)F-6082（ボデピーFL/C6のプロピオン酸サクシニミジルエステル（BODIPY FL propionic acid succinimidyl ester）の他に、当該化合物をオリゴヌクレオチドのアミン誘導体に結合させる試薬を含有するキット）を購入した。前記購入したオリゴヌクレオチドに当該キットを作用させて、本発明のボデピーFL/C6で標識したプライマー１を合成した。
【０１７５】
合成物の精製：得られた合成物を乾固し乾固物を得た。それを0.5M Na₂CO₃/NaHCO₃緩衝液（ｐＨ９．０）に溶解した。当該溶解物をＮＡＰ−２５カラム（ファルマシア社製）でゲルろ過を行い、未反応物を除去した。更に逆相HPLC（B gradient：１５〜６５％、２５分間）を以下の条件で行った。そして、溶出するメインピークを分取した。分取した画分を凍結乾燥して本発明のプライマー１を、最初のオリゴヌクレオチド原料２ｍＭより５０％の収率で得た。
【０１７６】

【０１７７】
実施例１７
プライマー２（Eu500R/forward：フォワード型）の調製：(5')CCAGCAGCCG CGGTAATAC(3')の塩基配列をもつオリゴデオキシリボヌクレオチドの５’末端に蛍光色素（BODIPY FL/C6）で標識したプライマー２を実施例１４と同様にして収率５０％で調製した。
【０１７８】
実施例１８
殺菌したニュトリエントブロス(NB)（Difco社製）液体培地５ml（組成：NB、0.08g/100ml）を含有する試験管を用いて、大腸菌JM109株を３７℃で一晩振蘯培養した。培養液１．５mlを１．５ml容量の遠心チューブで遠心分離し、菌体を得た。この菌体から、DNeasy Tissue Kit（キアゲン（QIAGEN）社、ドイツ国）を用いてゲノムＤＮＡを抽出した。その抽出は本キットのプロトコルに従った。その結果、17ng/μlのＤＮＡ溶液を得た。
【０１７９】
実施例１９
上記の大腸菌のゲノムＤＮＡ、プライマー１及び／又はプライマー２を使用して、ロシュ・ダイアグノスティックス株式会社発売のライトサイクラー^TMシステム（LightCycler^TM System）を用いて常法通りにＰＣＲ反応を行った。操作は当該システム機器の手順書に従った。
また、上記システムにおいてＰＣＲは、当該手順書に記されている核酸プローブ（FRET現象を利用する二個のプローブ）と通常のプライマー（蛍光色素で標識されていない通常のプライマー）の代りに本発明プライマー１及び／又は２を用いる以外は当該手順書通りに行った。
【０１８０】
ＰＣＲは次のコンポーネントのハイブリダイゼーション溶液中で行った。

尚、標的核酸である大腸菌１６ＳｒＤＮＡは、図９の説明欄に示される実験区の濃度で、また、プライマーは、同様に図９の説明欄に示される実験区のプライマー１及び／又は２の組合せで実験を行った。
【０１８１】
また、上記のTaq溶液は次の試薬の混合液である。

【０１８２】
尚、Taq 溶液、 Taq DNAポリメラーゼ溶液はロシュ・ダイアグノスティックス株式会社発売のＤＮＡマスターハイブリダイゼーションプローブ（DNA Master Hybridization Probes)キットのものである。特にTaq DNA ポリメラーゼ溶液は10×conc.（赤いキャップ）を１０倍に希釈して用いた。また、Taq スタートは、クローンテック社（ＵＳＡ）より販売されているTaq DNAポリメラーゼ用の抗体で、これを反応液に添加することで７０℃までTaq DNAポリメラーゼの活性を抑えることができる。即ち、ホット・スタート(hot start)を行うことができるものである。
【０１８３】

測定は、ライトサイクラー^TMシステムを用いて行った。その際、該システムにあるＦ１〜３の検出器にうち、Ｆ１の検出器を用い、その検出器のゲインは１０、励起強度は７５に固定した。
【０１８４】
その結果を図９及び１０に示した。図９及び１０から、蛍光色素の発光の減少が観察される時点のサイクル数と標的核酸の大腸菌１６ＳｒＤＮＡのコピー数が比例していることが分かる。尚、図においては、蛍光色素の発光の減少量を蛍光強度の減少値として表現した。
図１１は、サイクル数の関数として、大腸菌１６ＳｒＤＮＡのコピー数を表現した大腸菌１６ＳｒＤＮＡの検量線を示す。相関係数は０．９９７３で、極めてよい相関を示した。
以上の結果から分かるように、本発明の定量的ＰＣＲ方法を用いると標的核酸の当初のコピー数を測定できるようになる。即ち、標的核酸の濃度の測定ができる。
【０１８５】
実施例２０
実施例１９においては、本発明のプローブをプライマーとしてＰＣＲを行ったが、本実施例では従来法に用いるＦＲＥＴ現象を利用する二個のプローブの代わりに本発明のプローブを用いて下記の条件で本発明のＰＣＲを行った。
ａ）標的核酸：大腸菌の１６Ｓ−ｒＤＮＡ
ｂ）使用プライマー：
・フォワードプライマー E8F：(5')AGAGTTTGAT CCTGGCTCAG(3')
・リバースプライマー E1492R：(5')GGTTACCTTG TTACGACTT(3')
ｃ）使用プローブ：BODIPY FL-(5')CGGGCGGTGT GTAC(3')
ｄ）使用ＰＣＲ測定機器：ライトサイクラー^TMシステム
ｅ）ＰＣＲの条件：
変性反応 ：９５℃、１０秒（第一回のみ、６０秒間、９５℃）
アニーリング反応：５０℃、５秒
核酸伸長反応 ：７２℃、７０秒
全サイクル数 ：７０サイクル
【０１８６】

【０１８７】
その結果を図１２に示した。図から、蛍光色素の発光の減少が観察される時点のサイクル数と標的核酸の大腸菌１６ＳｒＤＮＡのコピー数が比例していることが分かる。
以上の結果から分かるように、本発明の定量的ＰＣＲ方法を用いると標的核酸の当初のコピー数を測定できるようになる。即ち、標的核酸の測定ができる。
【０１８８】
次に以下の実施例に、上記の本発明の定量的ＰＣＲ方法を用いて得られるデータを解析する本発明のデータ解析方法について記す。
実施例２１
ヒトゲノムＤＮＡ（ヒトβ−グロビン（globin）（TaKaRaカタログ商品番号 ９０６０）（TaKaRa株式会社製）（以下、ヒトゲノムＤＮＡという。）を標的核酸として、当該核酸の増幅のためのボデピー FL/C6で標識したプライマーを調製した。
【０１８９】
プライマーKM38+C（リバース型）の調製：（5')CTGGTCTCCT TAAACCTGTC TTG(3')の塩基配列をもつオリゴデオキシリボヌクレオチドを、ＤＮＡ合成機ABI394(Perkin Elmer社製、米国）を用いて合成し、更に当該オリゴデオキシリボヌクレオチドの５’末端のリン酸基をホスファターゼ処理してシトシンとし、そのシトシンの５’位の炭素ＯＨ基に、-(CH₂)₉-NH₂を結合したものを、ミドランド・サーテイファイド・レージンド・カンパニー社から購入した。更に、モレキュラープローブ社からリポーターキット（FluoReporter Kit)F-6082（ボデピーFL/C6のプロピオン酸サクシニミジルエステル(BODIPY FL propionic acid succinimidyl esters)の他に、当該化合物をオリゴヌクレオチドのアミン誘導体に結合させる試薬を含有するキット）を購入した。前記購入したオリゴヌクレオチドに当該キットを作用させて、本発明のボデピーFL/C6で標識したプライマーKM38+Cを合成した。
【０１９０】
合成物の精製：得られた合成物を乾固し乾固物を得た。それを0.5M Na₂CO₃/NaHCO₃緩衝液(pH9.0)に溶解した。当該溶解物をNAP-25カラム（ファルマシア社製）でゲルろ過を行い、未反応物を除去した。更に逆相HPLC(B gradient:15〜65%、25分間）を以下の条件で行った。そして、溶出するメインピークを分取した。分取した画分を凍結乾燥して本発明のプライマーKM38+Cを、最初のオリゴヌクレオチド原料2mMより５０％の収率で得た。
【０１９１】

【０１９２】
実施例２１
プライマーKM29（フォワード型）の調製：(5')GGTTGGCCAA TCTACTCCCA GG(3')の塩基配列をもつオリゴデオキシリボヌクレオチドを実施例１８と同様に合成した。
【０１９３】
比較実験例１
本比較実験例は、核酸伸長反応時の蛍光強度値を、熱変性反応時の蛍光強度値を用いて割る演算処理過程（数式（１）の処理）を有しないデータ解析用ソフトウエアの使用に係るものである。
上記のヒトゲノムＤＮＡ、プライマーKM38+C及びプライマーKM29を使用して、ライトサイクラー ^TMシステムを用いてＰＣＲ反応を行い、各サイクル毎の蛍光強度を測定した。
尚、本比較実施例のＰＣＲは、前記に説明した蛍光色素色素で標識したプライマーを用いるものであり、蛍光発光の増加でなく、減少を測定する新規なリアルタイム定量的ＰＣＲ方法である。データ解析は当該システムのソフトウエアを用いて行った。本比較実験例のＰＣＲは、当該手順書に記されている核酸プローブ（ＦＲＥＴ現象を利用する二個のプローブ）と通常のプライマー（蛍光色素で標識されていない通常のプライマー）の代りに本発明プライマーKM38+C及びKM29を用いる以外は当該装置の手順書通りに行った。
【０１９４】
ＰＣＲは次のコンポーネントのハイブリダイゼーション溶液中で行った。

尚、ヒトゲノムＤＮＡは、図１３の簡単な説明欄に示される実験区の濃度で実験を行った。MgCl₂の最終濃度は2mMであった。
【０１９５】
また、上記のTaq溶液は次に試薬の混合液である。

【０１９６】
尚、Taq溶液、 Taq DNA ポリメラーゼ溶液はロシュ・ダイアグノスティックス株式会社発売のＤＮＡマスターハイブリダイゼーションプローブ（DNA Master Hybridization Probes）キットのものである。特にＴaq DNA ポリメラーゼ溶液は10×conc.（赤いキャップ）を１０倍に希釈して用いた。また、Ｔaqスタートは、クローンテック社（ＵＳＡ）より販売されているTaq DNA ポリメラーゼ用の抗体で、これを反応液に添加することで７０℃までTaq DNAポリメラーゼの活性を抑えることができる。即ち、ホット・スタートを行うことができるものである。
【０１９７】
反応条件は次の如くである。

測定は、ライトサイクラー^TMシステムを用いて行った。その際、該システムにあるＦ１〜３の検出器にうち、Ｆ１の検出器を用い、その検出器のゲインは１０、励起強度は７５に固定した。
【０１９８】
前記の如くにＰＣＲを行って、各サイクルの蛍光強度を実測した。その結果を図１３に示す。即ち、各コピー数のヒトゲノムＤＮＡについて、各サイクルの変性反応時及び核酸伸長反応時の蛍光強度を測定し、印字したものである。どのサイクルにおいても変性反応時には蛍光強度値は一定であるが、核酸伸長反応時には、２５サイクル目当たりから蛍光強度が減少しているのが観察される。そうして、減少はヒトゲノムＤＮＡのコピー数が多い順に起こることが分かる。
【０１９９】
図１３に示すようにヒトゲノムＤＮＡの各コピー数について初期のサイクル数の蛍光強度値が一様でなかった。それで、本比較例で使用するデータ解析方法に以下の過程（ｂ）〜（ｊ）を追加した。
（ｂ）１０サイクル目の蛍光強度値を１として各サイクルの蛍光強度値を換算する過程、即ち、下記の〔数式８〕による計算をする過程、
Ｃ_n＝Ｆ_n（７２）／Ｆ₁₀（７２） 〔数式８〕
ただし、Ｃ_n＝各サイクルにおける蛍光強度値の換算値、Ｆ_n（７２）＝各サイクルの７２℃の蛍光強度値、Ｆ₁₀（７２）＝１０サイクル目の７２℃における伸長反応後の蛍光強度値。
（ｃ）前記（ｂ）の過程で得られた各換算値を、サイクル数の関数として、デスプレー上に表示及び／又は印字する過程、
【０２００】
（ｄ）前記（ｂ）の過程で得られた各サイクルの換算値から下記の〔数式９〕による蛍光強度の変化率（減少率、消光率）を計算をする過程、
〔数式９〕
Ｆ_dn ＝ｌｏｇ₁₀｛１００−Ｃ_n×１００）｝
Ｆ_dn ＝２ｌｏｇ₁₀｛１−Ｃ_n｝
ただし、Ｆ_dn＝蛍光強度変化率（減少率、消光率）、Ｃ_n＝〔数式８〕で得られた値。
（ｅ）前記（ｄ）の過程で得られた各換算値を、サイクル数の関数として、デスプレー上に表示及び／又は印字する過程、
【０２０１】
（ｆ）前記（ｄ）の過程で処理されたデータを、スレッシュホールド（ｔｈｒｅｓｈｏｌｄ）としての０．５と比較し、その値に達したサイクル数を計数する過程、
（ｇ）前記（ｆ）の過程で計数した値をＸ軸に、反応開始前のコピー数をＹ軸にプロットしたグラフを作成する過程、
（ｈ）前記（ｇ）の過程で作成したグラフをデスプレー上に表示及び／又は印字する過程、
（ｉ）前記（ｈ）の過程で描かれた直線の相関係数又は関係式を計算する過程、
（ｊ）前記（ｉ）の過程で計算された相関係数又は関係式をデスプレー上に表示及び／又は印字する過程。
【０２０２】
上記のデータ解析用ソフトウエアを用いて、前記図１３で得られたデータを前記に引き続いて以下のように処理した。
図１４は、上記（ｂ）の過程で処理されたデータを印字した（前記（ｃ）過程）したものである。即ち、１０サイクル目の蛍光強度値を１として各サイクルの蛍光強度を換算し、その換算値を対応するサイクル数に対してプロットしたものである。
図１５は、前記（ｄ）の過程で処理したデータを印字した（前記（ｅ）過程）ものである。即ち、図１４の各プロット値から蛍光強度の減少率（消光率）を計算して、各計算値を各サイクル数に対してプロットしたものである。
【０２０３】
図１６は、前記（ｆ）の過程で処理したデータについて、前記（ｇ）の過程で作成したグラフを印字した（前記（ｈ）の過程）ものである。即ち、蛍光強度減少率＝０．５をスレッシュホールド（threshold）し、その値に達したサイクル数をＸ軸に、ヒトゲノムＤＮＡの反応開始前のコピー数をＹ軸にプロットしたグラフである。このグラフの直線の相関係数（Ｒ２）を前記（ｉ）の過程で計算し、印字した（前記（ｊ）の過程）もので、０．９５１４であった。このように、この相関係数では正確なコピー数を求めるは無理であった。
【０２０４】
実施例２３（本発明のデータ解析方法を用いてデータ処理がなされた実験例）
ＰＣＲは比較実験例１と同様に行った。
データ処理は、比較実験例１の（ｂ）の過程の前に下記の（ａ）の過程をおき、（ｂ）、（ｄ）の過程を以下のように変更する以外は比較実験例１と同様な過程で行った。
（ａ）各サイクルにおける増幅した核酸が本発明の核酸プローブである（蛍光色素で標識された）核酸プライマーとハイブリダイズしたときの反応系の蛍光強度値（即ち、核酸伸長反応時（７２℃）の蛍光強度値）を、核酸ハイブリッド複合体（増幅した核酸が核酸プライマーとハイブリダイズしたもの）が解離したときに測定された反応系の蛍光強度値（即ち、核酸熱変性反応完了時（９５℃）の蛍光強度値）で割る補正演算処理過程、即ち、実測の蛍光強度値を次の〔数式１〕で補正した。
ｆ_n＝ｆ_hyb,n／ｆ_den,n 〔数式１〕
［式中、ｆ_n＝サイクルの蛍光強度の補正値、ｆ_hyb,n＝各サイクルの７２℃の蛍光強度値、ｆ_den,n＝各サイクルの９５℃の蛍光強度値］
得られた値を各サイクル数に対してプロットしたのが図１７である。
【０２０５】
（ｂ）各サイクルにおける〔数式１〕における補正演算処理値を〔数式３〕に代入し、各サイクルにおける各サンプル間の蛍光変化率（減少率又は消光率）を算出する演算処理過程、即ち、下記の〔数式１０〕で演算処理する過程、
Ｆ_n＝ｆ_n／ｆ₂₅ 〔数式１０〕
［式中、Ｆ_n＝各サイクルの演算処理値、ｆ_n＝〔数式１〕で得られた各サイクルの値、ｆ₂₅＝〔数式１〕で得られた値で、サイクル数が２５回目のもの］。
〔数式１０〕は〔数式３〕において、ａ＝２５とした場合におけるものである。
【０２０６】
（ｄ）前記（ｂ）の過程で得られた各サイクルの演算処理値を〔数式６〕による蛍光強度の変化率（減少率又は消光率）の対数値を得るための演算処理に付す過程、即ち、下記の〔数式１１〕で演算処理する過程、
ｌｏｇ₁₀｛（１−Ｆ_n）×１００｝ 〔数式１１〕
［式中、Ｆ_n＝［数式１０］で得られた値］。
〔数式１１〕は〔数式６〕において、ｂ＝１０、Ａ＝１００とした場合におけるものである。
【０２０７】
上記の結果を図１８及び１９に示した。
図１８は、前記（ａ）及び（ｂ）の過程で処理された値をサイクル数に対してプロットし、印字したものである。
図１９は、図１８で得られた値を前記（ｄ）の過程のように処理して得られた値を、サイクル数に対してプロットし、印字したものである。
【０２０８】
次に、図１９のグラフを基に、前記（ｆ）、（ｇ）、及び（ｈ）の過程で処理した。即ち、図１９のグラフを基に比較実験例１と同様に、ｌｏｇ₁₀（蛍光強度変化率）のスレッシュホールド値として、０．１、０．３、０．５、０．７、０．９、１．２を選び、その値に達したサイクル数をＸ軸に、ヒトゲノムＤＮＡの反応開始前のコピー数をＹ軸にプロットし、検量線を描かせた。その結果を図２０に示した。これらの検量線について前記（ｉ）及び（ｊ）の過程で処理して求めた相関係数（Ｒ²）は、前記各スレッシュホールド値に対して、各々０．９９８、０．９９９、０．９９９３、０．９９８５、０．９９８９、０．９９８８であった。これらの相関係数から、スレッシュホールド値として０．５（相関係数０．９９９３）を採用することが望ましいことが認識できた。この相関係数をもつ検量線であれば、未知コピー数の核酸試料について反応開始前のコピー数を精度よく求めることができることが分かる。
【０２０９】
実施例２４（核酸の融解曲線分析及びＴｍ値分析の例）
本発明の新規なＰＣＲ法により増幅された核酸について、５１）低い温度から核酸が完全に変性するまで、温度を徐々に上げるあるいは下げる過程（例えば、５０℃から９５℃まで）、５２）前記５１）過程において、短い時間間隔（例えば、０．２℃〜０．５℃の温度上昇に相当する間隔）で蛍光強度を測定する過程、５３）前記５２）過程の測定結果を時間の関数としてデスプレー上に表示する過程、即ち、核酸の融解曲線を表示する過程、５４）前記５３）過程の融解曲線を一次微分する過程、５５）前記５４）過程の微分値（−ｄＦ／ｄＴ、Ｆ：蛍光強度、Ｔ：時間）をデスプレー上に表示する過程、５６）前記５５）から得られる微分値から変曲点を求める過程からなるソフトウエアを作成し、前記本発明のデータ解析用ソフトウエアに合体した。当該データ解析用ソフトウエアを記録したコンピューター読み取り可能な記録媒体をインストールした前記ライトサイクラー^TMシステムを用いて本発明の新規リアルタイム定量的ＰＣＲ反応を行い、核酸融解曲線の分析を行った。本発明においては、蛍光強度は温度が上がるごとに増加する。
【０２１０】
実施例２３と同じヒトゲノムＤＮＡの１コピーと１０コピーについて、実施例２１と同様のＰＣＲを行い、前記５１）、５２）、５３）、５４）及び５５）の過程で処理されたデータを印字したものが図２１である。１コピーと１０コピーの７５回目の増幅産物について、本実施例の５１）、５２）及び５３）の過程で処理した核酸融解曲線の図が図２２である。５４）の過程でこの曲線を微分し、５５）及び５６）の過程で変曲点（Ｔｍ値）を求めたものが図２３である。図２３から、１コピーと１０コピーの増幅産物のＴｍ値が異なる故に、各増幅産物は異なる産物であることが判明した。
【０２１１】
【発明の効果】
本発明は次のような効果を有する。
１）本発明の標的核酸の濃度を測定する方法、本発明の各種の核酸プローブ、特に2-O-アルキルオリゴヌクレオチド若しくは2-O-アルキレンオリゴヌクレオチド、2-O-ベンジルオリゴヌクレオチドなど化学的修飾オリゴヌクレオチドなどからなる本発明の核酸プローブ、またオリゴリボヌクレオチドとオリゴデオキシリボヌクレオチドが介在するキメリックオリゴヌクレオチドなどからなる本発明の核酸プローブ、それらの本発明の核酸プローブを含有若しくは付帯する、標的核酸の濃度を測定する測定キット、及び前記の本発明の核酸プローブを結合してなるＤＮＡチップなどの核酸チップ若しくは核酸デバイスを用いると、測定系から未反応の核酸プローブを除く等の操作をすることがないので、標的核酸の濃度を短時間でかつ簡便に測定できる。また、複合微生物系又は共生微生物系に適用すると、当該系における特定菌株の存在量を特異的かつ短時間に測定できる。また、本発明は標的核酸若しくは遺伝子のＳＮＰなどの多型又は変異などの解析若しくは測定する簡便な方法を提供している。
【０２１２】
２）また、本発明の定量的ＰＣＲ方法は、次のような効果を有する。
ａ．ＴａｑＤＮＡポリメラーゼによる標的核酸の増幅に阻害的に作用する因子が添加されていないことから、従来公知の特異性のある通常のＰＣＲと同様の条件で定量的ＰＣＲを行うことができる。
ｂ．また、ＰＣＲの特異性を高く保つことができるので、プライマーダイマーの増幅が遅くなることから、従来公知の定量的ＰＣＲと比較すると定量限界が約１桁のオーダー低くなる。
ｃ．複雑な核酸プローブを用意する必要がないので、それに要する時間と費用が節約できる。
ｄ．標的核酸の増幅効果も大きく、増幅過程をリアルタイムでモニタリングすることができる。
【０２１３】
３）また、リアルタイム定量的ＰＣＲ方法で得られたデータを解析する際、本発明のデータ解析方法を用いて、未知核酸コピー数の核酸試料について核酸のコピー数を求める検量直線を作成すると、検量線の相関係数は従来の方法により得られたものに較べて格段に高い。それで、本発明のデータ解析方法を用いると核酸の正確なコピー数を求めることができる。
【０２１４】
４）また、本発明のリアルタイム定量的ＰＣＲ方法によって得られたデータの解析方法に係るデータ解析用ソフトウエア、また、その解析方法の手順をプログラムとして記録したコンピュータ読み取り可能な記録媒体、また、それを用いたリアルタイム定量的ＰＣＲの測定若しくは解析装置を用いると、相関係数の高い検量直線を自動的に作成することができる。
【０２１５】
５）また、本発明の新規な核酸の融解曲線の分析方法を用いると、精度の高い、核酸のＴｍ値を求めることができる。更に、当該方法に係るデータ解析用ソフトウエア、また、その分析方法の手順をプログラムとして記録したコンピュータ読み取り可能な記録媒体、また、それを用いたリアルタイム定量的ＰＣＲの測定若しくは解析装置を用いると、正確なＴｍ値を求めることができる
【図面の簡単な説明】
【図１】実施例１で得た核酸プローブを用いて大腸菌の１６ＳｒＲＮＡの５´末端から数えて３３５から３５８番目の核酸塩基配列を測定した場合の蛍光強度測定データを示す図。
【図２】３５塩基鎖2-O-Meプローブの標的核酸へのハイブリダイゼーションに対する熱処理の効果
点線：rRNAを加熱処理後，標的核酸が添加された。
実線：非加熱処理rRNA．
【図３】プローブと標的核酸１６ＳｒＲＮＡのハイブリダイゼーションに対するプローブの塩基鎖の塩基数、ヘルパープローブ及びプローブの５’末端のリボースの２’位炭素ＯＨ基のメチル基修飾の効果
【図４】本発明方法によるｒＲＮＡ測定のための検量線
【図５】ＫＹＭ７株及びＫＹＭ８株の複合培養系のｒＲＮＡ量の時間経過についての本発明のＦＩＳＨ方法による分析
【図６】本発明のＤＮＡチップを説明する図。
【図７】本発明のＤＮＡチップを用いるＳＮＰｓ検出測定のための装置類を説明する図。
【図８】本発明のＤＮＡチップを用いるＳＮＰｓ検出測定の実験結果を示す図。
【図９】 ボデピーFL/C6で標識したプライマー１及び２を用いた定量的ＰＣＲ方法：サイクル数と蛍光色素の発光の減少量の関係を示す図。図中の▲１▼〜▲８▼なる記号は、下記の意味を表す。

【図１０】ボデピーFL/C6で標識したプライマー１及び２を用いた定量的ＰＣＲ方法：サイクル数と蛍光色素の発光の減少量の対数値の関係を示す図。図中の▲１▼〜▲８▼なる記号は、図９と同じ意味を表す。
【図１１】本発明の定量的ＰＣＲ方法を用いて作成した大腸菌１６ＳｒＤＮＡの検量線を示す図。ｎ：１０ⁿ
【図１２】上図は、ＦＲＥＴ現象を用いるリアルタイム定量的ＰＣＲ方法において使用する蛍光色素で標識した二つのプローブの代わりに、本発明の一つのプローブを用いてリアルタイム定量的ＰＣＲを行った場合の蛍光強度の減少率の変化を示す図である。下図は蛍光強度の減少が有為に観察され始めるサイクル数（threshold number：Ct値）を算出し、検量線を作成した図である。
【図１３】本発明の補正演算処理しない場合の、本発明のボデピーFL/C6で標識したプライマーを用いたリアルタイム定量的ＰＣＲによって得られた蛍光減少曲線を示す図。
■：標的核酸＝１０コピー；蛍光強度測定の反応系の温度＝７２℃
●：標的核酸＝１００コピー；蛍光強度測定の反応系の温度＝７２℃
▲：標的核酸＝１０００コピー；蛍光強度測定の反応系の温度＝７２℃
◆：標的核酸＝１００００コピー；蛍光強度測定の反応系の温度＝７２℃
□：標的核酸＝１０コピー；蛍光強度測定の反応系の温度＝９５℃
○：標的核酸＝１００コピー；蛍光強度測定の反応系の温度＝９５℃
△：標的核酸＝１０００コピー；蛍光強度測定の反応系の温度＝９５℃
◇：標的核酸＝１００００コピー；蛍光強度測定の反応系の温度＝９５℃
【図１４】各曲線の１０サイクル目の値を１として補正する以外は、図１３の曲線の場合と同様にしたリアルタイム定量的ＰＣＲによって得られた蛍光減少曲線を示す図。
■：標的核酸＝１０コピー；蛍光強度測定温度＝７２℃
●：標的核酸＝１００コピー；蛍光強度測定温度＝７２℃
▲：標的核酸＝１０００コピー；蛍光強度測定温度＝７２℃
◆：標的核酸＝１００００コピー；蛍光強度測定温度＝７２℃
５：標的核酸＝１００００コピー；蛍光強度測定温度＝７２℃
【図１５】図１４の各曲線の各プロット値について、［数式９］の蛍光強度減少率（変化率）を計算し、計算値をプロットした曲線を示す図。
■：標的核酸＝１０コピー
●：標的核酸＝１００コピー
▲：標的核酸＝１０００コピー
◆：標的核酸＝１００００コピー
【図１６】図１５のデータから求めたヒトゲノムＤＮＡの検量直線を示す図。
ｙ＝ヒトβ−グロビン遺伝子コピー数
ｘ＝サイクル数（Ｃｔ）
Ｒ²＝相関係数
【図１７】図１３の各サイクルの測定値を〔数式１〕で補正演算処理した値を各サイクル数に対してプロットした曲線を示す図。
■：標的核酸＝１０コピー
●：標的核酸＝１００コピー
▲：標的核酸＝１０００コピー
◆：標的核酸＝１００００コピー
【図１８】図１７の各サイクルの演算処理値を〔数式３〕で演算処理した値をサイクル数に対してプロットした曲線を示す図。
■：標的核酸＝１０コピー
●：標的核酸＝１００コピー
▲：標的核酸＝１０００コピー
◆：標的核酸＝１００００コピー
【図１９】図１８の各サイクルの演算処理値を〔数式６〕の式で演算処理した値をサイクル数に対してプロットした曲線を示す図。
■：標的核酸＝１０コピー
●：標的核酸＝１００コピー
▲：標的核酸＝１０００コピー
◆：標的核酸＝１００００コピー
【図２０】図１８の各log（蛍光変化率）値からＣｔ値の候補として、０．１、０．３、０．５、０．７、０．９、１．２を適当に選んで、それに対応する検量直線を描かせた場合の図。尚、各検量線における相関係数を下記に示した。
▲：log₁₀（蛍光変化率）＝０．１；相関係数＝０．９９８
■：log₁₀（蛍光変化率）＝０．３；相関係数＝０．９９９
●：log₁₀（蛍光変化率）＝０．５；相関係数＝０．９９９３
△：log₁₀（蛍光変化率）＝０．７；相関係数＝０．９９８５
□：log₁₀（蛍光変化率）＝０．９；相関係数＝０．９９８９
○：log₁₀（蛍光変化率）＝１．２；相関係数＝０．９９８８
【図２１】１コピー及び１０コピーのヒトゲノムＤＮＡについて、本発明のボデピーFL/C6で標識したプライマーを用いてリアルタイム定量的ＰＣＲを行った場合の蛍光減少曲線を示す図。ただし、〔数式１〕の補正演算処理を施した。
１：標的核酸＝０コピー
２：標的核酸＝１コピー
３：標的核酸＝１０コピー
【図２２】図２１に示されるＰＣＲの増幅産物についての核酸の融解曲線分析を行った場合の核酸の融解曲線を示す図。
１：標的核酸＝０コピー
２：標的核酸＝１コピー
３：標的核酸＝１０コピー
【図２３】図２２の曲線を微分して得られた、Ｔｍ値を示す曲線を示す図（谷がＴｍ値）。
２：標的核酸＝１コピー
３：標的核酸＝１０コピー[0001]
BACKGROUND OF THE INVENTION
  The present invention relates to a method for measuring the concentration of a target nucleic acid. Specifically, in various methods for measuring the concentration of various nucleic acids using a nucleic acid probe labeled with a fluorescent dye,Chimeric oligonucleotides that are labeled with a fluorescent dye at the 3 ′ end or 5 ′ end and contain oligoribonucleotides and oligodeoxyribonucleotides ( chimeric oligonucleotide )Nucleic acid probeThe probeBefore the hybridization between the target nucleic acid and the target nucleic acid, a helper probe for increasing the hybridization efficiency is added to the hybridization reaction system, the hybridization reaction is performed, and the fluorescence intensity before and after the hybridization is measured. Related to the method.
[0002]
[Prior art]
Conventionally, various methods for measuring a nucleic acid concentration using a nucleic acid probe labeled with a fluorescent dye are known. The method includes the following.
(1) Dot blotting method
In this method, a nucleic acid probe labeled with a target nucleic acid and a fluorescent dye is hybridized on the membrane, then an unreacted nucleic acid probe is washed away, and only the fluorescent dye molecule labeled on the nucleic acid probe hybridized with the target nucleic acid is used. The fluorescence intensity is measured.
[0003]
(2) Intercalator method (Glazer et al., Nature 359: 959, 1992)
This method is a method of measuring the amount of increase in light emission because a certain type of fluorescent dye called an intercalator emits strong light when it gets stuck in the double strand of a nucleic acid. Examples of the fluorescent dye include ethidium bromide (Experimental Medicine, Vol. 15, No. 7, pages 46-51, 1997, Yodosha), SYBR R Green (LightCycler)^TM System; pamphlet issued on April 5, 1999, published by Roche Diagnostics Co., Ltd.).
[0004]
(3) Method using FRET (fluorescence energy transfer) (Mergney et al., Nucleic Acid Res., 22: 920-928, 1994.)
This method consists of hybridizing two nucleic acid probes to a target nucleic acid. The two nucleic acid probes are each labeled with a different fluorescent dye. One fluorescent dye of the two probes can transmit energy to the fluorescent dye of the other probe through the FRET phenomenon to emit light. The two probes are designed to hybridize so that the fluorescent dyes face each other and 1-9 bases apart. Therefore, when the two nucleic acid probes are hybridized to the target nucleic acid, the latter fluorescent dye emits light, and the intensity of the light emission is proportional to the replication amount of the target nucleic acid.
[0005]
(4) Molecular beacon method (Tyagi et al., Nature Biotech., 14: 303-308, 1996.)
The nucleic acid probe used in this method is labeled with a reporter dye at one end of the nucleic acid probe and a quencher dye at the other end. Since both ends thereof are complementary to each other in the base sequence, the base sequence is designed so as to form a hairpin stem as a whole probe. Due to Forster resonance energy, the light emission of the reporter dye is suppressed by the quencher dye in a state of floating in the liquid due to its structure. However, since the hairpin structure is broken when hybridized to the target nucleic acid, the distance between the reporter dye and the quencher dye increases, so that the Forster resonance energy does not move. As a result, the reporter dye emits light.
[0006]
(5) Davis method (Davis et al., Nucleic acids Res. 24: 702-706, 1996)
Davis created a probe with a fluorescent dye attached to the 3 'end of the oligonucleotide via a spacer with 18 carbon atoms. This was applied to flow cytometry. It has been reported that 10-fold fluorescence intensity can be obtained when hybridizing than when a fluorescent dye is directly bound to the 3 'end.
These methods include various nucleic acid measurement methods, FISH method (fluorescent in situ hybridization assays), PCR method, LCR method (ligase chain reaction), SD method (strand displacement assays), competitive hybridization method (competitive hybridization), etc. It is applied to and has made remarkable progress.
[0007]
These methods are generally used at present, but after performing a hybridization reaction between a nucleic acid probe labeled with a fluorescent dye and a target nucleic acid, it is necessary to wash away the non-hybridized nucleic acid probe from the reaction system. Has an unfavorable procedure. It is clear that the removal of such procedures leads to a reduction in measurement time, ease of measurement, and accuracy of measurement. Therefore, development of a nucleic acid measurement method that does not have such a procedure has been desired.
[0008]
[Problems to be solved by the invention]
In view of the above situation, an object of the present invention is to provide a method for measuring the concentration of a target nucleic acid in a shorter time, more simply, and more accurately in a method for measuring the concentration of a target nucleic acid using a nucleic acid probe labeled with a fluorescent dye. Is to provide.
[0009]
[Means for Solving the Problems]
  In order to solve the above problems, the present inventors have repeatedly studied a method for measuring the concentration of nucleic acid using a nucleic acid probe. As a result, when a nucleic acid probe labeled with a fluorescent dye is hybridized to the target nucleic acid, there is a phenomenon that the emission of the fluorescent dye decreases (fluorescence quenching phenomenon), and the decrease is remarkable in a specific dye, In particular, it has been discovered that the degree of reduction depends on the type or base sequence of the base to which the fluorescent dye is bound. In the measurement method,The 3 ′ terminal base is G or C and the 3 ′ terminal 3 ′ carbon OH group, or the 5 ′ terminal base is G or C and the 5 ′ terminal phosphate group, or the 5 ′ terminal A phosphoric acid group is treated with phosphatase, a 5'-position carbon OH group is labeled with a fluorescent dye, and a chimeric oligonucleotide containing oligoribonucleotides and oligodeoxyribonucleotides ( chimeric oligonucleotide )Before the hybridization between the nucleic acid probe and the target nucleic acid, a helper probe for increasing the hybridization efficiency is added to the hybridization reaction system and the hybridization reaction is performed. Furthermore, it was discovered that the concentration of the target nucleic acid can be measured more simply and more accurately. The present invention has been completed based on such findings.
[0010]
  That is, the present invention
  1) Nucleic acid probe labeled with any fluorescent dye of 6-joe, BODIPY TMR, BODIPY FL, BODIPY FL / C3, Alexa 488, or Alexa 532The 3 ′ terminal base is G or C and the 3 ′ terminal 3 ′ carbon OH group, or the 5 ′ terminal base is G or C and the 5 ′ terminal phosphate group, or 5 ′. The phosphate group at the terminal is treated with phosphatase, the carbon OH group at the 5 ′ position is labeled with a fluorescent dye,As an oligonucleotideTheIt consists of a chimeric oligonucleotide containing rigoribonucleotide and oligodeoxyribonucleotide, and when hybridized to the target nucleic acid, the base pair of hybridization is a pair of G (guanine) and C (cytosine) at the terminal portion. The base sequence of the probe is designed so that at least one pair is formed, and when hybridized to the target nucleic acid, the labeled fluorescent dye reduces the light emission, or the nucleic acid probe is a single nucleic acid probe. Alternatively, the 2 'carbons of multiple riboses are chemically modified2'-O-methyl oligonucleotide, 2'-O-ethyl oligonucleotide, 2'-O-butyl oligonucleotide, 2'-O-ethylene oligonucleotide, or 2'-O-benzyl oligonucleotideIn order to further increase the efficiency of hybridization to the hybridization sequence region of the nucleic acid probe before hybridization of the nucleic acid probe, which intervenes in the chain, to the target nucleic acid,Helper probes composed of deoxyribonucleotides as oligonucleotides, orA helper probe consisting of oligoribonucleotides as oligonucleotidesIsThe 2 'carbon of ribose was chemically modified2'-O-methyl oligonucleotide, 2'-O-ethyl oligonucleotide, 2'-O-butyl oligonucleotide, 2'-O-ethylene oligonucleotide, or 2'-O-benzyl oligonucleotideIs added in the chain (provided that “the 2′-position carbon of ribose is not chemically modified and the 2′-position carbon of ribose is not chemically modified) Not a combination of the above-mentioned helper probes "or" a combination of a probe and a helper oligonucleotide having a diphosphate ester backbone, an alkyl or aryl phosphate ester backbone "), or hybridization of the nucleic acid probe to a target nucleic acid. A method for measuring the concentration of a target nucleic acid, characterized by measuring a decrease in light emission of a fluorescent dye before and after hybridization, or
[0011]
  2) The aboveTarget nucleic acid is RNAThe method for measuring the concentration of the target nucleic acid according to 1) above, or
[0012]
  3)After heat treatment at 80-100 ° C. for 1-15 minutes, the nucleic acid probe and the target nucleic acid are hybridized.Said2) For measuring the concentration of the target nucleic acid according to
  4) The aboveThe helper probe is Cellulomonas sp. It hybridizes to 16S rRNA of KYM-7 strain, and its nucleotide sequence is (5 ′) TCCTTTGAGT TCCCGGCCGG A (3 ′) or (5 ′) CCCTGGTCGT AAGGGCCCATTGATGTGAC GT (3 ′), and the nucleic acid probe Cellulomonas sp. And its nucleotide sequence is (5 ′) CATCCCCACC TTCCTCCGAG TTGACCCCGG CAGTC (3 ′) or Agrobacterium sp. And its nucleotide sequence is (5 ′) CATCCCCACC TTCCTCTCGGG CTTATCACCCG GCAGTC (3 ′)Said2) For measuring the concentration of the target nucleic acid according to
[0013]
  5) SaidBase sequence (5 ′) TCCTTTGAGT TCCCGGCCGG A (3 ′) is modified with a methyl group on the 2′-position carbon OH group of the central 8 bases (9 to 16 bases counted from the 5 ′ end) (Ether-linked) oligoribonucleotide bodySaid4) For measuring the concentration of the target nucleic acid according to
[0014]
  6) Said base sequence(5 ′) CCCTGGTCGT AAGGGCCATG ATGACTTGAC GT (3 ′)Is an oligoribonucleotide body in which the OH group at the 2'-position of ribose in the middle 8 bases (9 to 16 bases counted from the 5 'end) is modified (ether-linked) with a methyl group.4) For measuring the concentration of the target nucleic acid according to
  7) Said base sequence(5 ′) CATCCCCACC TTCCTCCCAG TTGACCCCGG CAGTC (3 ′)But the middle 8 bases (counted from the 5 'end)17base~24The oligoribonucleotide body in which the OH group at the 2'-position carbon of ribose in the base portion) is modified with a methyl group (ether bond)4) For measuring the concentration of the target nucleic acid according to
[0015]
  8) The base sequence (5 ') CATCCCCACC TTCCTCCCGAG TTGACCCCGG CAGTC (3')9Base content (17 to 2 bases counting from the 5 'end5The oligoribonucleotide body in which the OH group at the 2'-position carbon of ribose in the base portion) is modified with a methyl group (ether bond)4) For measuring the concentration of the target nucleic acid according to
[0016]
  9) SaidWhen the nucleic acid probe is hybridized to the target nucleic acid, the base of the target nucleic acid is one base away from the terminal base of the target nucleic acid hybridized to the probe, and one G (guanine) base is present in the target nucleic acid. The base sequence is designedSaid1) For measuring the concentration of the target nucleic acid according to
  10)In the nucleic acid probe, the 3 ′ carbon hydroxyl group of ribose or deoxyribose at the 3 ′ end is phosphorylated.The method for measuring the concentration of the target nucleic acid as described in 1) above, or
  11)The fluorescence intensity value of the reaction system obtained after the target nucleic acid is hybridized with a nucleic acid probe labeled with a fluorescent dye and the probe-nucleic acid hybrid complex thus formed is dissociated. Correct byThe method for measuring the concentration of the target nucleic acid as described in 1) above, or
[0017]
  12)The target nucleic acid is derived from a microorganism obtained by pure separation or from an animal.The method for measuring the concentration of the target nucleic acid as described in 1) above, or
  13)The target nucleic acid is a nucleic acid contained in a complex microbial system or a symbiotic microbial system or in a cell homogenate.The method for measuring the concentration of the target nucleic acid as described in 1) above,
I will provide a.
[0037]
DETAILED DESCRIPTION OF THE INVENTION
Next, the present invention will be described in more detail with reference to preferred embodiments.
The first feature of the present invention is a method for measuring the concentration of a target nucleic acid using a nucleic acid probe labeled with a fluorescent dye, wherein the fluorescent dye before and after hybridization is generated when the nucleic acid probe is hybridized to the target nucleic acid. The purpose is to measure the decrease in luminescence.
In the present invention, the probe-nucleic acid hybrid complex refers to a compound (complex) in which a nucleic acid probe labeled with the fluorescent dye of the present invention is hybridized with a target nucleic acid. For the sake of simplicity, hereinafter, it is abbreviated as a nucleic acid hybrid complex.
A fluorescent dye-nucleic acid complex refers to a complex in which a fluorescent dye is bound to a target nucleic acid. For example, a state in which an intercalator is bound in a double-stranded nucleic acid can be mentioned.
In the present invention, DNA, RNA, cDNA, mRNA, rRNA, XTPs, dXTPs, NTPs, dNTPs, nucleic acid probe, helper nucleic acid probe (or nucleic acid helper probe, or simply helper probe), hybridization, hybridization, intercalator , Primer, annealing, extension reaction, heat denaturation reaction, nucleic acid melting curve, PCR, RT-PCR, RNA-primed PCR, Stretch PCR, reverse PCR, PCR using Alu sequence, multiplex PCR, PCR using mixed primers, PCR method using PNA, hybridization assay method, FISH (fluorescent in situ hybridization assays) method, PCR method (polymerase chain assays), LCR method (ligase chain reaction), SD method (strand displacement assays), Terms such as competitive hybridization, DNA chip, nucleic acid detection (gene detection) device, SNP (snip: single nucleotide substitution polymorphism), complex microbial system, etc. are currently used in molecular biology, genetic engineering It has the same meaning as a term generally used in microbial engineering and the like.
[0038]
In the present invention, measuring the concentration of the target nucleic acid means quantifying the concentration, quantitative detection, simple detection, or polymorphism / mutation of one or more types of nucleic acids in the measurement system. It means to analyze such as. In the case of multiple types of nucleic acids, quantitative detection of multiple types of nucleic acids at the same time, simple detection of multiple types of nucleic acids at the same time, or simultaneous analysis of polymorphisms and mutations of multiple types of nucleic acids, etc. Naturally, it is within the technical scope of the present invention.
The target nucleic acid concentration measuring device refers to various DNA chips and the like. Specific examples include various DNA chips. In the present invention, any type of DNA chip may be used as long as the nucleic acid probe of the present invention is applicable.
A method for measuring the concentration of a target nucleic acid using a nucleic acid probe labeled with a fluorescent dye (hereinafter simply referred to as the nucleic acid probe of the present invention or the probe of the present invention) (hereinafter simply referred to as a nucleic acid measurement method for the sake of simplicity). Means hybridization method, FISH method (fluorescent in situ hybridization assays), PCR method (polymerase chain assays), LCR method (ligase chain reaction), SD method (strand displacement assays), competitive It means measuring the concentration of a target nucleic acid by a method such as a hybridization method.
[0039]
Conventionally, in these methods, after adding a nucleic acid probe labeled with a fluorescent dye, unreacted fluorescent dye of the nucleic acid probe that has not hybridized to the target nucleic acid is removed from the measurement system by a method such as washing, The fluorescent dye labeled to the nucleic acid probe hybridized to the nucleic acid is directly emitted from the probe or indirectly applied to the probe (for example, by causing an enzyme to act) to emit light, and the This is a method for measuring the amount of luminescence. The present invention is characterized in that the target nucleic acid is measured without these complicated operations in these methods.
[0040]
In the present invention, the target nucleic acid refers to a nucleic acid or gene whose concentration is to be measured. Regardless of purification. Moreover, the magnitude | size of a density | concentration does not ask | require. Various nucleic acids may be mixed. For example, a specific nucleic acid intended for measurement of concentration in a complex microbial system (mixed system of RNA or gene DNA of a plurality of microorganisms) or a symbiotic microorganism system (mixed system of RNA or gene DNA of a plurality of animals and plants and / or a plurality of microorganisms) It is. When purification of the target nucleic acid is necessary, it can be performed by a conventionally known method. For example, it can be performed using a commercially available purification kit. Specific examples of the nucleic acid include DNA, RNA, PNA, oligodeoxyribonucleotides, oligoribonucleotides and the like, and chemically modified nucleic acids of the nucleic acids. 2'- as a chemically modified nucleic acidO-Methyl (Me) RNA and the like can be exemplified.
[0041]
In the present invention, a fluorescent dye generally labeled with a nucleic acid probe and used for nucleic acid measurement / detection can be conveniently used. However, when a nucleic acid probe labeled with a fluorescent dye is hybridized with a target nucleic acid, the probe is used. As the fluorescent dye labeled with, those that reduce the light emission are preferably used. For example, fluorescein or derivatives thereof (eg, fluorescein isothiocyanate (FITC) or derivatives thereof, such as Alexa 488, Alexa 532, cy3, cy5, EDANS (5- (2'-aminoethyl) amino- 1-naphthalene sulfonic acid)}, rhodamine 6G (R6G) or a derivative thereof (eg, tetramethylrhodamine isothiocyanate (TMRITC), x-rhodamine (x -rhodamine), Texas red, BODIPY FL (trade name; manufactured by Molecular Probes, USA), BODIPY FL / C3 (trade name; molecular probes) ), BODIPY FL / C6 (trade name; manufactured by Molecular Probes, USA), BODIP (BOD) IPY) 5-FAM (trade name; manufactured by Molecular Probes, USA), BODIPY TMR (trade name; manufactured by Molecular Probes, USA), or a derivative thereof (for example, BODIPY TR (trade name; manufactured by Molecular Probes, USA), BODIPY R6G (trade name; manufactured by Molecular Probes, USA), BODIPY 564 ( Trade names: Molecular Probes (USA), BODIPY 581 (Trade Name: Molecular Probes, USA), etc. Among these, FITC, EDANS, 6-joe, TMR, Alexa 488, Alexa 532, BODIPY FL / C3 (trade name; manufactured by Molecular Probes, USA), BODIPY FL / C6 (trade name) As a suitable example, a molecular probe (Molecular Probes), USA), etc., FITC, TMR, 6-jeo, BODIPY FL / C3 (trade name; manufactured by Molecular Probes), (US), BODIPY FL / C6 (trade name; manufactured by Molecular Probes, USA) can be mentioned as more preferable ones.
[0042]
The nucleic acid probe of the present invention to be hybridized to the target nucleic acid may be composed of oligodeoxyribonucleotides or oligoribonucleotides. Moreover, the chimeric oligonucleotide (chimeric oligonucleodite) containing both of them may be sufficient. Those oligonucleotides may be chemically modified. Chemically modified oligonucleotides may be interposed in the chain of the chimeric oligonucleotide.
[0043]
As the modification site of the oligonucleotide subjected to the above-mentioned chemical modification, the terminal hydroxyl group or terminal phosphate group of the oligonucleotide, the internucleoside phosphate site, the carbon at the 5-position of the pyrimidine ring, the nucleoside sugar (ribose or deoxyribose) ) Part. A ribose or deoxyribose moiety is preferable. Specifically, 2'-O-Alkyl oligoribonucleotides (2'-O-alkyloligoribonucleotides) (hereinafter 2'-O-Is abbreviated as 2-O-. ), 2-O-alkyleneoligoribonucleotides and 2-O-benzyloligoribonucleotides. The oligonucleotide is one in which the OH group at the 2'-position of one or more ribose at any position of the oligoribonucleotide is modified (by an ether bond) with an alkyl group, an alkylene group or a benzyl group. In the present invention, among 2-O-alkyl oligoribonucleotides, 2-O-methyl oligoribonucleotide, 2-O-ethyl oligoribonucleotide, 2-O-butyl oligoribonucleotide, 2-O Among O-benzyl oligoribonucleotides, among 2-O-alkylene oligoribonucleotides, 2-O-ethylene oligoribonucleotides and 2-O-benzyl oligoribonucleotides are particularly preferably 2-O- Methyl oligoribonucleotide (hereinafter simply abbreviated as 2-O-Me oligoribonucleotide) is used. By applying such chemical modification to the oligonucleotide, the affinity with the target nucleic acid is increased, and the hybridization efficiency of the nucleic acid probe of the present invention is improved. When the hybridization efficiency is increased, the rate of decrease in the fluorescence intensity of the fluorescent dye of the nucleic acid probe of the present invention is further improved, so that the accuracy of measurement of the concentration of the target nucleic acid is further improved.
In the present invention, the term oligonucleotide means oligodeoxyribonucleotide and / or oligoribonucleotide, and they are collectively referred to.
2-O-alkyl oligoribonucleotides, 2-O-alkylene oligoribonucleotides and 2-O-benzyl oligoribonucleotides can be synthesized by known methods (Nucleic Acids Research, 26, 2224-2229, 1998). . In addition, since GENSET (Inc.) (France) is commissioned synthesis, it can be easily obtained. The inventors of the present invention commissioned the company to synthesize the compound and conducted an experiment to complete the present invention.
[0044]
The nucleic acid probe of the present invention in which modified RNA such as 2-O-methyl oligoribonucleotide (hereinafter simply referred to as 2-O-Me oligoribonucleotide) is interposed in oligodeoxyribonucleotide is mainly used. Preferred results are obtained when used for the measurement of RNA, particularly rRNA.
When RNA is measured using the nucleic acid probe of the present invention, the sample RNA solution is subjected to 80 to 100 ° C., preferably 90 to 100 ° C., and optimally 93 to 97 ° C. before hybridization with the probe. In order to improve hybridization efficiency, it is preferable to destroy the higher order structure of RNA by heat treatment for 1 to 15 minutes, preferably 2 to 10 minutes, and optimally 3 to 7 minutes.
[0045]
Furthermore, in order to further increase the hybridization efficiency of the nucleic acid probe of the present invention to the hybridization sequence region, it is preferable to add a helper probe to the hybridization reaction solution. In this case, the oligonucleotide of the helper probe may be an oligodeoxyribonucleotide, an oligoribonucleotide, or an oligonucleotide that has undergone the same chemical modification as described above. As an example of the oligonucleotide, (5 ′) TCCTTTGAGT TCCCGGCCGG A (3 ′) as a forward type, (5 ′) CCCTGGTCGT AAGGGCCATG ATGACTTGAC GT () as a backward type or reverse ward type 3 '). Preferable examples of the chemically modified oligonucleotide include 2-O-alkyl oligoribonucleotides, particularly 2-O-Me oligoribonucleotides.
When the base chain of the nucleic acid probe of the present invention is 35 bases or less, the use of a helper probe is particularly effective. When the nucleic acid probe of the present invention having more than 35 base chains is used, it may be necessary only to heat denature the target RNA.
As described above, when the nucleic acid probe of the present invention is hybridized to RNA, the hybridization efficiency increases, so the fluorescence intensity decreases according to the amount of RNA in the reaction solution, and the RNA is reduced to a final RNA concentration of about 150 pM. It becomes possible to measure.
Thus, the present invention is also a measurement kit for measuring the concentration of a target nucleic acid, which contains or accompanies the above-mentioned helper probe in a measurement kit for measuring the concentration of a target nucleic acid that contains or is accompanied by the nucleic acid probe of the present invention.
[0046]
In RNA measurement by a conventional hybridization method using a nucleic acid probe, an oligodeoxyribonucleotide body or oligoribonucleotide body has been used as a nucleic acid probe. Since RNA has a strong high-order structure itself, the hybridization efficiency between the probe and the target RNA is poor, and the quantitativeness is poor. For this reason, the conventional method has the complexity of performing a hybridization reaction after denaturing RNA and then immobilizing it on the membrane. In contrast, the above-described method of the present invention uses a ribose moiety-modified nucleic acid probe having a high affinity for a specific structure portion of RNA, and thus can perform a hybridization reaction at a higher temperature than conventional methods. . Therefore, the above-described adverse effects of RNA higher-order structure can be overcome only by heat denaturation treatment as a pretreatment and the combined use of a helper probe. Thereby, in the method of the present invention, the hybridization efficiency is substantially 100%, and the quantitativeness is improved. Moreover, it becomes much simpler than the conventional method.
[0047]
The number of bases of the probe of the present invention is 5 to 50, preferably 10 to 25, particularly preferably 15 to 20. When it exceeds 50, the permeability of the cell membrane is deteriorated when used in the FISH method, and the application range of the present invention is narrowed. If it is less than 5, non-specific hybridization is likely to occur, resulting in a large measurement error.
[0048]
The base sequence of the probe is not particularly limited as long as it specifically hybridizes to the target nucleic acid. Preferably, when a nucleic acid probe labeled with a fluorescent dye hybridizes to a target nucleic acid,
(1) The base sequence of the probe is 1 to 3 bases away from the terminal base portion of the target nucleic acid hybridized to the probe so that at least one G (guanine) is present in the base sequence of the target nucleic acid. Designed base sequence,
(2) The base sequence of the probe is designed so that a plurality of base pairs of the nucleic acid hybrid complex form at least one pair of G (guanine) and C (cytosine) at the terminal portion of the probe. A base sequence is preferred.
[0049]
The oligonucleotide of the nucleic acid probe of the present invention can be produced by a general method for producing an oligonucleotide. For example, it can be produced by a chemical synthesis method, a microbial method using a plasmid vector, a phage vector or the like (Tetrahedron letters, 22, 1859-1862, 1981; Nucleic acids Research, 14, 6227-6245, 1986) ). In addition, it is preferable to use a commercially available nucleic acid synthesizer (for example, ABI394 (manufactured by Perkin Elmer, USA)).
[0050]
In order to label the oligonucleotide with a fluorescent dye, any desired labeling method known in the art can be used (Nature Biotechnology, 14, 303-308, 1996; Applied and Environmental Microbiology, 63). 1143-1147, 1997; Nucleic acids Research, 24, 4532-4535, 1996). For example, when a fluorescent dye molecule is bonded to the 5 ′ end, first, as a spacer on the phosphate group at the 5 ′ end, for example, — (CH₂)_nIntroduce -SH. These introducers are commercially available and may be purchased commercially (Midland Certified Reagent Company). In this case, n is 3-8, preferably 6. A labeled oligonucleotide can be synthesized by binding a fluorescent dye having SH group reactivity or a derivative thereof to this spacer. The oligonucleotide labeled with the fluorescent dye synthesized in this way can be purified by chromatography such as reverse phase to obtain the nucleic acid probe of the present invention.
[0051]
A fluorescent dye can also be bound to the 3 'end of the oligonucleotide. In this case, as a spacer to the OH group at the 3'-position C of ribose or deoxyribose, for example,-(CH₂)_n-NH₂Is introduced. Since these introductions are also commercially available in the same manner as described above, commercial products may be purchased (Midland Certified Reagent Company). In addition, by introducing a phosphate group, as a spacer to the OH group of the phosphate group, for example,-(CH₂)_nIntroduce -SH. In these cases, n is 3-8, preferably 4-7. A labeled oligonucleotide can be synthesized by binding a fluorescent dye having reactivity to an amino group or SH group or a derivative thereof to the spacer. The oligonucleotide labeled with the fluorescent dye synthesized in this way can be purified by chromatography such as reverse phase to obtain the nucleic acid probe of the present invention. When introducing this amino group, kit reagents (for example, Uni-link aminomodifier (CLONTECH, USA), FluoReporter Kit F-6082, F-6083, F-6084, F-10220 ( In any case, it is convenient to use a molecular probe (Molecular Probes, USA). Then, a fluorescent dye molecule can be bound to the oligoribonucleotide according to a conventional method. It is also possible to introduce fluorescent dye molecules into the probe nucleic acid chain (ANALYTICAL BIOCHEMISTRY 225, 32-38 (1998)).
[0052]
Although the nucleic acid probe of the present invention can be prepared as described above, the preferred probe form is one in which the 3 ′ or 5 ′ end is labeled with a fluorescent dye, and the labeled end base is G or C. It is what is. When the 5 ′ end is labeled and the 3 ′ end is not labeled, the OH group at the 3′-position carbon of the ribose or deoxyribose at the 3′-end is a phosphate group, etc., and the 2′-position carbon of the ribose at the 3 ′ end The OH group may be modified with a phosphate group or the like without any limitation.
[0053]
The nucleic acid probe of the present invention can be suitably used not only for nucleic acid measurement but also for a method for analyzing or measuring polymorphism or / and mutation of a target nucleic acid. In particular, a device for measuring a target nucleic acid concentration is provided by applying to a device for measuring a target nucleic acid concentration {DNA chip (protein nucleic acid enzyme, 43, 2004-2011, 1998)}. Further, a method for analyzing or measuring polymorphism or / and mutation of a target nucleic acid using the device is a very convenient method. That is, in the hybridization with the nucleic acid probe of the present invention, the fluorescence intensity varies depending on whether a GC pair is formed. Therefore, the polymorphism or / and mutation of the target nucleic acid can be analyzed or measured by hybridizing the nucleic acid probe of the present invention to the target nucleic acid and measuring the luminescence intensity. Specific methods are described in Examples 14 and 15. In this case, the target nucleic acid may be an amplified product obtained by one of various nucleic acid amplification methods, or may be an extracted product. Moreover, the target nucleic acid does not ask | require the kind. However, it is sufficient that a guanine base or cytosine base is present in the chain or at the end. If there is no guanine base or cytosine base in the chain or at the end, the fluorescence intensity does not decrease. Therefore, according to the method of the present invention, mutation or substitution of G → A, G ← A, C → T, C ← T, G → C, G ← C, that is, single nucleotide polymorphism (SNP). Polymorphism such as can be analyzed or measured. Currently, polymorphism analysis is currently performed by determining the base sequence of a target nucleic acid using the Maxam-Gilbert method or dideoxy method.
[0054]
Therefore, by including the nucleic acid probe of the present invention in a measurement kit for analyzing or measuring polymorphism and mutation of the target nucleic acid, polymorphism or / and mutation of the target nucleic acid can be obtained. It can be suitably used as a measurement kit for analysis or measurement.
[0055]
Reaction system when a target nucleic acid is hybridized with a nucleic acid probe of the present invention when analyzing data obtained by a method for analyzing or measuring polymorphism or / and mutation of the target nucleic acid of the present invention If the process of correcting the fluorescence intensity value of the above by the fluorescence intensity value of the reaction system when the above is not hybridized, the processed data becomes highly reliable.
Thus, the present invention provides a data analysis method for a method of analyzing or measuring polymorphism or / and mutation of a target nucleic acid.
[0056]
Further, the present invention is characterized in that the fluorescence of the reaction system when the target nucleic acid is hybridized with the nucleic acid probe of the present invention in a measuring apparatus for analyzing or measuring polymorphism or / and mutation of the target nucleic acid. Analyzing the target nucleic acid polymorphism and / or mutation characterized by having means to correct the intensity value based on the fluorescence intensity value of the reaction system when the above is not hybridized Or it is a measuring device to measure.
[0057]
In addition, the present invention is characterized in that when analyzing data obtained by a method for analyzing or measuring polymorphism or / and mutation of a target nucleic acid, the target nucleic acid hybridizes with the nucleic acid probe of the present invention. A computer-readable recording of a procedure for causing a computer to execute a process of correcting the fluorescence intensity value of the reaction system with the fluorescence intensity value of the reaction system when the above is not hybridized. It is a recording medium.
[0058]
The nucleic acid probe of the present invention may be fixed on the surface of a solid (support layer), for example, the surface of a slide glass. In this case, it is preferable that the end not labeled with the fluorescent dye is fixed. This format is now called a DNA chip. Can be used for gene expression monitoring, base sequence determination, mutation analysis, polymorphism analysis such as single nucleotide polymorphism (SNP) . Of course, it can also be used as a nucleic acid measuring device (chip).
[0059]
In order to bind the nucleic acid probe of the present invention to the surface of a slide glass, for example, a slide glass coated with a polycation such as polylysine, polyethyleneimine, polyalkylamine, a slide glass introduced with an aldehyde group, a slide introduced with an amino group First prepare the glass. And i) a slide glass coated with a polycation is reacted with a phosphate group of the probe, ii) a slide glass introduced with an aldehyde group is reacted with a probe introduced with an amino group, iii) an amino group Can be achieved by introducing PDC (pyridinium dichromate), reacting with an amino group or aldehyde group introduced probe (Fodor, PA, et al., Science, 251, 767-773 (1991); Schena , M., et al., Proc. Natl. Acad. Sci., USA, 93, 10614-10619 (1996); McGal, G., et al., Proc. Natl. Acad. Sci., USA, 93, 13555-13560 (1996); Blanchad, AP, et al., Biosens. Bioelectron., 11, 687-690 (1996)).
[0060]
The device in which the nucleic acid probes of the present invention are arrayed and bound in an array on a solid surface makes nucleic acid measurement more convenient.
In this case, many types of target nucleic acids can be simultaneously measured by creating a device in which many nucleic acid probes of the present invention having different base sequences are individually bound on the same solid surface.
In this device, for each probe, at least one temperature sensor and a heater are provided on the surface opposite to the surface to which the solid nucleic acid probe of the present invention is bound, and the solid region to which the probe is bound is the optimum temperature condition. It is preferably designed so that the temperature can be adjusted.
[0061]
In this device, a probe other than the nucleic acid probe of the present invention, for example, two different fluorescent dyes in one molecule, and when not hybridized to the target nucleic acid, the interaction of the two fluorescent dyes A nucleic acid probe having a structure designed to be quenched or luminescent but to be luminescent or quenched when hybridized to a target nucleic acid, ie, the molecular beacon (Tyagi et al., Nature Biotech., 14: 303- 308, 1996) and the like can be suitably used. Therefore, such a device is also included in the technical scope of the present invention.
[0062]
The basic operation of the measurement method using the device of the present invention is to simply place a solution containing a target nucleic acid such as mRNA, cDNA or rRNA on a solid surface to which a nucleic acid probe is bound, and hybridize. Thereby, the amount of fluorescence changes according to the amount of target nucleic acid, and the target nucleic acid can be measured from the amount of fluorescence change. Moreover, the concentration of many target nucleic acids can be measured at a time by binding many kinds of the nucleic acid probes of the present invention having different base sequences on one solid surface. Therefore, it is a novel DNA chip because it can be used for measuring a target nucleic acid in exactly the same application as a DNA chip. Under optimal reaction conditions, nucleic acids other than the target nucleic acid do not change the amount of fluorescence emitted, and therefore, there is no need to wash the unreacted nucleic acid. Furthermore, by controlling the temperature for each nucleic acid probe of the present invention with a microheater, it is possible to control the optimum reaction conditions for each probe, so that accurate concentration measurement can be performed. In addition, the dissociation curve between the nucleic acid probe of the present invention and the target nucleic acid can be analyzed by continuously changing the temperature with a micro heater and measuring the amount of fluorescence during that time. From the difference in the dissociation curve, the property of the hybridized nucleic acid can be determined and the SNP can be detected.
[0063]
A conventional device for measuring the concentration of a target nucleic acid binds (fixes) a nucleic acid probe not modified with a fluorescent dye to a solid surface, hybridizes the target nucleic acid labeled with the fluorescent dye, and then hybridizes. The target nucleic acid not washed out was washed away, and the fluorescence intensity of the remaining fluorescent dye was measured.
In order to label a target nucleic acid with a fluorescent dye, for example, when a specific mRNA is targeted, the following steps are taken: (1) All mRNA extracted from cells is extracted. (2) Then, a reverse transcriptase is used to synthesize cDNA while incorporating a nucleoside modified with a fluorescent dye. In the present invention, such an operation is not necessary.
[0064]
A number of various probes are spotted on the device, but the optimum hybridization conditions, such as temperature, of nucleic acids that hybridize to the probes are different. Therefore, originally, it is necessary to perform the hybridization reaction and the washing operation under optimum conditions for each probe (each spot). However, since this is physically impossible, hybridization is performed at the same temperature for all the probes, and washing is performed at the same temperature with the same washing solution. As a result, the nucleic acid expected to be hybridized does not hybridize, and even if it is hybridized, the hybridization is not strong, so that it is easily washed away. For these reasons, the quantitativeness of nucleic acids was low. In the present invention, this washing operation is not necessary, and thus there is no such disadvantage. Further, by providing a micro heater at the bottom of the spot and controlling the hybridization temperature, the hybridization reaction can be performed at the optimum temperature for each probe of the present invention. Therefore, the present invention has a greatly improved quantitativeness.
[0065]
In the present invention, by using the above-described nucleic acid probe or device of the present invention, the concentration of the target nucleic acid can be measured easily and specifically in a short time. The measurement method is described below.
In the measurement method of the present invention, first, the nucleic acid probe of the present invention is added to the measurement system and hybridized with the target nucleic acid. The method can be carried out by conventional known methods (Analytical Biochemistry, 183, 231-244, 1989; Nature Biotechnology, 14, 303-308, 1996; Applied and Environmental Microbiology, 63, 1143-1147, 1997). For example, the hybridization conditions are a salt concentration of 0 to 2 molar, preferably 0.1 to 1.0 molar, and a pH of 6 to 8, preferably 6.5 to 7.5.
[0066]
The reaction temperature is preferably within the range of Tm value ± 10 ° C. of the nucleic acid hybrid complex obtained by hybridizing the nucleic acid probe of the present invention to a specific site of the target nucleic acid. By doing so, non-specific hybridization can be prevented. Non-specific hybridization occurs when the temperature is lower than Tm-10 ° C, and no hybridization occurs when the temperature exceeds Tm + 10 ° C. The Tm value can be determined in the same manner as the experiment necessary for designing the nucleic acid probe of the present invention. That is, an oligonucleotide that hybridizes with the nucleic acid probe of the present invention (with a base sequence complementary to the nucleic acid probe) is chemically synthesized with the nucleic acid synthesizer or the like, and the Tm value of the nucleic acid hybrid complex with the nucleic acid probe Is measured in the usual way.
[0067]
The reaction time is 1 second to 180 minutes, preferably 5 seconds to 90 minutes. When the time is less than 1 second, the number of unreacted nucleic acid probes of the present invention in hybridization increases. Also, there is no particular meaning if the reaction time is too long. The reaction time is greatly influenced by the nucleic acid species, that is, the length of the nucleic acid or the base sequence.
As described above, the nucleic acid probe of the present invention is hybridized to the target nucleic acid. Then, before and after hybridization, the amount of emission of the fluorescent dye is measured with a fluorometer, and the amount of decrease in emission is calculated. Since the magnitude of the decrease is proportional to the concentration of the target nucleic acid, the concentration of the target nucleic acid can be determined.
[0068]
The concentration of the target nucleic acid in the reaction solution is preferably 0.1 to 10.0 nM. The concentration of the nucleic acid probe of the present invention in the reaction solution is preferably 1.0 to 25.0 nM. When preparing a calibration curve, it is desirable to use the nucleic acid probe of the present invention at a ratio of 1.0 to 2.5 with respect to the target nucleic acid.
[0069]
In fact, when measuring an unknown concentration of target nucleic acid in a sample, a calibration curve is first created under the above conditions. Then, the corresponding nucleic acid probe of the present invention having a plurality of concentrations is added to the sample, and the decrease in the fluorescence intensity value is measured for each. Then, the probe concentration corresponding to the maximum value of the measured decrease in fluorescence intensity is set as a preferable probe concentration. The quantitative value of the target nucleic acid is obtained from the calibration curve with the decrease value of the fluorescence intensity measured with a probe having a preferred concentration.
[0070]
The principle of the nucleic acid measurement method of the present invention is as described above, but the present invention is not limited to various nucleic acid measurement methods such as FISH method, PCR method, LCR method, SD method, competitive hybridization method, TAS method, etc. Applicable to.
[0071]
An example is given below.
a) When applied to FISH method
That is, in the present invention, various types of microorganisms are mixed, or one or more types of microorganisms are mixed with cells derived from animals and plants, and cannot be isolated from each other (complex microbial system, symbiotic microorganism system). It can be suitably applied to the measurement of nucleic acids such as intracellular or cell homogenates. The microorganism here is a general microorganism, and is not particularly limited. For example, eukaryotic microorganisms, prokaryotic microorganisms, mycoplasma, viruses, rickettsias and the like can be mentioned. The target nucleic acid referred to in this system is a nucleic acid having a base sequence having specificity for cells of a strain to be examined, for example, how it is active in these microbial systems. For example, a specific sequence of 5SrRNA, 16SrRNA or 23SrRNA of a specific strain or their gene DNA.
[0072]
By adding the nucleic acid probe of the present invention to a complex microorganism system or a symbiotic microorganism system, and measuring the amount of 5SrRNA, 16SrRNA or 23SrRNA of the specific strain or their gene DNA, the abundance of the specific strain in the system is measured. it can. The method of measuring the abundance of a specific strain by adding the nucleic acid probe to a complex microbial or symbiotic microbial homogenate, and measuring the amount of decrease in fluorescence emission of the fluorescent dye before and after hybridization. Within the target range.
[0073]
The measurement method is as follows, for example. That is, before adding the nucleic acid probe of the present invention, the temperature, salt concentration, and pH of the complex microorganism system or symbiotic microorganism system are adjusted to the above conditions. Specific strains in complex or symbiotic microbial systems are 10 cells⁷-10¹²Pieces / ml, preferably 10⁹-10^TenIt is preferable to adjust to the unit / ml. It can be performed by dilution or concentration by centrifugation or the like. 10 cells⁷When the number is less than 1 / ml, the fluorescence intensity is weak and the measurement error becomes large. 10¹²When the number exceeds 1 / ml, the fluorescence intensity of the complex microbial system or the symbiotic microbial system is too strong, and the abundance of the specific microorganism cannot be measured quantitatively.
[0074]
The concentration of the nucleic acid probe of the present invention to be added depends on the number of cells of a specific strain in a complex microorganism system or a symbiotic microorganism system. Number of cells 1 × 10⁸The concentration is 0.1 to 10.0 nM, preferably 0.5 to 5 nM, more preferably 1.0 nM with respect to / ml. When it is less than 0.1 nM, the data does not accurately reflect the abundance of the specific microorganism. However, since the optimal amount of the nucleic acid probe of the present invention depends on the amount of the target nucleic acid in the cell, it cannot be generally stated.
[0075]
Next, in the present invention, the reaction temperature when hybridizing the nucleic acid probe to 5SrRNA, 16SrRNA or 23SrRNA of the specific strain or the gene DNA thereof is the same as described above. The reaction time is also the same as the above conditions.
Under the conditions as described above, the nucleic acid probe of the present invention is hybridized to 5S rRNA, 16S rRNA or 23S rRNA of a specific strain or gene DNA thereof, and the amount of decrease in the emission of the fluorescent dyes of the complex microorganism or the symbiotic microorganism before and after hybridization is reduced. taking measurement.
[0076]
The amount of decrease in the emission of the fluorescent dye measured as described above is proportional to the abundance of the specific strain in the complex microorganism system or the symbiotic microorganism system. This is because the amount of 5SrRNA, 16SrRNA or 23SrRNA or their gene DNA is proportional to the abundance of the specific strain.
[0077]
In the present invention, the components other than the microorganism in the complex microorganism system or the symbiotic microorganism system, unless the nucleic acid probe of the present invention and the 5S rRNA, 16S rRNA or 23S rRNA of the specific strain or their gene DNA are not inhibited. There is no particular limitation as long as the emission of the fluorescent dye of the nucleic acid probe of the present invention is not inhibited. For example, KH₂PO_Four, K₂HPO_Four, NaH₂PO_Four, Na₂HPO_FourPhosphates such as ammonium sulfate, ammonium nitrate, urea, etc., various salts of metal ions such as magnesium, sodium, potassium, calcium ions, sulfates of trace metal ions such as manganese, zinc, iron, cobalt ions, Various salts such as hydrochloride and carbonate, vitamins and the like may be appropriately contained. If the above-mentioned inhibition is observed, the cells may be separated from the cell system in which a plurality of microorganisms are mixed by an operation such as centrifugation and suspended again in a buffer solution system or the like.
[0078]
As the above buffer solution, various buffer solutions such as a phosphate buffer solution, a carbonate buffer solution, a Tris / hydrochloric acid buffer solution, a Tris / glycine buffer solution, a citrate buffer solution, and a Gut buffer solution can also be used. The concentration of the buffer solution is a concentration that does not inhibit the hybridization, the FRET phenomenon, and the luminescence of the fluorescent dye. Its concentration depends on the type of buffer. The pH of the buffer is 4-12, preferably 5-9.
[0079]
b) When applied to PCR method
Although any method can be applied as long as it is a PCR method, a case where it is applied to a real-time quantitative PCR method will be described below.
That is, in the real-time quantitative PCR method, PCR is performed using the specific nucleic acid probe of the present invention, and the decrease in the emission of the fluorescent dye before and after the reaction is measured in real time.
The PCR of the present invention means various methods of PCR. For example, RT-PCR, RNA-primed PCR, Stretch PCR, reverse PCR, PCR using Alu sequence, multiplex PCR, PCR using mixed primers, PCR method using PNA, and the like are also included. In addition, the term “quantitative” means not only the original quantitative measurement but also the quantitative measurement of the detection level as described above.
[0080]
As described above, the target nucleic acid refers to a nucleic acid whose abundance is to be measured. Regardless of purification. Moreover, the magnitude | size of a density | concentration does not ask | require. Various nucleic acids may be mixed. For example, a specific nucleic acid for amplification in a complex microorganism system (mixed system of RNA or gene DNA of a plurality of microorganisms) or a symbiotic microorganism system (mixed system of a plurality of animals and plants and / or RNA or gene DNA of a plurality of microorganisms). When purification of the target nucleic acid is necessary, it can be performed by a conventionally known method. For example, it can be performed using a commercially available purification kit.
[0081]
Conventionally known quantitative PCR methods include dATP, dGTP, dCTP, dTTP or dUTP, target nucleic acid (DNA or RNA), Taq polymerase, primer, and nucleic acid probe or intercalator labeled with a fluorescent dye in the presence of Mg ions. In addition, the target nucleic acid is amplified while repeating the temperature at low and high temperatures, and the increase in emission of the fluorescent dye in the amplification process is monitored in real time (Experimental Medicine, Vol. 15, No. 7, pp. 46-51, 1997). Year, Yodosha).
[0082]
The quantitative PCR method of the present invention is characterized in that the target nucleic acid is amplified using the nucleic acid probe of the present invention, and the amount of decrease in the emission of the fluorescent dye is measured in the amplification process. In the quantitative PCR of the present invention, the preferred nucleic acid probe of the present invention has 5 to 50 bases, preferably 10 to 25, particularly preferably 15 to 20, and an amplification product of the target nucleic acid during the PCR cycle. Anything may be used as long as it can hybridize with. In addition, either a forward type or a reverse type may be designed.
[0083]
For example, the following can be mentioned.
(1) The end portion, preferably the end, of the nucleic acid probe is labeled with the fluorescent dye of the present invention, and when the nucleic acid probe is hybridized to the target nucleic acid, the end or end labeled with the fluorescent dye of the probe The base sequence of the probe is designed so that at least one base of G (guanine) is present in the base sequence of the target nucleic acid at a distance of 1 to 3 bases from the terminal base of the target nucleic acid that has hybridized to.
(2) Among the above (1), the nucleic acid probe is labeled with a fluorescent dye at the 3 'end.
[0084]
(3) Among the above (1), the nucleic acid probe is labeled with a fluorescent dye at the 5 'end.
(4) When the nucleic acid probe is hybridized to the target nucleic acid, the probe is such that a plurality of base pairs of the nucleic acid hybrid complex forms at least one G (guanine) and C (cytosine) pair at the probe end portion. The nucleotide sequence is designed.
(5) In the above (4), the 3 'terminal base of the nucleic acid probe is labeled with G or C, and the 3' terminal is labeled with a fluorescent dye.
(6) Among the above (4), the 5 'terminal base of the nucleic acid probe is labeled with G or C, and the 5' terminal is labeled with a fluorescent dye.
(7) The nucleic acid probe of (1) to (6) above has a hydroxyl group at the 3′-position of ribose or deoxyribose at the 3 ′ end, or a hydroxyl group at the 3′-position or 2′-position of ribose at the 3 ′ end. It is phosphorylated.
(8) The oligonucleotide of the nucleic acid probe of (1) to (6) is chemically modified.
[0085]
In the case of the above (6), when the 3 ′ or 5 ′ end cannot be designed as G or C from the base sequence of the target nucleic acid, at the 5 ′ end of the oligonucleotide that is a primer designed from the base sequence of the target nucleic acid, Even if 5′-guanylic acid or guanosine or 5′-cytidylic acid or cytidine is added, the object of the present invention can be suitably achieved. Even if 5'-guanylic acid or 5'-cytidylic acid is added to the 3 'end, the object of the present invention can be preferably achieved. Therefore, in the present invention, the nucleic acid probe designed so that the base at the 3 ′ or 5 ′ end is G or C refers to the 5 ′ end of the probe in addition to the probe designed from the base sequence of the target nucleic acid. A probe obtained by adding 5′-guanylic acid or guanosine or 5′-cytidylic acid or cytidine, and a probe obtained by adding 5′-guanylic acid or 5′-cytidylic acid to the 5 ′ end of the probe. It is defined as including.
[0086]
In particular, the nucleic acid probe of the present invention (7) is designed not to be used as a primer. Instead of the two probes (labeled with fluorescent dyes) used in the real-time quantitative PCR method using the FRET phenomenon, PCR is carried out using one nucleic acid probe of the present invention. The probe is added to the PCR reaction system and PCR is performed. During the nucleic acid extension reaction, the probe hybridized to the target nucleic acid or the amplified target nucleic acid is decomposed by the polymerase and decomposed and removed from the nucleic acid hybrid complex. The fluorescence intensity value of the reaction system at this time or the reaction system in which the nucleic acid denaturation reaction has been completed is measured. In addition, the fluorescence of the reaction system in which the target nucleic acid or the amplified amplified target nucleic acid is hybridized with the probe (an annealing reaction or a reaction system during the nucleic acid extension reaction until the probe is removed from the nucleic acid hybrid complex by polymerase) Measure the intensity value. And the nucleic acid amplified by calculating the decreasing rate of the fluorescence intensity value from the former is measured. Fluorescence when the probe is completely dissociated from the target nucleic acid or amplified target nucleic acid by a nucleic acid denaturation reaction, or is decomposed and removed from the nucleic acid hybrid complex of the probe and the target nucleic acid or amplified target nucleic acid by a polymerase during nucleic acid extension. The intensity value is large. However, the reaction system in which the probe is sufficiently hybridized to the target nucleic acid or the amplified target nucleic acid, or the nucleic acid hybrid complex of the probe and the target nucleic acid or the amplified target nucleic acid by the polymerase during the nucleic acid extension reaction. From the former, the fluorescence intensity value of the reaction system until it is decomposed and removed is decreased. The decrease in fluorescence intensity value is proportional to the amount of nucleic acid amplified.
[0087]
In this case, the Tm of the nucleic acid hybrid complex when the probe is hybridized with the target nucleic acid is within the range of ± 15 ° C., preferably ± 5 ° C. of the Tm value of the primer nucleic acid hybrid complex ( It is desirable that the base sequence of the probe in 7) is designed. When the Tm value of the probe is -5 ° C., particularly less than −15 ° C. of the primer, the probe does not hybridize, so that the emission of the fluorescent dye does not decrease. On the other hand, when the Tm value of the primer exceeds + 5 ° C., particularly + 15 ° C., the probe hybridizes with a nucleic acid other than the target nucleic acid, so that the specificity of the probe is lost.
[0088]
Probes other than the above (7), particularly the probe of (6) are added as primers to the PCR reaction system. A PCR method using a primer labeled with a fluorescent dye is not yet known except the present invention. As the PCR reaction proceeds, the amplified nucleic acid is secondarily labeled with a fluorescent dye useful in the practice of the present invention. Therefore, the fluorescence intensity value of the reaction system in which the nucleic acid denaturation reaction has been completed is large, but in the reaction system in which the annealing reaction is completed or in the nucleic acid extension reaction, the fluorescence intensity of the reaction system is lower than the former fluorescence intensity. To do.
[0089]
The PCR reaction can be performed under the same reaction conditions as in a normal PCR method. Thus, the target nucleic acid can be amplified in a reaction system with a low Mg ion concentration (1-2 mM). Of course, the present invention can also be carried out in a reaction system in the presence of a high concentration (2 to 4 mM) of Mg ions used in conventionally known quantitative PCR.
[0090]
In the PCR method of the present invention, the Tm value can be determined by performing the PCR of the present invention and analyzing the nucleic acid melting curve of the amplified product. This method is a novel method for analyzing melting curves of nucleic acids. In this method, the nucleic acid probe used in the PCR method of the present invention or the nucleic acid probe of the present invention used as a primer can be suitably used.
In this case, by making the base sequence of the nucleic acid probe of the present invention complementary to the region containing SNP (snip; single nucleotide substitution polymorphism), the nucleic acid is dissociated from the nucleic acid probe of the present invention after the PCR is completed. By analyzing the curve, SNP can be detected from the difference in the dissociation curve. If a nucleotide sequence complementary to the sequence containing SNP is used as the sequence of the nucleic acid probe of the present invention, the Tm value obtained from the dissociation curve between the probe sequence and the sequence containing SNP is the dissociation curve from the sequence not containing SNP. Higher than the Tm value obtained from
[0091]
The second feature of the present invention is an invention of a method for analyzing data obtained by the real-time quantitative PCR method.
The real-time quantitative PCR method is currently a computer-readable record in which each procedure of a reaction apparatus for performing PCR, an apparatus for detecting luminescence of a fluorescent dye, a user interface, that is, a data analysis method is programmed and recorded. It is measured in real time on a medium (also known as Sequence Detection Software System) and an apparatus composed of a computer that controls and analyzes the data. Therefore, the measurement of the present invention is also performed with such an apparatus.
[0092]
First, a real-time quantitative PCR analyzer will be described below. The apparatus used in the present invention may be any apparatus as long as it can monitor PCR in real time. For example, ABI PRISM^TM 7700 Sequence Detection System SDS 7700 (Perkin Elmer Applied Biosytems, USA), Light Cycler^TM A system (Roche Diagnostics Inc., Germany) can be cited as a particularly suitable one.
[0093]
The PCR reaction apparatus is an apparatus that repeatedly performs heat denaturation reaction, annealing reaction, and nucleic acid extension reaction of a target nucleic acid (for example, the temperature can be repeated at 95 ° C., 60 ° C., and 72 ° C.). . The detection system includes an argon laser for fluorescence excitation, a spectrograph, and a CCD camera. Further, a computer-readable recording medium on which each procedure of the data analysis method is programmed and recorded is used by being installed in the computer, and the system is controlled via the computer and output from the detection system. A program for analyzing data is recorded.
[0094]
The data analysis program recorded on the computer-readable recording medium is a process for measuring the fluorescence intensity for each cycle, and the measured fluorescence intensity is displayed on the computer display as a function of the cycle, that is, as an amplification plot of PCR. The process of displaying, the process of calculating the PCR cycle number (threshold cycle number: Ct) at which the fluorescence intensity starts to be detected, the process of creating a calibration curve for obtaining the copy number of the sample nucleic acid from the Ct value, the data of each process, the plot value The process of printing. When PCR progresses exponentially, a linear relationship is established between the Log value of the copy number of the target nucleic acid at the start of PCR and Ct. Therefore, a copy number at the start of PCR of the target nucleic acid can be calculated by preparing a calibration curve using a known number of copies of the target nucleic acid and detecting Ct of the sample containing the target nucleic acid with an unknown copy number.
[0095]
A PCR-related invention such as the above data analysis method is a method for analyzing data obtained by the above-described real-time quantitative PCR method. Each feature is described below.
The first feature is that in the method of analyzing data obtained by the real-time quantitative PCR method, the amplified nucleic acid in each cycle is bound to the fluorescent dye, or the amplified nucleic acid is hybridized with the nucleic acid probe of the present invention. The fluorescence intensity value of the reaction system is obtained by combining the fluorescent dye and the nucleic acid in each cycle, that is, the fluorescent dye-nucleic acid complex, or the hybridized nucleic acid probe of the present invention and the target nucleic acid, that is, the nucleic acid. This is a calculation process in which correction is performed based on the fluorescence intensity value of the reaction system when the hybrid complex is dissociated, that is, a correction calculation process.
[0096]
The “reaction system when the amplified target nucleic acid is bound to the fluorescent dye or the amplified target nucleic acid is hybridized with the nucleic acid probe of the present invention” is specifically exemplified by 40 to 85 in each cycle of PCR. A reaction system for nucleic acid elongation reaction or annealing having a reaction temperature of 50 ° C., preferably 50 to 80 ° C. can be mentioned. And it means a reaction system in which the reaction is completed. The actual temperature depends on the length of the amplified nucleic acid.
The “reaction system when the fluorescent dye-nucleic acid complex or the nucleic acid hybrid complex is dissociated” means a reaction system for heat denaturation of nucleic acid in each cycle of PCR, specifically, reaction temperature. A system in which the reaction is completed at 90 to 100 ° C., preferably 94 to 96 ° C. can be exemplified.
[0097]
The correction calculation process in the correction calculation process may be any process that meets the object of the present invention. Specifically, the process according to the following [Formula 1] or [Formula 2] Can be exemplified.
f_n= F_{hyb, n}/ F_{den, n}          [Formula 1]
f_n= F_{den, n}/ F_{hyb, n}          [Formula 2]
[Where,
f_n: Correction calculation processing value in the n-th cycle calculated by [Equation 1] or [Equation 2],
f_{hyb, n}: In the n-th cycle, when the amplified nucleic acid is bound to the fluorescent dye, or when the amplified nucleic acid is hybridized with the nucleic acid probe of the present invention, the fluorescence intensity value of the reaction system,
f_{den, n}: In the n-th cycle, the fluorescence intensity value of the reaction system when the fluorescent dye-nucleic acid complex or the nucleic acid hybrid complex is dissociated]
In this process, the correction calculation processing value obtained by the above processing is displayed on the computer display and / or the value is similarly displayed and / or printed in the form of a graph as a function of each cycle number. Includes steps.
[0098]
The second feature is that the correction calculation processing value according to [Equation 1] or [Equation 2] in each cycle is substituted into the following [Equation 3] or [Equation 4], and the fluorescence change rate or the fluorescence change rate between the samples. Is a data analysis method for calculating and comparing them.
[0099]
F_n= F_n/ F_a          [Formula 3]
F_n= F_a/ F_n          [Formula 4]
[Where,
F_n: In the n-th cycle, the fluorescence change rate or the fluorescence change rate calculated by [Equation 3] or [Equation 4],
f_n: Correction calculation processing value according to [Formula 1] or [Formula 2] in the n-th cycle
f_a: Correction calculation processing value according to [Formula 1] or [Formula 2], f_nOf any number of cycles before the change is observed, but typically for example 10 to 40 cycles, preferably 15 to 30 cycles, more preferably 20 to 30 cycles Is adopted. ]
In this process, the calculated value obtained by the above process is displayed and / or printed on the computer display, or the comparison value or the value is similarly displayed and / or displayed in the form of a graph as a function of each cycle number. Although it includes a sub-step for printing, the sub-step may or may not be applied to the correction calculation processing value according to [Formula 1] or [Formula 2].
[0100]
The third feature is
(3.1) A process of performing arithmetic processing according to [Equation 5], [Equation 6] or [Equation 7] using the fluorescence change rate or the fluorescence change rate data calculated by [Equation 3] or [Equation 4]. ,
log_b(F_n), Ln (F_n[Formula 5]
log_b{(1-F_n) × A}, ln {(1-F_n) × A} [Formula 6]
log_b{(F_n−1) × A}, ln {(F_n−1) × A} [Formula 7]
[0101]
[Where,
A, b: Any numerical value, preferably an integer value, more preferably a natural number. When A = 100 and b = 10, {(F_n−1) × A} is expressed as a percentage (%).
F_n: [Fluorescence change rate or fluorescence change rate in n cycles calculated by [Equation 3] or [Equation 4]]
(3.2) An arithmetic processing step for obtaining the number of cycles in which the arithmetic processing value of (3.1) has reached a certain value;
(3.3) An arithmetic process for calculating a relational expression between the number of cycles in a nucleic acid sample of a known concentration and the number of copies of the target nucleic acid at the start of the reaction,
(3.4) An operation process for obtaining a copy number of a target nucleic acid at the start of PCR in an unknown sample,
Is a data analysis method. A process consisting of (3.1) → (3.2) → (3.3) → (3.4) is preferable.
[0102]
In each of the processes (3.1) to (3.3), the processing value obtained by each processing is displayed on a computer display and / or the value is expressed in the form of a graph as a function of the number of cycles. Similarly to the above, it may include a sub-step of displaying and / or printing. Since the arithmetic processing value obtained in the process (3.4) needs to be printed at least, the process includes a sub-step for printing. The arithmetic processing value obtained in the above (3.4) may be further displayed on a computer display.
It should be noted that the correction processing value according to [Formula 1] or [Formula 2] and the calculation processing value according to [Formula 3] or [Formula 4] are displayed on the computer display in the form of a graph as a function of the number of cycles. Since it may or may not be printed, the display and / or printing sub-steps may be added as necessary.
[0103]
The data analysis method is particularly effective when the real-time quantitative PCR method measures the amount of decrease in the emission of the fluorescent dye. A specific example is the above-described real-time quantitative PCR method of the present invention.
[0104]
A fourth feature of the real-time quantitative PCR analyzer is a real-time quantitative PCR measurement and / or analysis device having a calculation and storage means for executing the data analysis method for the real-time quantitative PCR method of the present invention. It is.
[0105]
A fifth feature of the present invention is that in the computer-readable recording medium in which each procedure of the data analysis method for analyzing PCR using a real-time quantitative PCR analyzer is programmed and the program is recorded, the data analysis of the present invention is performed. A computer-readable recording medium recording a program that allows a computer to execute each procedure of the method.
[0106]
A sixth feature is a novel nucleic acid measurement method using the data analysis method, measurement and / or analysis apparatus, and recording medium of the present invention in the nucleic acid measurement method.
[0107]
A seventh feature is a method for analyzing data obtained by the method for analyzing a melting curve of a nucleic acid of the present invention, that is, a method for obtaining a Tm value of a nucleic acid by performing the PCR method of the present invention.
That is, for a nucleic acid amplified by the PCR method of the present invention, the temperature is gradually increased from a low temperature until the nucleic acid is completely denatured (for example, from 50 ° C. to 95 ° C.). For example, the process of measuring fluorescence intensity at intervals corresponding to a temperature rise of 0.2 ° C. to 0.5 ° C., the process of displaying the measurement result on the display as a function of time, ie, the melting curve of the nucleic acid is displayed. The process, the process of differentiating this melting curve to obtain a differential value (-dF / dT, F: fluorescence intensity, T: time), the process of displaying the value as a differential value on the display, the inflection point from the differential value It is an analysis method that consists of the process of obtaining. In the present invention, the fluorescence intensity increases with increasing temperature. In the present invention, the process of dividing the fluorescence intensity value at the time of nucleic acid extension reaction in each cycle, preferably at the end of the PCR reaction, using the fluorescence intensity value at the heat denaturation reaction is added to the above process, More favorable results are obtained.
[0108]
An apparatus for measuring and / or analyzing real-time quantitative PCR according to the present invention, which is obtained by adding a method for analyzing the melting curve of the nucleic acid according to the present invention to the data analysis method of the novel PCR method according to the present invention, is also provided. Within the technical scope.
[0109]
Furthermore, one of the features of the present invention is a computer-readable recording medium storing a program that allows a computer to execute each step of the method for analyzing a melting curve of a nucleic acid of the present invention, Each of the methods for analyzing the melting curve of the nucleic acid of the present invention in a computer-readable recording medium recording a program that allows a computer to execute each procedure of the data analysis method of the PCR method of the present invention A computer-readable recording medium additionally recorded with a program that allows a computer to execute the procedure.
[0110]
The data analysis method, apparatus, and recording medium of the present invention can be used in various fields such as medicine, forensic medicine, anthropology, ancient biology, biology, genetic engineering, molecular biology, agriculture, plant breeding, and the like. . Also referred to as a complex microbial system or a symbiotic microbial system, such as microbial systems in which various types of microorganisms are mixed or at least one type of microorganism is mixed with cells of other animals and plants and cannot be isolated from each other. Can be suitably used. The microorganism referred to herein is a microorganism generally referred to, and is not particularly limited. For example, eukaryotic microorganisms, prokaryotic microorganisms, other mycoplasmas, viruses, rickettsias and the like can be mentioned.
[0111]
Further, by using one or more of the data analysis method, apparatus and recording medium of the present invention, the number of copies of 5S rRNA, 16S rRNA or 23S rRNA of a specific strain in a complex microorganism system or a symbiotic microorganism system or their gene DNA is quantified. By doing so, the abundance of the specific strain in the system can be measured. This is because the gene DNA copy number of 5SrRNA, 16SrRNA or 23SrRNA is constant depending on the strain. In the present invention, it is possible to perform the real-time quantitative PCR of the present invention using a complex microbial or symbiotic microbial homogenate to measure the abundance of a specific strain. This method is also within the technical scope of the present invention.
[0112]
【Example】
Next, the present invention will be described more specifically with reference to examples and comparative examples.
Example 1
E. coli (Escherichia coliThe nucleic acid probe which hybridizes with the nucleic acid base sequence of 16S rRNA of (3 ') CCGCTCACGC ATC (5') was prepared as follows.
[0113]
Nucleic acid probe preparation: (3 ′) CCGCTCACGC To the OH group at the 3′-position of deoxyribose at the 3 ′ end of an oligodeoxyribonucleotide having the base sequence of ATC (5 ′), — (CH₂)₇-NH₂The product was purchased from Medland Certified Reagent Company (USA). In addition to the Fluo Reporter Kit F-6082 (BODIPY FL propionic acid succinimidyl ester) from Molecular Probes, the compound is added to the oligonucleotide amine. A kit containing a reagent that binds to the derivative) was purchased. The kit was allowed to act on the purchased oligonucleotide to synthesize a nucleic acid probe labeled with bodepy FL used in this example.
[0114]
Purification of compound: The obtained synthesized product was dried to obtain a dried product. 0.5M NaHCO_Three/ Na₂CO_ThreeDissolved in buffer (pH 9.0). The lysate was subjected to gel filtration with a NAP-25 column (Pharmacia) to remove unreacted substances. Further, reverse phase HPLC (B gradient: 15 to 65%, 25 minutes) was performed under the following conditions. And the main peak which eluted was fractionated. The fraction thus collected was lyophilized to obtain a nucleic acid probe in a yield of 23% from 2 mM of the first oligonucleotide raw material.
[0115]

[0116]
Example 2
Using a 200 ml (sterilized) Erlenmeyer flask to which 50 ml of a sterilized Nutrient Broth (NB) (Difco) liquid medium (composition: NB, 0.08 g / 100 ml) was added, 37 strains of E. coli JM109 were obtained. Shake culture overnight at ° C. Next, an equivalent amount of 99.7% ethanol was added to the main culture solution. 2 ml of this ethanol-added culture was centrifuged with a 2.0 ml Eppendorf centrifuge tube to obtain bacterial cells. The cells were washed once with 100 μl of 30 mM phosphate buffer (soda salt) (pH: 7.2). The cells were suspended in 100 μl of the phosphate buffer containing 130 mM NaCl. The suspension was sonicated in ice-cooling for 40 minutes (output: 33 w, oscillation frequency: 20 kHz, oscillation method: oscillation for 0.5 seconds, pause for 0.5 seconds) to produce a homogenate.
[0117]
After the homogenate was centrifuged, the supernatant was collected and transferred to a fluorometer cell. The temperature was adjusted to 36 ° C. The nucleic acid probe solution pre-warmed to 36 ° C. was added to a final concentration of 5 nM. The Escherichia coli 16S rRNA and the nucleic acid probe were hybridized for 90 minutes while controlling the temperature at 36 ° C. The amount of luminescence of the fluorescent dye was measured with a fluorometer.
[0118]
The light emission amount of the fluorescent dye before hybridization is a value measured using a 30 mM phosphate buffer solution (soda salt) (pH: 7.2) containing 130 mM NaCl instead of the above supernatant. did. The amount of luminescence was measured by changing the ratio between the amount of the nucleic acid probe and the amount of the supernatant (excitation light 503 nm; measurement fluorescence color 512 nm). The results are shown in FIG. As can be seen from FIG. 1, the light emission of the fluorescent dye decreased as the amount ratio of the supernatant increased. That is, in the present invention, it can be seen that the amount of decrease in emission of the fluorescent dye increases in proportion to the amount of target nucleic acid hybridized to the nucleic acid probe.
[0119]
Example 3
Preparation of nucleic acid probe: (5 ′) CCCACATCGTTTTGTCTGGG (3 ′) hybridizing to 23S rRNA of Escherichia coli JM109 strain,-(CH₂)₇-NH₂In the same manner as in Example 1, the product was purchased from Medland Certified Reagent Company (USA). Further, in the same manner as in Example 1, in addition to the Fluo Reporter Kit F-6082 (BODIPY FL propionic acid succinimidyl ester), from the Molecular Probes Co., Ltd. Kits containing reagents that bind compounds to amine derivatives of oligonucleotides were purchased. The kit was allowed to act on the oligonucleotide to synthesize a nucleic acid probe labeled with bodepy FL. The obtained synthesized product was purified in the same manner as in Example 1 to obtain a nucleic acid probe labeled with bodepy FL at a yield of 25% from 2 mM of the first oligonucleotide raw material.
[0120]
Example 4
Pseudomonas paucimobilis (current name: Sphingomonas pusiomobils) (FERM P-5122) prepared in the same medium and culture conditions as in Example 2 on the cells of Escherichia coli JM109 obtained in Example 2 ) Was mixed at the same concentration with E. coli strain JM109 at an OD660 value to prepare a complex microorganism system. A homogenate was prepared by the same method as in Example 2 for the obtained mixed solution (the cell concentration of Escherichia coli JM109 strain was the same as in Example 2). Using the nucleic acid probe prepared in Example 3, an experiment similar to Example 2 was performed except that the excitation light was changed to 543 nm and the measurement fluorescence color was changed to 569 nm. As a result, the same result as in Example 2 was obtained. It was.
[0121]
Example 5
The base selectivity of the target nucleic acid in the fluorescence quenching phenomenon, that is, the base specificity of the present invention was examined. Ten types (poly a to poly j) of target synthetic deoxyribooligonucleotides (30 mer) shown below were prepared with a DNA synthesizer ABI394 (Perkin Elmer, USA).
[0122]
Furthermore, the following probe of the present invention labeled with bodepy FL at the 5 'end of the deoxyribooligonucleotide corresponding to the above synthetic DNA was prepared.
To the phosphate group at the 5 'end of the primer DNA corresponding to the above synthetic DNA,-(CH₂)₆-NH₂The product was purchased from Medland Certified Reagent Company (USA). Furthermore, in addition to the Fluo Reporter Kit F-6082 (BODIPY FL propionic acid succinidyl ester of Bodepy FL / C6) from Molecular Probes, A kit containing a reagent to be bound to an amine derivative was purchased. Probes a to d and f to h of the present invention labeled with the following Bodepy FL were synthesized by allowing the kit to act on the purchased primer DNA. Then, the degree to which the fluorescence of the fluorescent dye decreased when hybridized with the corresponding synthetic deoxyribooligonucleotide was examined under the following conditions, and the specificity of the probe of the present invention was examined.
[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]
The results are shown in Table 1. As can be seen from Table 1, when a nucleic acid probe labeled with a fluorescent dye is hybridized to a target DNA (oligodeoxyribonucleotide), at the terminal part, the terminal base part from which the probe and target DNA hybridized is 1 It is preferable that the base sequence of the probe is designed so that at least one base or more of G (guanine) exists in the base sequence of the target DNA at a distance of -3 bases. Further, when the nucleic acid probe labeled with a fluorescent dye is hybridized to the target DNA, the probe is so formed that a plurality of base pairs of the nucleic acid hybrid complex form at least one pair of G and C at the terminal portion. It can be seen from Table 1 that the base sequence is preferably designed.
[0130]
Example 6
A target nucleic acid having the following base sequence and a nucleic acid probe of the present invention were prepared. The influence of the number of G in the target nucleic acid and the number of G in the nucleic acid probe of the present invention was examined in the same manner as in the above example.
[0131]

[0132]

[0133]

[0134]

[0135]

[0136]
As can be seen from Table 2, it is recognized that the number of G in the target nucleic acid and the number of G in the probe of the present invention do not significantly affect the decrease in fluorescence intensity.
[0137]
Example 7
In the same manner as described above, a target nucleic acid having the following base sequence and a nucleic acid probe of the present invention were prepared. The nucleic acid probe of the present invention in this example is one in which the 5 'end of the oligonucleotide is labeled with bodepy FL / C6. The influence of the base species in the target nucleic acid and the base species in the nucleic acid probe of the present invention was examined in the same manner as in the above Example.
[0138]

[0139]

[0140]

[0141]
As can be seen from Table 3 and the above examples, (i) when the end of the probe of the present invention labeled with a fluorescent dye is composed of C and the target nucleic acid is hybridized to form a GC pair (ii) ) When the end of the probe of the present invention labeled with a fluorescent dye is composed of a base other than C, the target nucleic acid 3 is determined from the base pair of the base labeled with the fluorescent dye and the base of the target nucleic acid. 'When at least one G exists on the terminal side, the decrease rate of the fluorescence intensity is large.
[0142]
Example 8
The kind of the dye labeled on the nucleic acid probe of the present invention was examined in the same manner as in the above example. Note that the probe z of Example 7 was used as the probe of the present invention, and the oligonucleotide z of Example 7 was used as the target nucleic acid.
The results are shown in Table 4. As can be seen from the table, suitable fluorescent dyes for use in the present invention include FITC, BODIPY FL, BODIPY FL / C3, 6-joe, TMR and the like.
[0143]

[0144]
Example 9 (Experiment of effect of heat treatment of target nucleic acid (16SrRNA))
Preparation of the nucleic acid probe of the present invention: specifically hybridizes to the 16S rRNA base sequence of the KYM-7 strain corresponding to the base sequence of 1190 bases to 1190 bases of the 16S rRNA of Escherichia coli JM109 strain (5 ') CATCCCCACC TTCCT CCGAG TTGACCCCGG CAGTC (3 ') The base sequence of (35 base pairs) is modified with deoxyribonucleotides at the 1st to 16th and 25th to 35th bases, and the OH group at the 2' position carbon at the 17th to 24th bases with a methyl group (with an ether bond) Modified) ribooligonucleotides, and the OH group of the phosphate group at the 5 'end is-(CH₂)₇-NN₂Oligonucleotides modified and bound in the same manner as in Example 1 were purchased from Medland Certified Reagent Company, USA. In addition, 2-O-Me oligonucleotides used for 2-O-Me probes (probes consisting of 2-O-Me oligonucleotides are simply referred to as 2-O-Me probes) were synthesized by GENSET Corporation (France). It was obtained by consigning.
Furthermore, in the same manner as in Example 1, Fluo Reporter Kit F-6082 (BODIPY FL / C6 propionic acid succinimidyl ester) was obtained from Molecular Probes. In addition, a kit containing a reagent that binds the compound to an amine derivative of an oligonucleotide was purchased. The kit was allowed to act on the oligonucleotide to synthesize the nucleic acid probe of the present invention labeled with bodepy FL / C6. The obtained synthesized product was purified in the same manner as in Example 1 to obtain the nucleic acid probe of the present invention labeled with Bodepy FL / C6 at a yield of 23% from 2 mM of the first oligonucleotide raw material. This probe was named 35-base chain 2-O-Me probe.
[0145]
5 ′) TCCTTTGAGT TCCCGGCCGG A (3 ′) The oligoribonucleotide having the base sequence was synthesized using a DNA synthesizer in the same manner as described above to obtain a forward type helper probe. On the other hand, an oligoribonucleotide having the base sequence of (5 ′) CCCTGGTCGT AAGGGCCATG ATGACTTGAC GT (3 ′) was synthesized using a DNA synthesizer in the same manner as described above, and a backward type, that is, a reverse type helper probe. did.
[0146]
A 35-base chain 2-O-Me probe, a forward-type helper probe, and a reverse-type helper probe were each dissolved in a buffer solution having the composition shown below so as to have a concentration of 25 nM, and heated to 75 ° C. (probe solution).
The 16S rRNA was heat-treated at 95 ° C. for 5 minutes, added to the probe solution under the reaction conditions shown below, and then measured for fluorescence intensity with a fluorescence measuring instrument Perkin Elmer LS-50B. . The results are shown in FIG. A control using 16S rRNA not subjected to heat treatment was used as a control. It can be seen from FIG. 2 that the decrease in fluorescence intensity is large in the heat-treated experimental section. This result indicates that the 16S rRNA is heat-treated at 95 ° C. to thereby perform stronger hybridization with the probe of the present invention.

[0147]
Example 10 (Experiment on Contribution of 2′-O-Me Oligonucleotide and Helper Probe to Improvement of Hybridization Efficiency)
The following various nucleic acid probes of the present invention and various helper probes hybridizing to the 16S rRNA were prepared in the same manner as in Example 9. All 2-O-Me oligonucleotides used for 2-O-Me probes were obtained by entrusting synthesis to GENSET (France). Under the following conditions, the effects of the 2-O-Me probe of the present invention, the influence of the length of the base chain of the probe, and the effect of the helper probe are shown in the experiments shown in FIGS. 3A, 3B, 3D, and 3D below. The system was examined in the same manner as in Example 9. The results are shown in FIG.
From the figure, it can be seen that the 2-O-Me probe of the present invention contributes to the hybridization efficiency. Further, when the base chain of the 2-O-Me probe is short, the helper probe is useful for increasing the hybridization efficiency.
[0148]
1) 35-base chain 2-O-Me probe: The same probe as in Example 9,
2) 35-base DNA probe: a probe having the same base sequence as the 35-base 2-O-Me probe of 1) above, wherein the oligonucleotide is composed of deoxyribose,
3) 17-base chain 2-O-Me probe: The same nucleotide sequence as the 35-base chain 2-O-Me probe in 1) above, but 8 nucleotides from the 5 'end and 10 bases from the 3' end Removed probe,
4) 17-base DNA probe: a probe having the same base sequence as the 33-base DNA probe of 2) above, except that 16 base nucleotides are deleted from the 3 'end;
[0149]
5) Forward type 2-O-Me helper probe: OH group at the 2′-position carbon of ribose corresponding to the center 8 bases (9 bases to 16 bases from the 5 ′ end) of the forward type helper probe of Example 9 A helper probe modified with a methyl group (ether bond),
6) Reverse-type 2-O-Me helper probe: OH group at the 2′-position carbon of ribose corresponding to the center 8 bases (9 to 16 bases counted from the 5 ′ end) of the reverse-type helper probe of Example 9 A helper probe modified with a methyl group (ether bond),
7) Forward DNA helper probe: a helper probe having the same base sequence as that of the forward helper probe of Example 9, but having an oligonucleotide composed of deoxyribonucleotides,
8) Reverse-type DNA helper probe: a helper probe having the same base sequence as that of the reverse-type helper probe of Example 9 above, wherein the oligonucleotide is composed of deoxyribonucleotides,
9) 35 base oligoribonucleotide: (5 ') CATCCCCACC TTCCTCCGAG TTGA CCCCGG CAGTC (3') oligoribonucleotide having the base sequence,
10) 17-base-chain oligoribonucleotide: (5 ′) Oligoribonucleotide having a base sequence of CCTTCCTCCG AGTTGAC (3 ′).
[0150]

[0151]

[0152]

[0153]

[0154]

[0155]
Example 11 (Preparation of calibration curve for rRNA measurement)
The rRNA was heated at 95 ° C. for 5 minutes at various concentrations ranging from 0.1 to 10 nM, and the obtained nucleic acid solution was added to the reaction solution in advance under the following reaction conditions. After 1000 seconds, the fluorescence intensity was increased. The decrease was measured using a Perkin Elmer LS-50B. The results are shown in FIG. It can be seen from the figure that the calibration curve exhibits linearity at 0.1 to 10 nM. The following 35-base chain 2-O-Me probe is the same probe as in Example 9.

[0156]
Example 12 (FISH method)
Cellulomonas sp. KYM-7 (FERM P-11339) and Agrobacterium sp. KYM-8 (FERM P-16806) The following 35 or 36 bases of the present invention that hybridize to each rRNA Strand oligodeoxyribonucleotide 2-O-Me probes were prepared as described above. The base sequence of each probe is as follows.
[0157]
35-base oligodeoxyribonucleotide 2-O-Me probe for rRNA measurement of Cellulomonas sp. KYM-7:
(5 ') CATCCCCACC TTCCTCCGAG TTGACCCCGG CAGTC (3 ') (underlined part is modified with a methyl group).
A 36-base oligodeoxyribonucleotide 2-O-Me probe for rRNA measurement of Agrobacterium sp. KYM-8:
(5 ') CATCCCCACC TTCCTCTCGG CTTATCACCG GCAGTC (3 ') (underlined part is modified with methyl group).
[0158]
Cellulomonas sp. KYM-7 and Agrobacterium sp. KYM-8 were mixed and cultured in a medium having the following medium composition under the following culture conditions, and the culture was collected at each culture time. From them, rRNA was prepared using RNeasy Maxikit (QIAGEN). The rRNA was heated at 95 ° C. for 5 minutes, then added to the reaction solution in advance under reaction conditions, reacted at 70 ° C. for 1000 seconds, and then the fluorescence intensity was measured using Perkin Elmer LS-50B. The results are shown in FIG. The total rRNA was measured using RiboGreen total RNA Quantification Kit (company name: molecular probes, location name: Eugene, Oregon, USA).
As can be seen from the figure, the kinetics of rRNA in each strain was consistent with the kinetics of total rRNA. Moreover, the total amount of rRNA of each strain was consistent with the total rRNA. This indicates that the method of the present invention is an effective method in the FISH method.
[0159]
Medium composition (g / l): starch, 10.0; aspartic acid, 0.1; K₂HPO_Four, 5.0; KH₂PO_Four, 2.0; MgSO_Four・ 7H₂O, 0.2; NaCl, 0.1; (NH_Four)₂SO_Four; 0.1.
-100 ml of medium was dispensed into a 500 ml conical flask, and the flask was sterilized at 120 ° C for 10 minutes using an autoclave kettle.
-Culture conditions: The above strain was cultured in advance on a slant medium. One platinum loop of fungus was taken from the slant medium and inoculated into the medium of the sterilized conical flask. Stirring culture was performed at 30 ° C. and 150 rpm.
[0160]

[0161]
Hereinafter, Example 13 describes a method for analyzing or measuring polymorphisms and mutations of the target nucleic acid.
Example 13
Four types of oligonucleotides having the base sequences shown below were synthesized using the DNA synthesizer of Example 5. Further, in the same manner as in Example 5, the nucleic acid probe of the present invention having the following base sequence was synthesized. After the probe and each oligonucleotide were hybridized in solution, it was examined whether single base substitution could be evaluated from the change in fluorescence intensity. The base sequence of the nucleic acid probe of the present invention is designed to match 100% with the base sequence of the oligonucleotide when G exists at the 3 'end of any of the target oligonucleotides. The hybridization temperature was set at 40 ° C. at which 100% of all base-pairs between the probe and target oligonucleotide could be hybridized. The concentration of the probe and target oligonucleotide, the concentration of the buffer, the fluorescence measuring device, the fluorescence measuring conditions, the experimental operation, etc. are the same as in Example 5.
[0162]

[0163]
The results are shown in Table 5. From the table, the target oligonucleotide No. 1-3, no change was observed in the fluorescence intensity. In 4 a decrease of 84% was observed.
[0164]

[0165]
In the present invention, data (for example, data in columns A and B in Table 5) obtained by a method for analyzing or measuring a polymorphism and / or mutation of the target nucleic acid (for example, the target oligonucleotide Nos. 1 to 4) is analyzed. In the method, the fluorescence intensity value of the reaction system when the target nucleic acid is hybridized with the nucleic acid probe of the present invention (the above-described nucleic acid probe) is corrected by the fluorescence intensity value of the reaction system when not hybridized. Then, it means the calculation of (AB) / B in Table 3.
From the above results, it was revealed that when the target nucleic acid is double-stranded, substitutions of G → A, G ← A, C → T, C ← T, G → C, and G ← C can be detected.
[0166]
Example 14
FIG. 6 shows an example of the DNA chip model of the present invention. First, a modified probe prepared by introducing an amino group into the OH group at the 3′-position carbon of the 3′-terminal ribose of 3′TTTTTTTTGGGGGGGGC5′BODIPY FL / C6, which is the probe of the present invention prepared in Example 13, A surface-treated slide glass was prepared by treating the surface of the slide glass with a silane coupling agent having an epoxy group using the slide glass as a reactive group. The solution containing the above modified probe is converted into a DNA chip making apparatus GMS.^TMSpotted on the surface-treated glass slide with 417 ARRAYER (TAKARA). Then, the modified probe bound to the glass surface at the 3 'end. The slide glass was placed in a sealed container for about 4 hours to complete the reaction. Then, the glass slide was soaked twice in a 0.2% SDS solution and water for about 1 minute. Further boron solution (NaBH in 300 ml water)_FourDissolved 1.0g. ) For about 5 minutes. After immersing in water at 95 ° C. for 2 minutes, the reagent was quickly washed off by alternately immersing twice in a 0.2% SDS solution and water for about 1 minute. Dry at room temperature. Thus, the DNA chip of the present invention was prepared.
[0167]
Furthermore, by providing a minute temperature sensor and a heater as shown in the figure at the position corresponding to each spot of the modified probe on the lower surface of the glass, high performance could be imparted to the DNA chip of the present invention.
A case where target nucleic acid is measured using this DNA chip will be described. When the target nucleic acid is not hybridized to the probe, or when it does not form a GC pair at the fluorescent dye-labeled end, or 1 to 3 bases from the terminal base portion where the probe and the target nucleic acid are hybridized Aside from this, when at least one G (guanine) or cytosine (C) is not present in the base sequence of the target nucleic acid, the fluorescence intensity does not change. However, on the contrary, when the target nucleic acid is hybridized to the probe, or when a GC pair is formed at the fluorescent dye-labeled end even if it is hybridized, or the probe and the target nucleic acid are hybridized. When at least one G (guanine) or cytosine (C) is present in the base sequence of the target nucleic acid 1 to 3 bases away from the terminal base portion, the fluorescence intensity decreases. This fluorescence intensity is measured by the DNA chip analyzer GMS.^TMIt can be measured using a 418 Array Scanner (TAKARA).
[0168]
Example 15: Single nucleotide polymorphism (SNPs) detection experiment using the DNA chip of the present invention
I) Preparation of target nucleic acid: (5 ')AAACGATGTG GGAAGGCCCA GACAGAn oligodeoxyribonucleotide having a base sequence of CCAGG ATGTTGGCTT AGAAGCAGCC (3 ′) was synthesized using a DNA synthesizer ABI394 (Perkin Elmer, USA) to obtain a target nucleic acid.
II) Preparation of nucleic acid probe: The following 6 oligodeoxyribonucleotides having a base sequence that hybridizes to the sequence (underlined) of 15 bases from the 5 ′ end of the target nucleic acid were converted into a DNA synthesizer ABI394 (Perkin Elmer) And the United States). Then, the 3'-position OH group of deoxyribose at the 3 'end was aminated using 3'-Amino-Modifier C7 CPG (Glen Research, catalog number 20-2957). Further, the phosphate group at the 5 'end was labeled with BODIPY FL in the same manner as in Example 5.
[0169]
1) Probe 100 (100% match): (5 ') CCTTCCCACA TCGTTT (3'),
2) Probe-T (1 base mismatch): (5 ') CCTTCCCATA TCGTTT (3'),
3) Probe-A (1 base mismatch): (5 ') CCTTCCCAAA TCGTTT (3'),
4) Probe-G (1 base mismatch): (5 ') CCTTCCCAGA TCGTTT (3'),
5) Probe-TG (2-base mismatch): (5 ') CCTTCCCTGA TCGTTT (3'),
6) Probe-TGT (3-base mismatch): (5 ') CCTTCCCTGT TCGTTT (3'),
[0170]
III) Preparation of DNA chip
All DNA probes were dissolved in sterile distilled water to make a 1 μM concentration solution. A DNA chip slide glass (Black silylated slides) using a DNA microarrayer (manual chip arrayer consisting of DNA microarrayer No.439702, 32-pin type, and DNA slide index No.439701, Greiner). The probe solution was spotted on top of Greiner. The reaction was performed for 10 minutes after the spot was completed, and the probe was immobilized on a slide glass. Then, it was washed with 50 mM TE buffer (pH: 7.2). Each probe solution was spotted by 4 spots.
A schematic diagram of the DNA chip of the present invention is shown in FIG. When the probe of the present invention immobilized on a slide glass is not hybridized to the target nucleic acid, BODIPY FL is colored, but when it is hybridized, the color is less than that when it is not hybridized. . That is, it decreases. The slide glass is heated by a microheater (in the present invention, as shown below, it was performed with a transparent heating plate for a microscope (MP-10MH-PG, Kitasato Supply Co., Ltd.)).
[0171]
IV) SNPs detection measurement
A target nucleic acid solution {using 50 mM TE buffer (pH: 7.2)} having a concentration of 100 μM was placed on the DNA chip prepared as described above. Covered with a cover glass and sealed with a nail polish so that the target nucleic acid did not leak.
An outline of the apparatus for detection measurement is shown in FIG. First, a transparent heating plate for a microscope (MP-10MH-PG, Kitasato Supply Co., Ltd.) was placed on a sample stage of an Olympus upright microscope (AX80 type). The DNA chip of the present invention prepared above was placed on the plate, changed from 95 ° C. to 33 ° C. in increments of 3 ° C., and reacted for 30 minutes. The change in fluorescence intensity during the reaction process of each spot was measured in an image capture format with a cooled CCD camera (C5810 type, Hamamatsu Photonics).
The captured image was analyzed with an image analysis device (PC (NEC) with image analysis software (TPlab spectrum; Signal Analytics, Verginia) installed), the brightness of each spot was calculated, and the relationship between temperature and brightness was obtained. .
[0172]
The experimental results are shown in FIG. From the figure, it can be seen that the fluorescence intensity decreased for all probes. Therefore, the dissociation curve between the probe of the present invention and the target nucleic acid can be easily monitored by the method of the present invention. Further, since the difference in Tm value between the probe 100 that matches 100% with the target nucleic acid and the probe that mismatches by one base is 10 ° C. or more, both can be easily identified from the dissociation curve. That is, it can be seen that SNPs can be easily analyzed by using the DNA chip of the present invention.
[0173]
Hereinafter, Examples 16 to 19 describe the PCR method of the present invention.
Example 16
Using a 16S rRNA gene in the genomic DNA of E. coli as a target nucleic acid, a primer (labeled with BODIPY FL / C6) for amplification of the nucleic acid (the nucleic acid probe of the present invention) was prepared.
[0174]
Preparation of primer 1 (Eu800R: reverse type): Oligodeoxyribonucleotide having the base sequence of (5 ′) CATCGTTTAC GGCGTGGAC (3 ′) was synthesized using a DNA synthesizer ABI394 (manufactured by Perkin Elmer, USA). The phosphate group at the 5 ′ end of the oligodeoxyribonucleotide is treated with phosphatase to form cytosine, and the carbon OH group at the 5 ′ position of the cytosine has — (CH₂)₉-NH₂Was purchased from Midland Certified Raised Company (USA). In addition to the Fluo Reporter Kits F-6082 (BODIPY FL propionic acid succinimidyl ester) from Molecular Probes, the compound is converted to an amine derivative of oligonucleotide. A kit containing the reagent to be bound was purchased. The kit was allowed to act on the purchased oligonucleotide to synthesize Primer 1 labeled with Bodepy FL / C6 of the present invention.
[0175]
Purification of synthesized product: The obtained synthesized product was dried to obtain a dried product. 0.5M Na₂CO_Three/ NaHCO_ThreeDissolved in buffer (pH 9.0). The lysate was subjected to gel filtration with a NAP-25 column (Pharmacia) to remove unreacted substances. Further, reverse phase HPLC (B gradient: 15 to 65%, 25 minutes) was performed under the following conditions. And the main peak which eluted was fractionated. The fraction obtained was freeze-dried to obtain the primer 1 of the present invention in a yield of 50% from the initial oligonucleotide raw material of 2 mM.
[0176]

[0177]
Example 17
Preparation of primer 2 (Eu500R / forward: forward type): Primer 2 labeled with a fluorescent dye (BODIPY FL / C6) at the 5 ′ end of oligodeoxyribonucleotide having the base sequence of (5 ′) CCAGCAGCCG CGGTAATAC (3 ′) Prepared in 50% yield as in Example 14.
[0178]
Example 18
Using a test tube containing 5 ml of a sterilized neutral broth (NB) (manufactured by Difco) liquid medium (composition: NB, 0.08 g / 100 ml), E. coli strain JM109 was shaken and cultured overnight at 37 ° C. 1.5 ml of the culture solution was centrifuged with a 1.5 ml capacity centrifuge tube to obtain bacterial cells. From this microbial cell, genomic DNA was extracted using DNeasy Tissue Kit (QIAGEN, Germany). The extraction followed the protocol of this kit. As a result, a DNA solution of 17 ng / μl was obtained.
[0179]
Example 19
Light cycler released by Roche Diagnostics Inc. using the above genomic DNA of Escherichia coli, primer 1 and / or primer 2.^TMSystem (LightCycler^TM The PCR reaction was performed as usual using System). The operation followed the procedure manual for the system equipment.
In the above system, PCR is performed according to the present invention in place of the nucleic acid probes (two probes using the FRET phenomenon) and ordinary primers (ordinary primers not labeled with a fluorescent dye) described in the procedure. The procedure was followed except that primers 1 and / or 2 were used.
[0180]
PCR was performed in the following component hybridization solution.

The target nucleic acid E. coli 16SrDNA is the concentration of the experimental group shown in the explanatory column of FIG. 9, and the primer is also a combination of primers 1 and / or 2 of the experimental group similarly shown in the explanatory column of FIG. The experiment was conducted.
[0181]
The above Taq solution is a mixture of the following reagents.

[0182]
The Taq solution and Taq DNA polymerase solution are those of the DNA Master Hybridization Probes kit sold by Roche Diagnostics Inc. In particular, the Taq DNA polymerase solution was used by diluting 10 × conc. (Red cap) 10 times. Taq start is an antibody for Taq DNA polymerase sold by Clontech (USA), and the activity of Taq DNA polymerase can be suppressed to 70 ° C. by adding it to the reaction solution. That is, a hot start can be performed.
[0183]

Measurement is a light cycler^TMPerformed using the system. At that time, among the detectors F1 to F3 in the system, the detector of F1 was used, and the gain of the detector was fixed to 10 and the excitation intensity was fixed to 75.
[0184]
The results are shown in FIGS. 9 and 10, it can be seen that the number of cycles at which the decrease in the emission of the fluorescent dye is observed is proportional to the number of copies of the target nucleic acid E. coli 16SrDNA. In the figure, the amount of decrease in emission of the fluorescent dye is expressed as a decrease in fluorescence intensity.
FIG. 11 shows a calibration curve of E. coli 16SrDNA expressing the copy number of E. coli 16SrDNA as a function of cycle number. The correlation coefficient was 0.9973, indicating a very good correlation.
As can be seen from the above results, the initial copy number of the target nucleic acid can be measured by using the quantitative PCR method of the present invention. That is, the concentration of the target nucleic acid can be measured.
[0185]
Example 20
In Example 19, PCR was performed using the probe of the present invention as a primer, but in this example, the probe of the present invention was used in place of the two probes using the FRET phenomenon used in the conventional method under the following conditions. The PCR of the present invention was performed.
a) Target nucleic acid: 16S-rDNA of E. coli
b) Primers used:
-Forward primer E8F: (5 ') AGAGTTTGAT CCTGGCTCAG (3')
・ Reverse primer E1492R: (5 ') GGTTACCTTG TTACGACTT (3')
c) Probe used: BODIPY FL- (5 ') CGGGCGGTGT GTAC (3')
d) PCR measuring instrument used: Light cycler^TMsystem
e) PCR conditions:
Denaturation reaction: 95 ° C., 10 seconds (only once, 60 seconds, 95 ° C.)
Annealing reaction: 50 ° C, 5 seconds
Nucleic acid elongation reaction: 72 ° C., 70 seconds
Total number of cycles: 70 cycles
[0186]

[0187]
The results are shown in FIG. From the figure, it can be seen that the number of cycles at which the decrease in the emission of the fluorescent dye is observed is proportional to the number of copies of the target nucleic acid E. coli 16S rDNA.
As can be seen from the above results, the initial copy number of the target nucleic acid can be measured using the quantitative PCR method of the present invention. That is, the target nucleic acid can be measured.
[0188]
Next, the data analysis method of the present invention for analyzing data obtained by using the quantitative PCR method of the present invention will be described in the following examples.
Example 21
Human genomic DNA (human β-globin (TaKaRa catalog product number 9060) (TaKaRa Co., Ltd.) (hereinafter referred to as human genomic DNA) was labeled as a target nucleic acid with Bodepy FL / C6 for amplification of the nucleic acid. Primers were prepared.
[0189]
Preparation of primer KM38 + C (reverse type): (5 ′) Oligodeoxyribonucleotide having the base sequence of CTGGTCTCCT TAAACCTGTC TTG (3 ′) was synthesized using DNA synthesizer ABI394 (Perkin Elmer, USA) Further, the phosphate group at the 5 ′ end of the oligodeoxyribonucleotide is treated with phosphatase to form cytosine, and the carbon OH group at the 5 ′ position of the cytosine has — (CH₂)₉-NH₂The product was purchased from Midland Certified Raised Company. In addition to FluoReporter Kit F-6082 (BODIPY FL propionic acid succinimidyl esters of Bodepy FL / C6) from Molecular Probes, this compound is conjugated to an oligonucleotide amine derivative. A kit containing the reagent) was purchased. The kit was allowed to act on the purchased oligonucleotide to synthesize primer KM38 + C labeled with Bodepy FL / C6 of the present invention.
[0190]
Purification of the synthesized product: The obtained synthesized product was dried to obtain a dried product. 0.5M Na₂CO_Three/ NaHCO_ThreeDissolved in buffer (pH 9.0). The lysate was subjected to gel filtration with a NAP-25 column (Pharmacia) to remove unreacted substances. Further, reverse phase HPLC (B gradient: 15 to 65%, 25 minutes) was performed under the following conditions. And the main peak which eluted was fractionated. The collected fraction was freeze-dried to obtain the primer KM38 + C of the present invention in a yield of 50% from 2 mM of the first oligonucleotide raw material.
[0191]

[0192]
Example 21
Preparation of primer KM29 (forward type): An oligodeoxyribonucleotide having the base sequence of (5 ′) GGTTGGCCAA TCTACTCCCA GG (3 ′) was synthesized in the same manner as in Example 18.
[0193]
Comparative Experiment Example 1
This comparative experimental example is for the use of data analysis software that does not have a calculation process (processing of Equation (1)) that divides the fluorescence intensity value at the time of nucleic acid extension reaction by the fluorescence intensity value at the time of heat denaturation reaction. It is concerned.
Using the above human genomic DNA, primer KM38 + C and primer KM29, light cycler^TMA PCR reaction was performed using the system, and the fluorescence intensity for each cycle was measured.
The PCR of this comparative example uses a primer labeled with the fluorescent dye described above, and is a novel real-time quantitative PCR method that measures a decrease rather than an increase in fluorescence. Data analysis was performed using the system software. The PCR of this comparative experimental example uses the present invention in place of the nucleic acid probe (two probes using the FRET phenomenon) described in the procedure manual and a normal primer (a normal primer not labeled with a fluorescent dye). Except for using primers KM38 + C and KM29, the procedure was performed according to the procedure of the apparatus.
[0194]
PCR was performed in the following component hybridization solution.

The human genomic DNA was tested at the concentration in the experimental group shown in the simple explanation column of FIG. MgCl₂The final concentration of was 2 mM.
[0195]
Further, the above Taq solution is a mixed solution of reagents next.

[0196]
The Taq solution and Taq DNA polymerase solution are those of the DNA Master Hybridization Probes kit released by Roche Diagnostics Inc. In particular, Taq DNA polymerase solution was used by diluting 10 × conc. (Red cap) 10 times. Taq start is an antibody for Taq DNA polymerase sold by Clontech (USA), and the activity of Taq DNA polymerase can be suppressed to 70 ° C. by adding it to the reaction solution. That is, a hot start can be performed.
[0197]
The reaction conditions are as follows.

Measurement is a light cycler^TMPerformed using the system. At that time, among the detectors F1 to F3 in the system, the detector of F1 was used, and the gain of the detector was fixed to 10 and the excitation intensity was fixed to 75.
[0198]
PCR was performed as described above to measure the fluorescence intensity of each cycle. The result is shown in FIG. That is, for the human genomic DNA of each copy number, the fluorescence intensity at the time of denaturation reaction and nucleic acid extension reaction of each cycle was measured and printed. In any cycle, the fluorescence intensity value is constant during the denaturation reaction, but during the nucleic acid extension reaction, it is observed that the fluorescence intensity decreases from around the 25th cycle. Thus, it can be seen that the decrease occurs in order of increasing copy number of human genomic DNA.
[0199]
As shown in FIG. 13, the fluorescence intensity value at the initial cycle number was not uniform for each copy number of human genomic DNA. Therefore, the following steps (b) to (j) were added to the data analysis method used in this comparative example.
(B) The process of converting the fluorescence intensity value of each cycle by setting the fluorescence intensity value of the 10th cycle to 1, that is, the process of calculating by the following [Equation 8],
C_n= F_n(72) / F_Ten(72) [Formula 8]
However, C_n= Conversion value of fluorescence intensity value in each cycle, F_n(72) = Fluorescence intensity value at 72 ° C. for each cycle, F_Ten(72) = Fluorescence intensity value after extension reaction at 72 ° C. in the 10th cycle.
(C) a process of displaying and / or printing each converted value obtained in the process of (b) on the display as a function of the number of cycles;
[0200]
(D) A process of calculating the change rate (decrease rate, quenching rate) of the fluorescence intensity according to the following [Equation 9] from the converted value of each cycle obtained in the process of (b).
[Formula 9]
F_dn  = Log_Ten{100-C_n× 100)}
F_dn  = 2log_Ten{1-C_n}
However, F_dn= Fluorescence intensity change rate (decrease rate, extinction rate), C_n= Value obtained by [Formula 8].
(E) a process of displaying and / or printing each converted value obtained in the process of (d) on the display as a function of the number of cycles;
[0201]
(F) comparing the data processed in the step (d) with 0.5 as a threshold and counting the number of cycles that have reached that value;
(G) A process of creating a graph in which the value counted in the process of (f) is plotted on the X axis and the copy number before starting the reaction is plotted on the Y axis,
(H) a process of displaying and / or printing the graph created in the process of (g) on the display;
(I) a process of calculating a correlation coefficient or a relational expression of the straight line drawn in the process of (h),
(J) A process of displaying and / or printing the correlation coefficient or the relational expression calculated in the process of (i) on the display.
[0202]
Using the above data analysis software, the data obtained in FIG. 13 was processed as follows.
FIG. 14 shows the result of printing the data processed in the process (b) (process (c)). That is, the fluorescence intensity value of each cycle is converted with the fluorescence intensity value of the 10th cycle being 1, and the converted value is plotted against the corresponding number of cycles.
FIG. 15 shows the data processed in the process (d) printed (process (e)). That is, the decrease rate (quenching rate) of the fluorescence intensity is calculated from each plot value in FIG. 14, and each calculated value is plotted against each cycle number.
[0203]
FIG. 16 is a graph in which the graph created in the process (g) is printed for the data processed in the process (f) (process (h)). That is, it is a graph in which the fluorescence intensity reduction rate = 0.5 is thresholded, the number of cycles reaching that value is plotted on the X axis, and the number of copies of human genomic DNA before the reaction is plotted on the Y axis. The correlation coefficient (R2) of the straight line in this graph was calculated in the process (i) and printed (process (j)), which was 0.9514. Thus, it is impossible to obtain an exact copy number with this correlation coefficient.
[0204]
Example 23 (Experimental example of data processing using the data analysis method of the present invention)
PCR was performed in the same manner as in Comparative Experimental Example 1.
The data processing is the same as in Comparative Experiment Example 1 except that the following process (a) is performed before the process (b) in Comparative Experiment Example 1 and the processes (b) and (d) are changed as follows. The same process was performed.
(A) The fluorescence intensity value of the reaction system when the amplified nucleic acid in each cycle is hybridized with a nucleic acid primer (labeled with a fluorescent dye) that is the nucleic acid probe of the present invention (ie, at the time of nucleic acid extension reaction (72 ° C.)) The fluorescence intensity value of the reaction system measured when the nucleic acid hybrid complex (the amplified nucleic acid hybridized with the nucleic acid primer) was dissociated (ie, at the completion of the nucleic acid heat denaturation reaction (95 ° C.)). The process of correction calculation divided by (fluorescence intensity value)), that is, the actually measured fluorescence intensity value was corrected by the following [Equation 1].
f_n= F_{hyb, n}/ F_{den, n}          [Formula 1]
[Where f_n= Cycle fluorescence intensity correction value, f_{hyb, n}= 72 ° C fluorescence intensity value for each cycle, f_{den, n}= 95 ° C fluorescence intensity value of each cycle]
FIG. 17 shows the obtained values plotted against the number of cycles.
[0205]
(B) Substituting the correction calculation processing value in [Formula 1] in each cycle into [Formula 3], and calculating the fluorescence change rate (decrease rate or quenching rate) between the samples in each cycle, The process of calculating with the following [Equation 10],
F_n= F_n/ F_{twenty five}          [Formula 10]
[Where F_n= Arithmetic processing value of each cycle, f_n= Value of each cycle obtained by [Equation 1], f_{twenty five}= The value obtained by [Equation 1] and the number of cycles is 25th].
[Equation 10] is the case where a = 25 in [Equation 3].
[0206]
(D) The process of subjecting the calculation processing value of each cycle obtained in the process of (b) to the calculation processing for obtaining the logarithmic value of the change rate (decrease rate or quenching rate) of the fluorescence intensity according to [Formula 6], That is, the process of calculating the following [Formula 11],
log_Ten{(1-F_n) × 100} [Formula 11]
[Where F_n= [Value obtained by [Equation 10]].
[Formula 11] is obtained when b = 10 and A = 100 in [Formula 6].
[0207]
The above results are shown in FIGS.
FIG. 18 is a plot of the values processed in steps (a) and (b) plotted against the number of cycles.
FIG. 19 is a plot of values obtained by processing the values obtained in FIG. 18 as in the step (d), plotted against the number of cycles.
[0208]
Next, it processed in the process of said (f), (g), and (h) based on the graph of FIG. That is, based on the graph of FIG._TenAs the threshold value of (fluorescence intensity change rate), select 0.1, 0.3, 0.5, 0.7, 0.9, 1.2, and the number of cycles that reached that value on the X-axis, The copy number of human genomic DNA before the start of the reaction was plotted on the Y axis, and a calibration curve was drawn. The results are shown in FIG. Correlation coefficients (R) obtained by processing these calibration curves in the processes (i) and (j).²) Were 0.998, 0.999, 0.9993, 0.9985, 0.9989, and 0.9988, respectively, for the threshold values. From these correlation coefficients, it was recognized that it was desirable to adopt 0.5 (correlation coefficient 0.9993) as the threshold value. It can be seen that a calibration curve having this correlation coefficient can accurately determine the copy number before starting the reaction for a nucleic acid sample having an unknown copy number.
[0209]
Example 24 (Example of melting curve analysis and Tm value analysis of nucleic acid)
For the nucleic acid amplified by the novel PCR method of the present invention, 51) gradually increasing or decreasing the temperature from the low temperature until the nucleic acid is completely denatured (for example, from 50 ° C. to 95 ° C.), 52) 51 ) In the process, the fluorescence intensity is measured at a short time interval (for example, an interval corresponding to a temperature rise of 0.2 ° C. to 0.5 ° C.). 53) The measurement result of the 52) process is displayed as a function of time. The process shown above, that is, the process of displaying the melting curve of the nucleic acid, 54) the process of primary differentiation of the melting curve of the process 53), 55) the differential value of the process 54) (−dF / dT, F: fluorescence) Intensity, T: time) is displayed on the display, 56) software including a process of obtaining an inflection point from the differential value obtained from 55) is created, and the data analysis software of the present invention is created. Coalesced to A. The light cycler installed with a computer-readable recording medium recording the data analysis software^TMUsing the system, the novel real-time quantitative PCR reaction of the present invention was performed, and nucleic acid melting curves were analyzed. In the present invention, the fluorescence intensity increases with increasing temperature.
[0210]
The same PCR as in Example 21 was performed for 1 copy and 10 copies of the same human genomic DNA as in Example 23, and the data processed in the process of 51), 52), 53), 54) and 55) were printed. The thing is FIG. FIG. 22 is a diagram of nucleic acid melting curves obtained by treating the 1st and 10th copies of the 75th amplification product in the process of 51), 52) and 53) of this example. This curve is differentiated in the process of 54), and the inflection point (Tm value) is obtained in the processes of 55) and 56) is shown in FIG. From FIG. 23, it was found that each amplification product was a different product because the Tm values of the 1-copy and 10-copy amplification products were different.
[0211]
【The invention's effect】
The present invention has the following effects.
1) Method for measuring the concentration of the target nucleic acid of the present invention, various nucleic acid probes of the present invention, especially 2-O-alkyl oligonucleotides, 2-O-alkylene oligonucleotides, 2-O-benzyl oligonucleotides and other chemical modifications Nucleic acid probes of the present invention comprising oligonucleotides, etc., nucleic acid probes of the present invention comprising chimeric oligonucleotides intervening between oligoribonucleotides and oligodeoxyribonucleotides, and target nucleic acids containing or incidental to those nucleic acid probes of the present invention When using a measurement kit for measuring the concentration of DNA and a nucleic acid chip or nucleic acid device such as a DNA chip formed by binding the nucleic acid probe of the present invention, an operation such as removing an unreacted nucleic acid probe from the measurement system is performed. Therefore, the target nucleic acid concentration can be measured easily in a short time. That. Moreover, when applied to a complex microorganism system or a symbiotic microorganism system, the abundance of a specific strain in the system can be measured specifically and in a short time. The present invention also provides a simple method for analyzing or measuring polymorphisms or mutations such as target nucleic acids or gene SNPs.
[0212]
2) Further, the quantitative PCR method of the present invention has the following effects.
a. Since no factor that inhibits the amplification of the target nucleic acid by Taq DNA polymerase is added, quantitative PCR can be carried out under the same conditions as conventional PCR with known specificity.
b. In addition, since the specificity of PCR can be kept high, amplification of primer dimers is delayed, and the limit of quantification is about an order of magnitude lower than that of conventionally known quantitative PCR.
c. Since it is not necessary to prepare a complicated nucleic acid probe, the time and cost required for it can be saved.
d. The amplification effect of the target nucleic acid is also great, and the amplification process can be monitored in real time.
[0213]
3) In addition, when analyzing data obtained by the real-time quantitative PCR method, using the data analysis method of the present invention, a calibration line for obtaining the copy number of a nucleic acid for a nucleic acid sample having an unknown nucleic acid copy number is prepared. The correlation coefficient of the line is much higher than that obtained by the conventional method. Therefore, when the data analysis method of the present invention is used, the exact copy number of the nucleic acid can be obtained.
[0214]
4) Also, data analysis software according to a method for analyzing data obtained by the real-time quantitative PCR method of the present invention, a computer-readable recording medium recording the procedure of the analysis method as a program, and When a real-time quantitative PCR measurement or analysis apparatus using is used, a calibration line having a high correlation coefficient can be automatically created.
[0215]
5) Further, by using the novel method for analyzing a melting curve of a nucleic acid of the present invention, a highly accurate Tm value of a nucleic acid can be obtained. Furthermore, using data analysis software according to the method, a computer-readable recording medium in which the procedure of the analysis method is recorded as a program, and a real-time quantitative PCR measurement or analysis device using the software, Accurate Tm value can be obtained
[Brief description of the drawings]
1 is a graph showing fluorescence intensity measurement data when the 335th to 358th nucleobase sequence counted from the 5 ′ end of E. coli 16S rRNA was measured using the nucleic acid probe obtained in Example 1. FIG.
FIG. 2. Effect of heat treatment on hybridization of 35-base 2-O-Me probe to target nucleic acid
Dotted line: The target nucleic acid was added after heat treatment of rRNA.
Solid line: non-heat-treated rRNA.
FIG. 3 shows the effect of modification of the number of bases of the probe base chain and the methyl group modification of the 2′-position carbon OH group of the ribose at the 5 ′ end of the helper probe and the probe on the hybridization between the probe and the target nucleic acid 16SrRNA.
FIG. 4 is a calibration curve for rRNA measurement by the method of the present invention.
FIG. 5 shows the time course of rRNA amount in the combined culture system of KYM7 strain and KYM8 strain by the FISH method of the present invention.
FIG. 6 illustrates a DNA chip of the present invention.
FIG. 7 is a diagram for explaining devices for SNPs detection measurement using the DNA chip of the present invention.
FIG. 8 is a diagram showing experimental results of SNPs detection measurement using the DNA chip of the present invention.
FIG. 9 shows a quantitative PCR method using primers 1 and 2 labeled with bodepy FL / C6: the relationship between the number of cycles and the amount of decrease in emission of the fluorescent dye. Symbols (1) to (8) in the figure represent the following meanings.

FIG. 10 shows a quantitative PCR method using primers 1 and 2 labeled with bodepy FL / C6: the relationship between the number of cycles and the logarithmic value of the amount of decrease in emission of the fluorescent dye. Symbols (1) to (8) in the figure have the same meaning as in FIG.
FIG. 11 is a diagram showing a calibration curve of E. coli 16S rDNA prepared using the quantitative PCR method of the present invention. n: 10ⁿ
FIG. 12 shows the case where real-time quantitative PCR is performed using one probe of the present invention instead of two probes labeled with a fluorescent dye used in the real-time quantitative PCR method using the FRET phenomenon. It is a figure which shows the change of the decreasing rate of fluorescence intensity. The figure below shows the calibration curve created by calculating the number of cycles (threshold number: Ct value) at which a decrease in fluorescence intensity begins to be observed.
FIG. 13 is a graph showing a fluorescence decrease curve obtained by real-time quantitative PCR using a primer labeled with Bodepy FL / C6 of the present invention when the correction calculation processing of the present invention is not performed.
■: target nucleic acid = 10 copies; temperature of reaction system for fluorescence intensity measurement = 72 ° C.
●: target nucleic acid = 100 copies; temperature of reaction system for fluorescence intensity measurement = 72 ° C.
▲: target nucleic acid = 1000 copies; temperature of reaction system for fluorescence intensity measurement = 72 ° C.
◆: Target nucleic acid = 10000 copies; temperature of reaction system for fluorescence intensity measurement = 72 ° C.
□: target nucleic acid = 10 copies; temperature of reaction system for fluorescence intensity measurement = 95 ° C.
○: target nucleic acid = 100 copies; temperature of reaction system for fluorescence intensity measurement = 95 ° C.
Δ: target nucleic acid = 1000 copies; temperature of reaction system for fluorescence intensity measurement = 95 ° C.
◇: target nucleic acid = 10000 copies; temperature of reaction system for fluorescence intensity measurement = 95 ° C.
14 is a graph showing a fluorescence decrease curve obtained by real-time quantitative PCR in the same manner as the curve in FIG. 13, except that the value at the 10th cycle of each curve is corrected to 1. FIG.
■: target nucleic acid = 10 copies; fluorescence intensity measurement temperature = 72 ° C.
●: target nucleic acid = 100 copies; fluorescence intensity measurement temperature = 72 ° C.
▲: target nucleic acid = 1000 copies; fluorescence intensity measurement temperature = 72 ° C.
◆: Target nucleic acid = 10000 copies; fluorescence intensity measurement temperature = 72 ° C.
5: target nucleic acid = 10000 copies; fluorescence intensity measurement temperature = 72 ° C.
FIG. 15 is a diagram showing a curve obtained by calculating the fluorescence intensity reduction rate (change rate) of [Formula 9] for each plot value of each curve in FIG. 14 and plotting the calculated values.
■: Target nucleic acid = 10 copies
●: Target nucleic acid = 100 copies
▲: Target nucleic acid = 1000 copies
◆: Target nucleic acid = 10000 copies
16 is a diagram showing a calibration straight line of human genomic DNA obtained from the data of FIG.
y = human β-globin gene copy number
x = cycle number (Ct)
R²= Correlation coefficient
FIG. 17 is a diagram showing a curve in which values obtained by correcting and calculating the measured values of each cycle in FIG.
■: Target nucleic acid = 10 copies
●: Target nucleic acid = 100 copies
▲: Target nucleic acid = 1000 copies
◆: Target nucleic acid = 10000 copies
18 is a diagram showing a curve in which values obtained by calculating the calculation processing values of each cycle in FIG. 17 by [Equation 3] are plotted against the number of cycles.
■: Target nucleic acid = 10 copies
●: Target nucleic acid = 100 copies
▲: Target nucleic acid = 1000 copies
◆: Target nucleic acid = 10000 copies
FIG. 19 is a diagram showing a curve obtained by plotting the values obtained by calculating the calculation processing values of the respective cycles in FIG. 18 using the formula [6] with respect to the number of cycles.
■: Target nucleic acid = 10 copies
●: Target nucleic acid = 100 copies
▲: Target nucleic acid = 1000 copies
◆: Target nucleic acid = 10000 copies
20 appropriately selects 0.1, 0.3, 0.5, 0.7, 0.9, and 1.2 as Ct value candidates from each log (fluorescence change rate) value of FIG. The figure at the time of having drawn the calibration straight line corresponding to it. In addition, the correlation coefficient in each calibration curve is shown below.
▲: log_Ten(Fluorescence change rate) = 0.1; correlation coefficient = 0.998
■: log_Ten(Fluorescence change rate) = 0.3; correlation coefficient = 0.999
●: log_Ten(Fluorescence change rate) = 0.5; correlation coefficient = 0.993
Δ: log_Ten(Fluorescence change rate) = 0.7; correlation coefficient = 0.985
□: log_Ten(Fluorescence change rate) = 0.9; correlation coefficient = 0.9989
Y: log_Ten(Fluorescence change rate) = 1.2; correlation coefficient = 0.99888
FIG. 21 is a graph showing fluorescence decrease curves when real-time quantitative PCR is performed on 1-copy and 10-copy human genomic DNA using a primer labeled with Bodepy FL / C6 of the present invention. However, the correction calculation processing of [Formula 1] was performed.
1: Target nucleic acid = 0 copy
2: Target nucleic acid = 1 copy
3: Target nucleic acid = 10 copies
22 is a diagram showing a melting curve of a nucleic acid when a nucleic acid melting curve analysis is performed on the PCR amplification product shown in FIG. 21. FIG.
1: Target nucleic acid = 0 copy
2: Target nucleic acid = 1 copy
3: Target nucleic acid = 10 copies
FIG. 23 is a diagram showing a curve indicating a Tm value obtained by differentiating the curve of FIG. 22 (valley is a Tm value).
2: Target nucleic acid = 1 copy
3: Target nucleic acid = 10 copies

Claims (13)

In a nucleic acid probe labeled with a fluorescent dye of any one of 6-joe, BODIPY TMR ™, BODIPY FL ™, BODIPY FL / C3 ™, Alexa 488 ™, Alexa 532 ™ , 3 'The terminal base is G or C and the 3' carbon OH group at the 3 'end, or the 5' terminal base is G or C and the 5 'terminal phosphate group, or the 5' terminal phosphorus acid phosphatased, and the carbon OH groups of the 5'-position, and labeled with a fluorescent dye, as an oligonucleotide consists texture Rick oligonucleotides comprising oligoribonucleotides and oligodeoxyribonucleotides (chimeric oligonucleotide), high in the target nucleic acid When the hybridization is performed, the base pair of hybridization forms at least one pair of G (guanine) and C (cytosine) at the terminal portion. When the nucleotide sequence of the nucleic acid probe is designed and hybridized to the target nucleic acid, the labeled fluorescent dye reduces the emission of the nucleic acid probe, or the nucleic acid probe is a carbon at the 2 ′ position of one or more riboses. Chemically modified 2′-O-methyl oligonucleotide, 2′-O-ethyl oligonucleotide, 2′-O-butyl oligonucleotide, 2′-O-ethylene oligonucleotide, or 2′-O-benzyl In order to further increase the efficiency of hybridization to the hybridization sequence region of the nucleic acid probe in the hybridization reaction system before the nucleic acid probe that intervenes the oligonucleotide in the chain is hybridized to the target nucleic acid. Consists of deoxyribonucleotides as oligonucleotides And which helper probes, or as an oligonucleotide, a helper probe is 2'-O- methyl oligonucleotide carbon at the 2 'position of the ribose is chemically modified comprising a oligoribonucleotides, 2'-O- ethyl oligo A helper probe is added which intervenes nucleotides, 2′-O-butyl oligonucleotides, 2′-O-ethylene oligonucleotides, or 2′-O-benzyl oligonucleotides in the strand (provided that “the 2 ′ position of ribose A combination of the above-described nucleic acid probe in which carbon is not chemically modified and the above-mentioned helper probe in which the carbon at the 2 ′ position of ribose is not chemically modified ”or“ a probe and a helper oligonucleotide are diphosphates ” DNA with an ester backbone, alkyl or aryl phosphate ester backbone Excluding “combinations”. ), A method for measuring the concentration of a target nucleic acid, wherein the nucleic acid probe is hybridized to a target nucleic acid, and the amount of decrease in light emission of the fluorescent dye before and after hybridization is measured.   The method for measuring the concentration of a target nucleic acid according to claim 1, wherein the target nucleic acid is RNA. The method for measuring the concentration of a target nucleic acid according to claim 2 , wherein the nucleic acid probe and the target nucleic acid are hybridized after heat treatment at 80 to 100 ° C for 1 to 15 minutes. The helper probe is cellulomonas sp. Hybridizes to the 16SrRNA of KYM-7 strain, the nucleotide sequence of Katsuso is a (5 ') TCCTTTGAGT TCCCGGCCGG A ( 3') or (5 ') CCCTGGTCGT AAGGGCCATG ATGACTTGAC GT (3'), the nucleic acid The probe is Cellulomonas sp. Of is for the rRNA measurements, the base sequence of Katsuso is, (5 ') CATCCCCACC TTCCTCCGAG TTGACCCCGG CAGTC (3') or Agrobacterium sp. The method for measuring the concentration of a target nucleic acid according to claim 2 , wherein the nucleotide sequence is (5 ') CATCCCCACC TTCCTCTCGGG CTTATCACCGCAGTC (3'). The base sequence (5 ′) TCCTTTGAGT TCCCGGCCGG A (3 ′) is an 8-base central OH group (9 to 16 bases counted from the 5 ′ end) of the 2′-position carbon OH group in the methyl group. The method for measuring the concentration of a target nucleic acid according to claim 4 , which is a modified (ether-linked) oligoribonucleotide body. The base sequence (5 ′) CCCTGGTCGT AAGGGCCATG ATGACTTGAC GT (3 ′) has a OH group at the 2′-position carbon of ribose in the middle 8 bases (9 to 16 bases counted from the 5 ′ end), a methyl group The method for measuring the concentration of a target nucleic acid according to claim 4 , which is an oligoribonucleotide body modified with (ether bond). The base sequence (5 ′) CATCCCCACC TTCCTCCGAG TTGACCCCGG CAGTC (3 ′) is the central 8 bases (17 to 24 bases counted from the 5 ′ end) of the 2′-position OH group of the ribose with a methyl group. The method for measuring the concentration of a target nucleic acid according to claim 4 , which is a modified (ether-linked) oligoribonucleotide body. The base sequence (5 ') CATCCCCACC TTCCTCCCGAG TTGACCCCGG CAGTC (3') is an OH group at the 2'-position carbon of ribose of the central 9 bases (17 to 25 bases counted from the 5 'end) with a methyl group. The method for measuring the concentration of a target nucleic acid according to claim 4 , which is a modified (ether-linked) oligoribonucleotide body. The nucleic acid probe when hybridized to target nucleic acid, as off 1 salt group from the terminal base of a target nucleic acid hybridized to the probe, G (guanine) to the nucleotide sequence of the target nucleic acid is present one base, The method for measuring the concentration of a target nucleic acid according to claim 1, wherein the base sequence of the probe is designed. The nucleic acid probe, the 3 'end of the ribose or 3 deoxyribose' method of measuring the concentration of a target nucleic acid according to claim 1, hydroxyl group of carbon is phosphorylated.   The fluorescence intensity value of the reaction system obtained after the target nucleic acid is hybridized with a nucleic acid probe labeled with a fluorescent dye is obtained after the probe-nucleic acid hybrid complex thus formed is dissociated. The method for measuring the concentration of a target nucleic acid according to claim 1, wherein the concentration is corrected by the above.   The method for measuring the concentration of a target nucleic acid according to claim 1, wherein the target nucleic acid is derived from a microorganism obtained by pure separation or from an animal.   The method for measuring the concentration of a target nucleic acid according to claim 1, wherein the target nucleic acid is a nucleic acid contained in a cell of a complex microorganism system or a symbiotic microorganism system or in a cell homogenate.

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