JP4217318B2 - Method and apparatus for quantitative analysis of target nucleic acid - Google Patents

Description

【００４２】
数式１において、ｙはポリマーＤＮＡ成分の割合、τ_mono＝Ｗｏ²／４Ｄ_mono 、τ_poly＝Ｗｏ²／４Ｄ_poly、Ｓ＝Ｗｏ／Ｚｏ（ここで、Ｗｏは焦点付近の微小 視野に形成される略円柱状の測定領域（図２（Ｂ）参照）の体積要素の径であり、２Ｚｏはその長さを意味する）、Ｄ_mono及びＤ_polyはそれぞれモノマーＤＮＡ及びポリマーＤＮＡの並進拡散係数である。
【００４３】
データ解析は、さらに、データとモデル間の正規化平均二乗偏差を計算するために非線形最小二乗パラメター法を用いて行った。所望のＰＣＲサイクルを経て増幅された分子の総数Ｔは、式２；Ｔ＝（Ｎ／Ｖｃ）Ｖｃにより計算することで、定量化データとして出力するようにした。ここで、Ｖｏは体積要素、Ｖｃはスピンカラムによって収集された溶液の体積である。ＦＣＳ計測中、平均蛍光強度も測定した。その強度は体積要素中で増幅されたＤＮＡに取り込まれたフロロフォアー（Ｆｌｕ−ｄＵＴＰ）の総数を反映している。平均蛍光強度の計測速度をＤＮＡ分子数Ｎｙで割った値は、ＤＮＡ分子の平均計測速度Ｃ／Ｍとして採用した。この値は、ＰＣＲ産物の実効的な標識濃度として用いることができるものであった。
【００４４】
（３）測定領域の算定
測定領域の体積要素は、基準となる試料としての、ローダミン６Ｇ、フルオレッセイン（ｆｌｕｏｒｅｓｃｅｉｎ）、及びＦｌｕ−ｄＵＴＰの拡散時間の測定から定めた。フォーカシングされた付近に設けられた測定領域（図２（Ｂ）参照）における長さと径の比率は、式１におけるＳの値から求められ、約５であった。また、体積要素Ｖ₀は、１×１０^-15ｌと計算された。
【００４５】
（４）ＰＣＲによるＦｌｕ−１１−ｄＵＴＰの取込み
ＰＣＲにおける蛍光標識ヌクレオシドの作用効果を明らかにするために、Ｆｌｕ−ｄＵＴＰの濃度を１０％から５０％まで変化させて調べてみた。図３は、アガロース電気泳動における増幅産物４ｋｂの特性を示す写図である。即ち、５０サイクルの核酸増幅反応後の検液としてのＰＣＲ反応混合液３μｌを７％アガロースゲルにかけた。ここで、図３（Ａ）は、エチジューム・ブロマイド（ｅｔｈｉｄｉｕｍ ｂｒｏｍｉｄｅ）染色前、図３（Ｂ）は、エチジューム染色後であ る。また、図中Ｍは、分子マーカー（ＨａｅIII−ｄｉｇｅｓｔｅｄファイＸ１７４ＤＮＡ）を示す。また、図中、「０」、「１０」、「２０」、「３０」、「４０」および「５０」は、Ｆｌｕ−ｄＵＴＰとｄＵＴＰ＋ｄＴＴＰの％比を示している。ＦＮは、蛍光ヌクレオシド（組み込まれていないヌクレオシド）である。また、図３（Ａ）におけるゲルから発する蛍光の測定にはオレンジ色光学フィルターＹａ３を用い、図３（Ｂ）における測定には赤色フィルターＲ１を用いた。
【００４６】
図３（Ａ）によれば、５０％のＦｌｕ−ｄＵＴＰ存在下においても、４０００ｂｐの蛍光ＰＣＲ産物を合成することが分かる。標識ＤＮＡの蛍光強度は、Ｆｌｕ−ｄＵＴＰの濃度と共に増加した。増幅ＤＮＡの総生産量はエチジューム染色して示すことが出来るが、この実験では、図３（Ｂ）に示すように、産物量はＦｌｕ−ｄＵＴＰがない場合と同じであることが示された。
【００４７】
（５）ＰＣＲにおける蛍光強度と増幅産物量
図４は、ＰＣＲにおける、Ｆｌｕ−ｄＵＴＰの％比の関数として得た、標識濃度と産物量の関係を示すグラフである。図中、四角印は、ＤＮＡ１分子当たりの平均計測値Ｃ／Ｍを示す。また、黒丸印は、観察視野である測定領域におけるＤＮＡ分子数を示す。図４によると、分子当たりの蛍光強度Ｃ／ＭがＦｌｕ−ｄＵＴＰの増加に連れて増加していることが分かった。この結果は、標識濃度はＦｌｕ−ｄＵＴＰの濃度に依存する、ことを示した。かつまた、ＤＮＡ分子の数は、実験したＦｌｕ−ｄＵＴＰの濃度範囲内ではほとんど一定であった。この結果は、図３に示したゲル電気泳動の解析と一致した。これは、フルオレッセインがＤＮＡ合成酵素の働きを妨げない、ことを示している。モノマーとしての遊離のＦｌｕ−ｄＵＴＰとＰＣＲ産物の各Ｃ／Ｍ値を比較すると、４０００ｂｐのＤＮＡ鎖の中に取込まれたＦｌｕ−ｄＵＴＰモノマーの数が分かる。それは修飾されたヌクレオシドが隣同士に存在している可能性を示している。
【００４８】
（６）ＦＣＳによるＰＣＲのモニター
図５は、ＰＣＲ反応の過程における自己相関関数の推移を示すグラフであり、相関曲線の典型的な時間変化を示している。図中、丸印は１０サイクル、三角印は２０サイクル、逆三角印は３０サイクル、菱形印は４０サイクル、四角印は５０サイクルの核酸増幅反応をそれぞれ終了したときに、測定したものである。
図５から分かるように、自己相関関数のゼロ時間遅れの値（ｙ切片）はＰＣＲサイクルと共に減少している。この結果は、ＤＮＡ分子数の増加に起因している。
【００４９】
図６は、各自己相関関数をゼロ時間遅れのところで規格化したグラフである。図６では、比較の為にＦｌｕ−ｄＵＴＰの曲線が、黒丸印で示されている。また、図中、実線は１０サイクル、破線は２０サイクル、点線は３０サイクル、１点鎖線は４０サイクル、２点鎖線は５０サイクルの核酸増幅をそれぞれ終えたときの値である。図６から分かるように、１０サイクル後に得られた自己相関関数（実線部分）は、Ｆｌｕ−ｄＵＴＰのそれとは明らかな違いがある。この自己相関関数は、簡単な１成分モデルに当てはめることは出来なかったが、上記の式１に示したような２成分モデルに当てはめることが出来た。また、５０サイクルのＰＣＲを実行した検液においてさえ、短い拡散時間成分が検出出来た。短い成分はスピンカラムを通り抜けた未反応Ｆｌｕ−ｄＵＴＰとして定義される。
【００５０】
図７は、ＰＣＲサイクル数の関数としてＰＣＲ反応溶液中（5μｌ）における 増幅ＤＮＡ分子数を示すグラフである。増幅されたＤＮＡ分子の総数を、上記の式２によって計算した。実験上、ラムダフアージＤＮＡの初期量を各種検討した。図中、四角印は２００ｐｇ（３．７５×１０⁶コピー）のラムダフアージＤＮ Ａを初期量とした場合を示し、以下、丸印は２０ｐｇ（３．７５×１０⁵コピー ）、三角印は２ｐｇ（３．７５×１０⁴コピー）、逆三角印は０．２ｐｇ（３． ７５×１０³コピー）をそれぞれ示す。
【００５１】
図８は、鋳型の初期数の関数としての２０サイクル後のＤＮＡ分子数を示したグラフである。ＤＮＡがＰＣＲによって増幅された場合には、ＤＮＡ分子数はプラトー効果に達する前に指数関数的に増加する。鋳型分子の初期数は、１０サイクル後に１０２４（＝２¹⁰）倍に増幅され得るので、３．７５×１０⁶コピー（ ２００ｐｇ相当）のＤＮＡは３．７５×１０⁹コピーという結果となる。しかし ながら、初期コピー数が３．７５×１０⁶の時、ＦＣＳ計測で１０サイクル後に ７．４４×１０⁹コピー計測され、同様に、初期コピー数が３．７５×１０⁵の時には１０サイクル後に０．３１×１０⁹コピー計測された。このように、ＦＣＳ 計測の定量性は、増幅サンプルの早期段階では忠実度が低いように見える。これは、検出領域において増幅ＤＮＡが少なく、多くの取込まれていない標識ヌクレオシドが分布している結果といえる。
【００５２】
ＦＣＳを用いたこの直接標識ベースのＰＣＲ定量は１０サイクルの増幅後でも、１０³レベルの標的ＤＮＡ初期濃度の検出が可能であるが、図８から分かるよ うに、標的ＤＮＡの初期濃度が２０サイクル後の方がより良く推定出来る。この図ではＤＮＡ分子の初期数と産物の数の関係は直線的であるので、ＦＣＳ計測により目的ＤＮＡ分子数の定量が可能である。また、データは省略するが、別の曲線を用いて同様の関係が５００ｂｐの別のＤＮＡ配列で検出出来ることも確認できた。さらに、種々の実験では、ＰＣＲ２０サイクル、５μｌの試料溶液が用いられた。試料溶液量を増加すれば、もっと精密で且つ高い再現性を得る事が出来ることはいうまでもない。このことは診断応用においては最も重要なことである。それ故、時間や資源を節約するためには、良好な再現性を提供出来るようなサイクル数を実験を通じて適宜選択するのが好ましい。
【００５３】
上述した実験結果に示すように、本発明では、直接標識ＰＣＲに対する分子計数方法としてＦＣＳを用いることに初めて成功した。また、４０００ｂｐの標的核酸に対しても、５０％のｄＴＴＰをＦｌｕ−ｄＵＴＰに置き換えてもＰＣＲが阻害されないことを証明した。例えば５００ｂｐ、１０００ｂｐ、２０００ｂｐといったもっと短い標的核酸に対しては、より強い蛍光バンドが５０熱サイクル後のゲル電気泳動で検出された。この方法は、標識試薬として通常のＰＣＲに用いられる基質分子に蛍光等の測定可能な標識分子に結合させたものを用いるので、他の通常のＰＣＲの工程と同様に、通常の研究室で容易に応用出来る。ここで述べたＦＣＳアッセイは、ＰＣＲ条件のほんの少しの変更と、５μｌ程の少量の検液としての反応溶液と、少数回のＰＣＲサイクルが必要なだけである。ＤＮＡ分子に取込まれた蛍光標識分子は、どの様な遺伝子マーカーや、感染マーカーにも結合させることが出来、既に確立されたＰＣＲ法のノウハウを有効に利用しながら、各種様々な生物の核酸分子を増幅出来る。
【００５４】
本発明によるＦＣＳベースの定量的ＰＣＲは、多くの分野で有利である。というのは、このテクニックは高感度であり、時間や労力を節約し、そして反応成分の構成とその使用手順が極めて簡単であるからである。また、ＦＣＳの測定は非侵襲的であり、ＦＣＳで測定した検液を、更に次の実験、例えば蛍光ハイブリダイゼーションやＦＩＳＨ解析にも都合よく使用することが出来ることも述べておきたい。さらに、ＦＣＳは、均一溶液中の制限酵素消化過程を測定する事もできる。それゆえ、ここで紹介したように、高密度に標識したＤＮＡを用いることによって、点突然変異やＲＥＬＦといった分析が、カラムクロマトグラフィーやゲルクロマトグラフィーのような分離方法を何ら用いることなく、増幅後引き続きＦＣＳで検出出来るのである。これらのＦＣＳベースのＰＣＲ定量法は診断やスクリーニング実験に応用され、さらには１分子検出ベースの新しい分野を開くものである。
【００５５】
以上のように、ＦＣＳにより生物科学における２つの重要なパラメーターを得ることが出来る。それは検出領域（１０^-15ｌ）における分子の平均数と分子の 並進拡散係数である。本発明では、ＦＣＳを１分子計測の道具として用いてＰＣＲ産物の解析を行った。ＰＣＲによるＤＮＡの増幅過程がプラトーに達する前に、特定のＤＮＡ塩基配列の定量を行う事が出来る。ＰＣＲにＦＣＳを応用出来るようにする為に、酵素的増幅サイクルに用いる基質としてフルオレッセイン１１−ｄＵＴＰを用い、かかる標識したヌクレオシド存在下でＰＣＲを行うことによって、４０００ｂｐのひょう標的ＤＮＡのラベリングを行うことが出来た。１０サイクル後には、ＦＣＳ検出領域における蛍光性分子の拡散時間の増加によって増幅ＤＮＡの検出が出来た。ＰＣＲ検出の微少な検出領域の体積（５μｌ）中の標的ＤＮＡの初期分子数は、３．７５×１０⁶コピーから３．７５１０³コピーの範囲であった。
【００５６】
蛍光エネルギー転移解析のような蛍光クエンチングに基づいたＰＣＲ定量法が以前に報告されている（Ｈｅｉｄ，Ｃ．Ａ．ら、ＧｅｎｏｍｅＲｅｓ．、６、９８６−９９４、１９９６、Ｌｉｖａｋ，Ｋ．Ｊ．、ＰＣＲ ＭｅｔｈｏｄＡｐｐ ｌ．、４、３５７−３６２、１９９５）。この技法は市販されているが、第１、第２のＰＣＲプライマ・ペアーの他に、従来技術として述べたＮＡＳＢＡ法とかＡＰＥＸ法と同じく、一つのプローブ・シーケンスの中に２種類の色素を含んでいる。プローブのオリゴ塩基設計にあたっては、プライマの塩基配列と競合しない塩基配列を考えるだけでなく、融解温度（Ｔｍ）も考慮する必要がある。しかも、ドナーとアクセプター色素の位置も蛍光強度の増加効率及びポリメラーゼの５'ヌクレアーゼ活性に影響する。もう一つのゆらぎ分光法（蛍光クロスコリレ ーション分光法）が均一溶液中における特定目標遺伝子の検出法として報告されている（Ｃａｓｔｒｏ，Ａ．ら、Ａｎａｌ．Ｃｈｅｍ．、６９、３９１５−３９２０、１９９７）。クロスコリレーション法では非常に低濃度の目標配列を検出出来るけれど、この方法は依然として、実験溶液中では２種類の蛍光プローブを必要とする。
【００５７】
簡便性の観点からは、ＦＣＳに基づいたＰＣＲ定量法はエネルギー転移法およびクロスコリレーション法に比べて、重要な利点を有する。ＰＣＲの再現性と感度は、酵素のタイプ（ＰｏｌＩタイプ或いはタイプ）、プライマ配列、及び反応条件に強く依存する。一度これらの条件が決定されると、ＦＣＳに基づいたＦＡ−ＰＣＲはプライマ比をわずかに変えるだけで、通常のＰＣＲアッセイにすぐに応用出来る。それ故、ルーチン的ＰＣＲ条件はＦＡ−ＰＣＲの条件に容易に変更出来る。しかも、ＲＴ−ＰＣＲ（逆転写ＰＣＲ）のような他の増幅方法も、この方法に変更することが出来、将来的には一個の細胞中のｍＲＮＡを定量することも出来る。
【００５８】
ゲル電気泳動及びカラムクロマトグラフィーではＰＣＲ産物はプライマと副産物から分離される。しかしながら、目標産物は電気泳動とゲル物質からの抽出課程で稀釈され、さらにヌクレアーゼと接触する機会もあり、変成することもある。逆にＦＣＳでは測定後のサンプル回収に便利であり、それはサンプル液滴はカバーグラスの上に置くだけであり、ＦＣＳは非浸襲性であるため、ＰＣＲ溶液は稀釈されることがなく、測定中に変成することもない。ゲル電気泳動の分解能は高く、１塩基の長さの違いを検出出来るが通常１時間程度の泳動時間を要するのに対して、ＦＣＳは非常な高速測定であり（実施例では１分間であるが、ＦＣＳ装置の設計に応じて、適宜、それ１分より長くてもよく、好ましくは１分未満であってもよい）、例えば臨床検査やスクリーニング検査のような大量検体検査では利点がある。ＦＣＳ検査後のサンプルは直ちにゲル電気泳動等で詳細に解析出来、ＦＣＳ測定結果を確認したり、塩基配列を解析したり出来る。かつまた、回収されたサンプルは後に続く実験に使用することが出来る。たとえば、ハイブリダイゼーションプローブとして用いることが出来る。もしも、ＦＣＳ測定後に増幅サンプルを回収する必要がなければ、キャリーオーバーによる汚染の危険性もなく廃棄出来る。というのは、ＦＣＳは非接触型の測定法だからである。
【００５９】
ＦＣＳは非侵襲性であり、直接測定を可能にするので、ＰＣＲサンプル量はＦＣＳが必要とする測定量で決まる。発明者による今回の測定は１０μＬのサンプル量で行ったが、理論的には１０^−１５Lの体積要素レベルまで減らすことが出 来る。しかし、ＰＣＲ量は１μＬまで減らすことが出来れば、他の方法と比べて、経済面で大きな利点と言える。かつまた、ＦＣＳは蛍光顕微鏡を基礎としており、検出領域を対物レンズ上におくことが出来るため、ｉｎ−ｓｉｔｕＰＣＲによる特定のＲＮＡやＤＮＡの定量に用いることが出来る。
【００６０】
発明者は、ＰＣＲ溶液の解析に２成分モデルを使用して、良好な結果を得た。しかし、他の実験では多くのＤＮＡ種が存在するかもしれない。そのような場合には、ＤＮＡの種は従来のゲル電気泳動法で良く分離出来る。観測されたＦＣＳデータのモデル関数からの相関曲線の違いから溶液中に分布する他の成分の存在が示唆することができるが、単純な２成分モデルはそのような場合は限界があると思われる。その結果、もっと詳細な定量解析が必要とされる場合は、ＣＯＮＴＩＶＮ（Ｍａｘ−ＰｌａｎｋＩｎｓｔｉｔｕｔｅ製）のようなコンピュータープログラムを上述した本発明の方法に沿うように若干改訂するようにして、分布関数を得る多成分ＦＣＳ解析法を開発するのが好ましい。
【００６１】
本発明における測定工程は、物理的分離手段を用いることもなく、非侵襲的且つ非接触的に測定でき、使用するサンプル量も少ない。比較的少ないＰＣＲサイクル数で測定する場合のように、標的核酸分子の初期量が明らかに少ないような測定条件で分析する場合には、測定工程に先だって、増幅過程で取り込まれなかった標識分子をゲルろ過等の適宜のＢｏｕｎｄ／Ｆｒｅｅ分離手段によって除去してから、上記非侵襲的且つ非接触的な測定を実行すれば、容易にＳ／Ｎ比を高めて感度上昇できるという有利な一面も有する。このような少量のサンプルの物理的取り扱いは毛細管を使用したり、薄いカバーグラスや、フィルムをかぶせたガラス表面上の小さな穴を用いて行うことが出来、リアルタイム解析に用いることも出来る。これらの特性とさらなる改良により、増幅課程のモニターは容易に（リアルタイムモニタリング）、自動検出システム（ロボティックス）も可能であることは明白である。我々はＦＣＳに基づいたＦＡ−ＰＣＲ定量法は感度、定量精度、簡便性において利点を有すると結論する。この方法は迅速スクリーニングのみならず、分子診断の新時代を開くものと期待している。
【００６２】
【発明の効果】
本発明によれば、基質分子に標識分子を標識してＰＣＲによる増幅反応を行うとともに、標識分子の動き易さを測定するようにしたので、試料中の標的核酸の存在量を簡単な構成で正確に定量的に検出できる。
【図面の簡単な説明】
【図１】図１は、本発明の方法を実施するための装置の一例を示す概略図。
【図２】図２は、図１の装置の一部拡大図。
【図３】図３は、アガロース電気泳動における増幅産物４ｋｂの特性を示す写図。
【図４】図４は、ＰＣＲにおける、Ｆｌｕ−ｄＵＴＰの％比の関数として得た、標識濃度と産物量の関係を示すグラフ。
【図５】図５は、ＰＣＲ反応の過程における自己相関関数の推移を示すグラフ。
【図６】図６は、各自己相関関数をゼロ時間遅れのところで規格化したグラフ。
【図７】図７は、ＰＣＲサイクル数の関数としてＰＣＲ反応溶液中（5μｌ）における 増幅ＤＮＡ分子数を示すグラフ。
【図８】図８は、鋳型の初期数の関数としての２０サイクル後のＤＮＡ分子数を示したグラフ。
【符号の説明】
１…蛍光顕微鏡、
２…フォトマルチプライヤ、
３…データ処理装置、
４…表示装置、
１１…試料含有液、
１２…試料台、
１３…スライドガラス、
１４…蓋、
１５…対物レンズ、
１６…レーザー発生装置。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a technique for analyzing a nucleic acid molecule present in a sample, and more particularly to an analysis method and apparatus for detecting a target nucleic acid molecule based on a reaction result by a nucleic acid amplification reaction. The present invention is applied to a detection method in which the presence / absence of an amplification reaction and / or the reaction amount is obtained by effectively using tracking data relating to a single nucleic acid molecule. Therefore, the present invention can be effectively applied to any purpose for obtaining information related to the number of target nucleic acid molecules.
[0002]
[Prior art]
Detection of nucleic acids such as DNA and RNA and measurement of molecular weight are the most important analytical means in biochemical and molecular biology research. Recently, it has become an important analytical tool in gene diagnosis and gene therapy. For example, in the PCR (polymerase chain reaction) method, which is very important as a gene amplification method, the final detection of the amplified gene is carried out by fractionation for each chain length (molecular weight) using gel electrophoresis. After separation, the presence or absence of a fraction at a specific position is measured.
[0003]
In the presence of a DNA polymerase as a nucleic acid synthase, a primer for initiating replication, and four types of bases (dATP, dGTP, dTTP, dCTP) as substrates during replication, the target nucleic acid molecule is When the treatment is carried out under conditions allowing a polymerization reaction, nucleic acid amplification may occur in which a single strand DNA derived from a target nucleic acid is used as a template (template DNA) and a complementary strand is synthesized to form a double strand (dsDNA). Are known.
[0004]
In the PCR method, the total amount of amplified dsDNA is measured by a fluorometer as the total amount of fluorescence emitted from the entire test solution in an appropriate container or plate. The total fluorescence thus measured corresponds to the target DNA after being sufficiently amplified. However, the amplification reaction in PCR is a reaction process under contradictory temperature conditions, that is, a temperature rising stage in which a sample is placed under high temperature in order to denature the dsDNA into two separate ssDNAs, Since it includes a temperature lowering step for lowering the temperature in order to hybridize with the pair of primers described above, it is difficult to quantitatively track the state of amplification. Therefore, with the conventional PCR method, it was impossible to quantitatively analyze the total amount during or before amplification of the target nucleic acid.
[0005]
In recent years, a technique has been developed that measures the fluctuation motion of a labeled molecule in a liquid and accurately measures the minute motion of each target molecule using an autocorrelation function. This type of method is particularly called correlation fluorescence spectroscopy (abbreviated as FCS) because a fluorescent molecule as a labeled molecule is measured by an optical instrument. The calculation method of measurement data on biological materials using FCS has been reported using FCS in a hybridization reaction between a labeled nucleic acid probe and a target nucleic acid molecule (Kinjo, M., Rigler, R., Nucleic Acids Research). 23, 1795-1799, 1995).
[0006]
Several reports have recently been made on the detection of target genes using FCS. NASBA (Nucleic Acid Sequence Based Amplification) was combined with FCS and proved to be an effective detection method for the diagnosis of HIV (Human Immunodefective Virus) in serum (Oehlenschlager, F. et al., Proc. Nat. -12816, 1996). This NASBA method is a modification of PCR, but requires two primers and a separate fluorescently labeled primer as a probe in the presence of three enzymes, T7 RNA polymerase, reverse transcriptase, and RNase H. To do.
[0007]
A simpler method than the NASBA method (amplified probe extension detection method using FCS, APEX (Amplified Probe Extension)) has been reported from the same group (Walter, NG et al., Proc. Natl. Acad. Sci. USA, 93, 12805). -12810, 1996) Still, in addition to the forward and reverse primer sets, a separate fluorescently labeled primer is required as a probe. A time shift is observed after 26 cycles.
[0008]
[Problems to be solved by the invention]
However, both the NASBA method and the APEX method require a labeled probe that is different from the normal nucleic acid amplification material, which complicates the reaction conditions and makes it difficult to stably control the analysis accuracy. is there. In addition, since the labeled molecule is labeled with one fluorescent label, among the probes labeled with the labeled molecule, the signal derived from the label emitted from the non-hybridized probe significantly reduces the S / N ratio. The smaller the initial concentration of the target nucleic acid molecule, the lower the detection sensitivity. In addition, many PCR cycles must be run before it can be detected.
[0009]
An object of the present invention is to provide an analysis method and apparatus capable of quantifying the abundance of a target nucleic acid molecule with an accurate and simple configuration in view of the actual situation of such a nucleic acid detection method.
Another object of the present invention is to enable quantitative determination with high sensitivity even with a small copy number. Another object of the present invention is to enable detection of the presence or absence of a target nucleic acid molecule even with a small amount of sample. Another object of the present invention is to make it possible to know the amplification process in PCR at the molecular level.
[0010]
[Means for solving the problems and effects]
In the present invention, when fluorescein-11-dUTP, which is a nucleotide labeled with a fluorescent molecule, is mixed with a nucleic acid-containing sample and a DNA polymerase and nucleic acid amplification is performed, the movement state of the fluorescent dye molecules incorporated in the amplification process is It is based on the discovery that changes can be proved by image analysis. In addition, the present invention is based on the discovery that when the motion state of a fluorescent dye molecule is evaluated with an autocorrelation function, a quantitative result can be obtained. Furthermore, according to the present invention, the labeled substrate molecule that was not incorporated into the target nucleic acid molecule during the nucleic acid amplification process was separated by a gel filtration column, etc., and there was no problem in measurement, and a further increase in sensitivity was achieved. Based on discovery.
[0011]
That is, the method for analyzing a target nucleic acid of the present invention is a method for analyzing a target nucleic acid using a nucleic acid amplification reaction by a test solution containing a primer, a substrate molecule, a nucleic acid synthase, and a target nucleic acid molecule. A step of performing a nucleic acid amplification reaction with a test solution containing a substrate molecule labeled with a label molecule that can be generated, a step of measuring a signal from the labeled molecule in a test solution that has been subjected to nucleic acid amplification, and based on the measured signal And a step of evaluating the ease of movement of the labeled molecule in the liquid, and a step of quantifying the target nucleic acid molecule based on the evaluation result.
[0012]
Here, the measurement step preferably includes a step of measuring the abundance of the labeled molecule in a predetermined measurement region. Further, in this measuring step, it is preferable to measure the movement amount within a predetermined time a plurality of times. In addition, the method further includes a step of removing the labeled substrate molecule that was not involved in the nucleic acid amplification reaction between the step of performing the amplification reaction and the measurement step, and the measurement step is measured without contact with the test solution. In this case, the sensitivity can be easily increased, and it is a non-contact measurement, which is preferable in that the measurement accuracy is not lowered and a result with good reproducibility can be maintained.
[0013]
Moreover, it is preferable that an evaluation process includes the process converted into the statistical data which expresses the change of a movement amount based on several measurement data. Further, it is preferable that the converting step includes a step of performing an operation using an autocorrelation function. The quantitative step preferably includes a step of determining the presence or absence of a labeled molecule incorporated during nucleic acid amplification based on the evaluation result. The quantification step preferably includes a step of determining the number of target nucleic acid molecules incorporated during nucleic acid amplification based on the evaluation result.
[0014]
The target nucleic acid analyzer of the present invention is a target nucleic acid analyzer using a nucleic acid amplification reaction by a test solution containing a primer, a substrate molecule, a nucleic acid synthase (= polymerase or reverse transcriptase), and a target nucleic acid molecule. A holding means for holding a test solution containing a substrate molecule labeled with a labeled molecule capable of generating a measurable signal, and a measurement for measuring the signal from the labeled molecule after the nucleic acid amplification reaction with respect to the held test solution Means, evaluation means for evaluating the ease of movement of the labeled molecule in the liquid based on the measured signal, and data output means for outputting data obtained by quantifying the target nucleic acid molecule based on the evaluation result. It is a feature.
[0015]
Here, it is preferable that the measuring means has an optical system that performs the measurement in the minute visual field provided in the diffraction limited region near the focal point. Alternatively, the measurement means preferably has a microscope for performing measurement in a microscopic field formed by a confocal optical system. Furthermore, the diffraction limited region is preferably formed by an aperture having an average diameter of 30 ± 20 μm. The diffraction limited region is preferably formed by an aperture having an average diameter of 20 ± 10 μm.
[0016]
Moreover, it is preferable that the minute visual field is a substantially cylindrical region having an average radius of 200 ± 50 nm and an average length of 2000 ± 500 nm on the optical axis.
[0017]
The evaluation means preferably includes means for storing a plurality of measurement data obtained within a predetermined time and an operation means for calculating the stored plurality of measurement data using an autocorrelation function. Further, the evaluation means has means for storing measurement data relating to a plurality of labeled molecules obtained within a predetermined measurement region, and a calculation means for calculating the stored measurement data for each label molecule using an autocorrelation function. Is preferred.
[0018]
Further, it is preferable that the data output means includes conversion means for converting into statistical data expressing temporal position changes related to a plurality of tracking data based on a result calculated by the autocorrelation function.
[0019]
DETAILED DESCRIPTION OF THE INVENTION
In the present invention, at least one of the substrate molecules incorporated in the nucleic acid amplification process in the PCR method is labeled with a label molecule that can generate a measurable signal. The substrate molecule having such a labeled molecule is in a free state without binding during the nucleic acid hybridization reaction step between the target nucleic acid and the primer for initiating synthesis. In the nucleic acid synthesis reaction subsequent to the hybridization reaction, when the nucleic acid synthase acts on the primer bound to the target nucleic acid molecule, the primer tries to extend and successively takes in substrate molecules necessary for synthesis. Thus, the label molecules incorporated together with the substrate molecules during the nucleic acid amplification are less likely to move in the liquid than the free label molecules.
[0020]
Therefore, at least a part of the sample-containing test solution that has undergone the effective number of PCR cycles is quantitatively collected or attached to the measurement of the labeled molecule while being kept in the container or the like. Here, the test solution holding means may be a concave container such as a test tube, a well, or a petri dish, or a flat plate-like support such as a slide glass. In some cases, it may be held by being spotted directly on the surface of the measuring means.
[0021]
The measurement method may be selected as appropriate as long as the measurement is related to the change in the size of the labeled molecule accompanying amplification. Gel electrophoresis, capillary electrophoresis, flow cytometer for fluorescence measurement, image cytometer, fluorescence microscope, etc. Can be used effectively. By performing the measurement process in a three-dimensional microscopic field, free micromotion of the target molecule in the sample-containing liquid can be measured with high accuracy in the natural state according to the result of the PCR reaction. On the other hand, when the measurement is performed in a two-dimensional field of view, the measurement accuracy is low because the three-dimensional free movement of the labeled molecule cannot be captured as in the Brownian motion. In forming a fine field of view, it can be obtained by optical design to converge light from a halogen lamp or emit from an aperture (pinhole, optical fiber end face, etc.) having an extremely small average radius, It is preferable to use convergent light by a laser beam.
[0022]
A measurement field with a very small depth of field can be obtained by forming a small field of view of the measurement process using a confocal optical system. Output data can be supplied to the measuring means.
[0023]
Since the microscopic field is a diffraction limited region near the focal point, individual labeled molecules can be measured with a high S / N ratio.
[0024]
Since the diffraction limitation is formed by pinholes having an average diameter of 15 ± 5 μm, measurement data from a small number of selected labeled molecules can be efficiently obtained.
[0025]
Since the minute region is a substantially cylindrical region having an average radius of 200 ± 50 nm and an average length of 2000 ± 500 nm on the optical axis, it is possible to efficiently acquire free minute motion relating to the labeled molecule aimed in the measurement visual field. it can.
[0026]
In the present invention, the movement speed of the label molecules entering and exiting the minute measurement visual field is measured by using an increase or decrease or disappearance of the output intensity measured from one or more individual label molecules in a predetermined space as an index. Therefore, it is preferable that the kind of the label molecule for labeling the substrate molecule is a label material that maintains a substantially constant output at a plurality of measurement points. Examples of such a labeled molecule material include a luminescent substance, a fluorescent substance, a magnetic substance, a radioactive substance, and the like. In particular, for the luminescent substance and the fluorescent substance, it is preferable to select a dye that emits light or fluorescence for a long time. If a labeling molecule having a luminescent dye or a fluorescent dye is used, measurement at the molecular level can be performed with an optically easy configuration. Examples of fluorescent dyes include various known ones such as DAPI, FITC, rhodamine, Cy3, CY3.5, Cy5, Cy5.5, and Cy7.
[0027]
When the measurement process measures the ease of movement of fluorescent molecules in the liquid, the fluorescence data can be received by a fluorescence measuring means such as a photomultiplier or a photodiode. It is advantageous for the fluorescence measurement means to have a measurement mode in which a single photon can be measured, in order to perform individual measurements on fluorescent molecules.
[0028]
In the case where the measurement step is to measure the fluctuation movement of the labeled molecule in the liquid, the present invention can be made a more effective embodiment, and the minute movement of each target molecule can be accurately measured. it can. In measuring the fluctuation motion, it is preferable to perform calculation using an autocorrelation function. In particular, when fluorescence is used as a labeling molecule, it is preferable to employ autocorrelation fluorescence spectroscopy (abbreviated as FCS). The calculation method of measurement data on biological materials using FCS has been reported using FCS in a hybridization reaction between a labeled nucleic acid probe and a target nucleic acid molecule (Kinjo, M., Rigler, R., Nucleic Acids Research). 23, 1795-1799, 1995).
[0029]
An outline of an apparatus for performing FCS is shown in FIG. This FCS apparatus receives an inverted fluorescence microscope 1 using a confocal optical system, a photomultiplier 2 for measuring fluorescence from a sample, and measurement data, and performs calculation using an autocorrelation function to quantify or A data processing device 3 that performs graphing and a display device 4 that displays calculation results on a screen are provided.
[0030]
As shown in FIG. 1, the sample-containing liquid 11 can be easily set by spotting on the slide glass 13 placed on the sample table 12. In this apparatus, in particular, since a very small amount of the sample-containing liquid 11 is used, a lid 14 for preventing evaporation of moisture is placed on the slide glass 13. The lid 14 is preferably made of a material having as low light transmittance as possible, so that airtightness and light shielding properties can be obtained at the same time. However, it is preferable to use an inner surface of the lid that has as low a light reflectivity as possible so as to prevent reflection of excitation light. An objective lens 15 set so as to be focused in the sample-containing liquid 11 is disposed directly below the portion of the slide glass 13 where the sample-containing liquid 11 is located.
[0031]
The fluorescent microscope 1 may be an epi-illumination type. In the epi-illumination type, the sample-containing liquid 11 may be spotted directly on the lower surface of the objective lens 15. In addition, the laser generator 16 that is a light source of the fluorescence microscope 1 uses an argon (Ar) ion laser in FIG. 1, but depending on the type of fluorescence, a krypton argon (Kr-Ar) ion laser, helium neon Various changes may be made to a (He—Ne) laser, a helium cadmium (He—Cd) laser, or the like. Further, various operations such as loading and unloading of the slide glass 13 in the fluorescence microscope 1, spotting of the sample-containing liquid on the slide glass 13 and the opening and closing of the lid 14 may be appropriately automated as necessary.
[0032]
FIG. 2 is an enlarged view showing a measurement portion of the fluorescence microscope 1 of FIG. In FIG. 2A, a minute visual field region 20 is formed by the positional relationship between the slide glass 13 and the objective lens 15 having a predetermined numerical aperture (FA = 1.2 in the figure). As shown in FIG. 2 (B), the minute visual field region 20 actually has a substantially cylindrical visual field extending vertically from the focal point of the laser beam having a volume (in the figure, an intermediate constricted portion). is doing. The visual field region 20 is defined by the length Z on the optical axis and the average radius W with the focus as the reference position. In such a fluorescence measurement in the micro region 20, noise derived from fluorescent molecules other than the vicinity of the focal point in the sample-containing liquid 11 is effectively reduced by reducing it to a minimum region where the micro motion of the fluorescent molecule can be traced. This contributes to accurate measurement of each fluorescent molecule.
[0033]
The present invention can be applied to genetic disease diagnosis, parentage testing, crime investigation, gene therapy, molecular biological research, drug development, and the like based on the various embodiments described above. The above-described embodiment also includes a changeable invention without departing from the gist of the present invention. For example, in the above-described embodiment, the measurement apparatus has been described as being capable of measurement in the same environment as that under microscope observation on the sample stage of the fluorescence microscope. However, the apparatus disclosed in European Patent No. 640828A1 If the optical design is made so that the laser beam emitted from the objective lens can be focused on the sample-containing liquid inside the thermal cycler, the sample-containing liquid can be amplified without being transferred onto the slide glass. The reaction can be measured continuously. In addition, the method of obtaining multiple data within the elapsed time in a PCR cycle is either when obtaining multiple data continuously or intermittently within a certain time after a predetermined cycle, or when obtaining an appropriate number of measurement data for different cycles. possible.
[0034]
Examples of the present invention will be described below. However, the present invention is not limited to these examples. Based on the gist of the present invention, various examples within the obvious range according to the technical level at the time of filing of the present invention. It can be changed.
[0035]
【Example】
(1) PCR in the presence of fluorescently labeled nucleosides
0.1 Uμl ^-1 Pol I type DNA synthase (AmpliTaq Gold, Perkin-Elmer), 1X buffer I (Perkin-Elmer), 200 μM dATP, dGTP and dCTP, 120 μM dTTP, 80 μM fluorescein 11-dUTP (FluoroGreen, AmerFlumerAmerFraction) -DUTP), and a mixed solution containing 0.8 μM forward and reverse primers as a test solution, and 5 μl and 25 μl of this test solution were subjected to standard nucleic acid amplification. .
[0036]
200 pg / 5 μl of full-length lambda phage DNA (48.5 kb) was used as a template. The length of the target DNA was 4000 bp. As for the base sequence of the primer used for nucleic acid amplification, the forward primer was GATGAGTTCGGTCCCGTACAACT (base number = 7131-7153), and the reverse primer was CTTAACCAGTGCGCTGAGTGAACT (base number = 11098-11120).
[0037]
As a PCR apparatus for performing a nucleic acid amplification reaction, a programmable thermal control system (PC700, Astec) was used, and an oil-free reaction tube (Takara Shuzo) was used as a PCR container for storing each of the above-mentioned test solutions. That is, after first performing a denaturation treatment at 96 ° C. for 15 minutes (initial transformation treatment), an annealing treatment by incubation at 55 ° C. for 15 minutes, an extension reaction by incubation at 65 ° C. for 4 minutes, and further at 96 ° C. Each round of PCR consisting of a denaturation treatment with a 1.5 minute incubation was performed. The PCR container was taken out at a predetermined PCR cycle and stored at −20 ° C. until the next treatment.
[0038]
In order to remove the unincorporated nucleoside, distilled water was added so that the mixed solution of each test solution subjected to PCR reaction was 50 μl, and applied to a MicroSpin column (S-400HR, Pharmacia Biotec). When the volume Vc of each solution collected with this column was measured by weight, it was distributed between 55 μl and 60 μl. The solution was subjected to the next FCS measurement without further purification. The molecular weight of one base pair was assumed to be 660 Da. Therefore, the molecular weight of lambda phage DNA is 32 × 10 ⁶ I was able to calculate Da.
[0039]
(2) FCS measurement
FCS measurement was performed using the FCS apparatus as shown in FIG. That is, a part of the test solution subjected to the nucleic acid amplification reaction is used as a sample, and the sample droplet is spotted on a sample slide and placed on a sample stage of a commercially available FCS device (ConfoCor, CarlZissJenGmbH), and the magnification is 40 times. CW Ar that has passed through the lens by the objective lens (C-Apochromat, NA = 1.2) ⁺ Excited by the laser beam, the emitted light is measured in a single photon counting mode with SPCM-200-PQ (EG & G) which is an avalanche diode (APD). The measured fluorescence signal was evaluated by analyzing with a digital correlator ALV5000 / E (ALV GmbH). In actuality, 20 μl of the sample droplet for measurement was spotted on the cover glass, and a small box was placed on the sample table so as to cover the cover glass in order to prevent evaporation during measurement. The sample volume in the focal region was determined from the diffusion count value of rhodamine 6G. The volume element was determined using the concentration of a solution of fluorescein and Flu-dUTP.
[0040]
The fluorescence signal measured for about 1 minute was stored in a sequential storage unit, and programmed to be analyzed by applying it to the fluorescence autocorrelation function G (t) for evaluation. This fluorescent autocorrelation function G (t) is expressed by the average number N of fluorescent molecules in the measurement region, the translation time τ of the free labeled substrate molecule (Flu-11-dUTP) as a mononucleoside (monomer DNA). _mono And the translation time τ of the incorporated labeled substrate molecule as polymer DNA _poly Based on the method of Rigler et al. (Refer to Fluorescence Spectroscopy-New Methods and Applications, Springer, Berlin, 13-24, InJ. R. Lakowicz (Ed.), 1992).
[0041]
[Expression 1]

[0042]
In Equation 1, y is the ratio of the polymer DNA component, τ _mono = Wo ² / 4D _mono , Τ _poly = Wo ² / 4D _poly , S = Wo / Zo (Wo is the diameter of the volume element of the substantially cylindrical measurement region (see FIG. 2B) formed in the minute visual field near the focal point, and 2Zo means the length thereof. D), D _mono And D _poly Are translational diffusion coefficients of monomer DNA and polymer DNA, respectively.
[0043]
Data analysis was further performed using a non-linear least square parameter method to calculate the normalized mean square deviation between the data and the model. The total number T of the molecules amplified through the desired PCR cycle was calculated by Equation 2; T = (N / Vc) Vc, and was output as quantification data. Where Vo is the volume element and Vc is the volume of the solution collected by the spin column. During the FCS measurement, the average fluorescence intensity was also measured. Its intensity reflects the total number of fluorophores (Flu-dUTP) incorporated into the DNA amplified in the volume element. The value obtained by dividing the average fluorescence intensity measurement speed by the number of DNA molecules Ny was adopted as the average measurement speed C / M of DNA molecules. This value could be used as an effective labeling concentration for PCR products.
[0044]
(3) Calculation of measurement area
The volume element of the measurement region was determined from the measurement of the diffusion times of rhodamine 6G, fluorescein, and Flu-dUTP as reference samples. The ratio of the length to the diameter in the measurement region (see FIG. 2B) provided in the vicinity of the focusing was obtained from the value of S in Equation 1, and was about 5. The volume element V ₀ Is 1 × 10 ^-15 l was calculated.
[0045]
(4) Uptake of Flu-11-dUTP by PCR
In order to clarify the action effect of fluorescently labeled nucleosides in PCR, the concentration of Flu-dUTP was changed from 10% to 50% and examined. FIG. 3 is a copy showing the characteristics of the amplified product 4 kb in agarose electrophoresis. That is, 3 μl of a PCR reaction mixture as a test solution after 50 cycles of nucleic acid amplification reaction was applied to a 7% agarose gel. Here, FIG. 3 (A) is before ethidium bromide staining, and FIG. 3 (B) is after ethidium staining. In the figure, M indicates a molecular marker (HaeIII-digested Phi X174DNA). In the figure, “0”, “10”, “20”, “30”, “40”, and “50” indicate% ratios of Flu-dUTP and dUTP + dTTP. FN is a fluorescent nucleoside (an unincorporated nucleoside). Further, an orange optical filter Ya3 was used for measurement of fluorescence emitted from the gel in FIG. 3A, and a red filter R1 was used for measurement in FIG. 3B.
[0046]
FIG. 3A shows that a 4000 bp fluorescent PCR product is synthesized even in the presence of 50% Flu-dUTP. The fluorescence intensity of the labeled DNA increased with the concentration of Flu-dUTP. Although the total production amount of the amplified DNA can be shown by ethidium staining, in this experiment, as shown in FIG. 3B, the product amount was shown to be the same as the case without Flu-dUTP.
[0047]
(5) Fluorescence intensity and amplification product amount in PCR
FIG. 4 is a graph showing the relationship between the label concentration and the product amount obtained as a function of the% ratio of Flu-dUTP in PCR. In the figure, square marks indicate the average measured value C / M per DNA molecule. Black circles indicate the number of DNA molecules in the measurement region that is the observation field. According to FIG. 4, it was found that the fluorescence intensity C / M per molecule increased with increasing Flu-dUTP. This result indicated that the label concentration depends on the concentration of Flu-dUTP. Moreover, the number of DNA molecules was almost constant within the concentration range of Flu-dUTP studied. This result was consistent with the gel electrophoresis analysis shown in FIG. This indicates that fluorescein does not interfere with the function of DNA synthase. Comparing the C / M values of free Flu-dUTP as a monomer and the PCR product, the number of Flu-dUTP monomers incorporated into the 4000 bp DNA strand can be seen. It indicates the possibility that a modified nucleoside exists next to each other.
[0048]
(6) PCR monitoring by FCS
FIG. 5 is a graph showing the transition of the autocorrelation function in the course of the PCR reaction, and shows a typical time change of the correlation curve. In the figure, the circle mark is measured when the nucleic acid amplification reaction of 10 cycles, the triangle mark is 20 cycles, the inverted triangle mark is 30 cycles, the diamond mark is 40 cycles, and the square mark is 50 cycles.
As can be seen from FIG. 5, the zero time delay value (y-intercept) of the autocorrelation function decreases with the PCR cycle. This result is due to the increase in the number of DNA molecules.
[0049]
FIG. 6 is a graph in which each autocorrelation function is normalized at a zero time delay. In FIG. 6, the Flu-dUTP curve is shown by a black circle for comparison. In the figure, the solid line represents 10 cycles, the broken line represents 20 cycles, the dotted line represents 30 cycles, the one-dot chain line represents 40 cycles, and the two-dot chain line represents values obtained after 50 cycles of nucleic acid amplification. As can be seen from FIG. 6, the autocorrelation function (solid line portion) obtained after 10 cycles is clearly different from that of Flu-dUTP. This autocorrelation function could not be applied to a simple one-component model, but could be applied to a two-component model as shown in Equation 1 above. In addition, a short diffusion time component could be detected even in a test solution in which 50 cycles of PCR were performed. The short component is defined as unreacted Flu-dUTP that has passed through the spin column.
[0050]
FIG. 7 is a graph showing the number of amplified DNA molecules in the PCR reaction solution (5 μl) as a function of the number of PCR cycles. The total number of amplified DNA molecules was calculated according to Equation 2 above. In the experiment, various initial amounts of lambda phage DNA were examined. In the figure, the square mark is 200 pg (3.75 × 10 ⁶ Copy) Lambda forage DN A is shown as the initial amount, and the circle is 20 pg (3.75 × 10 5) below. ^Five Copy), triangle mark is 2pg (3.75 × 10 ^Four Copy), inverted triangle mark is 0.2 pg (3.75 × 10 ^Three Each).
[0051]
FIG. 8 is a graph showing the number of DNA molecules after 20 cycles as a function of the initial number of templates. When DNA is amplified by PCR, the number of DNA molecules increases exponentially before reaching the plateau effect. The initial number of template molecules is 1024 after 10 cycles (= 2 ^Ten ) 3.75 × 10 ⁶ The DNA of the copy (equivalent to 200 pg) is 3.75 × 10 ⁹ The result is a copy. However, the initial number of copies is 3.75 × 10 ⁶ At the time of FCS measurement, after 10 cycles, 7.44 × 10 ⁹ The number of copies is measured, and similarly, the initial number of copies is 3.75 × 10 ^Five In the case of 0.31 × 10 after 10 cycles ⁹ The copy was measured. Thus, the quantification of the FCS measurement appears to have low fidelity at an early stage of the amplified sample. This is a result of a small amount of amplified DNA in the detection region and a distribution of many unincorporated labeled nucleosides.
[0052]
This direct label-based PCR quantification using FCS can be performed even after 10 cycles of amplification. ^Three The target DNA initial concentration can be detected, but as can be seen from FIG. 8, the initial target DNA concentration can be estimated better after 20 cycles. In this figure, since the relationship between the initial number of DNA molecules and the number of products is linear, the number of target DNA molecules can be determined by FCS measurement. Although the data is omitted, it was confirmed that the same relationship could be detected with another DNA sequence of 500 bp using another curve. In addition, in various experiments, PCR 20 cycles, 5 μl of sample solution was used. Needless to say, if the amount of the sample solution is increased, more precise and higher reproducibility can be obtained. This is most important in diagnostic applications. Therefore, in order to save time and resources, it is preferable to appropriately select the number of cycles that can provide good reproducibility through experiments.
[0053]
As shown in the experimental results described above, the present invention has succeeded for the first time in using FCS as a molecular counting method for directly labeled PCR. Further, it was proved that PCR was not inhibited even when 50% of dTTP was replaced with Flu-dUTP even for a 4000 bp target nucleic acid. For shorter target nucleic acids, eg 500 bp, 1000 bp, 2000 bp, a stronger fluorescence band was detected by gel electrophoresis after 50 thermal cycles. Since this method uses a substrate molecule that is used for normal PCR as a labeling reagent and bound to a label molecule that can be measured such as fluorescence, it can be easily used in a normal laboratory as in other normal PCR processes. It can be applied to. The FCS assay described here requires only a slight change in PCR conditions, a reaction solution as small as 5 μl of test solution, and a few PCR cycles. Fluorescently labeled molecules incorporated into DNA molecules can be bound to any genetic marker or infection marker, and nucleic acids from various organisms can be used while making effective use of established PCR know-how. Amplify molecules.
[0054]
The FCS-based quantitative PCR according to the present invention is advantageous in many fields. This is because this technique is highly sensitive, saves time and effort, and the composition of the reaction components and the procedures for their use are very simple. It should also be mentioned that the measurement of FCS is non-invasive, and the test solution measured by FCS can be used conveniently for further experiments such as fluorescence hybridization and FISH analysis. Furthermore, FCS can also measure the restriction enzyme digestion process in a homogeneous solution. Therefore, as introduced here, by using high-density labeled DNA, analysis such as point mutation and RELF can be performed after amplification without using any separation method such as column chromatography or gel chromatography. It can still be detected by FCS. These FCS-based PCR quantification methods are applied to diagnosis and screening experiments, and further open a new field based on single molecule detection.
[0055]
As described above, two important parameters in biological science can be obtained by FCS. It is the detection area (10 ^-15 The average number of molecules and translational diffusion coefficient of molecules in l). In the present invention, PCR products were analyzed using FCS as a tool for single molecule measurement. Before the DNA amplification process by PCR reaches a plateau, a specific DNA base sequence can be quantified. In order to be able to apply FCS to PCR, fluorescein 11-dUTP is used as a substrate used in the enzymatic amplification cycle, and PCR is performed in the presence of such a labeled nucleoside to label the 4000 bp hail target DNA. I was able to do it. After 10 cycles, amplified DNA could be detected by increasing the diffusion time of fluorescent molecules in the FCS detection region. The initial number of molecules of target DNA in the volume (5 μl) of the minute detection region of PCR detection is 3.75 × 10 ⁶ 3.71010 from copy ^Three The range of the copy.
[0056]
PCR quantification methods based on fluorescence quenching such as fluorescence energy transfer analysis have been previously reported (Heid, CA, et al., Genome Res., 6, 986-994, 1996, Livak, KJ, PCR Method App l., 4, 357-362, 1995). Although this technique is commercially available, in addition to the first and second PCR primer pairs, two kinds of dyes are included in one probe sequence as in the case of the NASBA method or APEX method described as the prior art. It is out. In designing the oligo base of the probe, it is necessary to consider not only the base sequence that does not compete with the primer base sequence, but also the melting temperature (Tm). Moreover, the positions of the donor and acceptor dyes also affect the efficiency of increasing the fluorescence intensity and the 5 ′ nuclease activity of the polymerase. Another fluctuation spectroscopy (fluorescence cross-correlation spectroscopy) has been reported as a method for detecting a specific target gene in a homogeneous solution (Castro, A. et al., Anal. Chem., 69, 3915-3920, 1997). Although the cross-correlation method can detect very low concentrations of target sequences, this method still requires two fluorescent probes in the experimental solution.
[0057]
From the viewpoint of simplicity, the PCR quantification method based on FCS has significant advantages over the energy transfer method and the cross-correlation method. The reproducibility and sensitivity of PCR strongly depends on the type of enzyme (PolI type or type), primer sequence, and reaction conditions. Once these conditions are determined, FCS-based FA-PCR can be readily applied to conventional PCR assays with only a slight change in primer ratio. Therefore, routine PCR conditions can be easily changed to FA-PCR conditions. In addition, other amplification methods such as RT-PCR (reverse transcription PCR) can also be changed to this method, and mRNA in a single cell can be quantified in the future.
[0058]
In gel electrophoresis and column chromatography, PCR products are separated from primers and byproducts. However, the target product is diluted in the electrophoresis and extraction process from the gel material, and also has the opportunity to come into contact with nucleases and may be denatured. Conversely, FCS is convenient for sample collection after measurement, because the sample droplet is only placed on the cover glass and FCS is non-invasive, so the PCR solution is not diluted and the measurement is There is no metamorphosis inside. The resolution of gel electrophoresis is high, and the difference in length of one base can be detected, but usually it takes about 1 hour of migration time, whereas FCS is a very high speed measurement (in the example, it is 1 minute) Depending on the design of the FCS device, it may be longer than 1 minute, preferably less than 1 minute, as appropriate), for example in large volume testing such as clinical and screening tests. The sample after the FCS test can be immediately analyzed in detail by gel electrophoresis or the like, and the FCS measurement result can be confirmed or the base sequence can be analyzed. Also, the collected sample can be used for subsequent experiments. For example, it can be used as a hybridization probe. If it is not necessary to recover the amplified sample after the FCS measurement, it can be discarded without the risk of contamination due to carryover. This is because FCS is a non-contact measurement method.
[0059]
Since FCS is non-invasive and allows direct measurement, the amount of PCR sample is determined by the amount of measurement required by FCS. This measurement by the inventor was performed with a sample volume of 10 μL. ^-15 It can be reduced to L volume element level. However, if the amount of PCR can be reduced to 1 μL, it can be said that it is a great economic advantage compared with other methods. Moreover, since FCS is based on a fluorescence microscope and a detection region can be placed on an objective lens, it can be used for quantification of specific RNA or DNA by in-situ PCR.
[0060]
The inventor obtained good results using a two-component model for analysis of PCR solutions. However, there may be many DNA species in other experiments. In such cases, the DNA species can be well separated by conventional gel electrophoresis. The difference in the correlation curve from the model function of the observed FCS data can indicate the presence of other components distributed in the solution, but the simple two-component model seems to be limited in such cases . As a result, when a more detailed quantitative analysis is required, a distribution program is obtained by slightly revising a computer program such as CONTIBN (manufactured by Max-Plant Institute) so as to follow the method of the present invention described above. It is preferred to develop a multi-component FCS analysis method.
[0061]
The measurement process in the present invention can be performed non-invasively and non-contact without using a physical separation means, and the amount of sample used is small. When analyzing under measurement conditions where the initial amount of target nucleic acid molecule is clearly small, such as when measuring with a relatively small number of PCR cycles, labeled molecules that have not been incorporated in the amplification process prior to the measurement step are used. If the non-invasive and non-contact measurement is performed after removal by an appropriate Bound / Free separation means such as gel filtration, the S / N ratio can be easily increased to increase the sensitivity. . Such physical handling of a small amount of sample can be performed using a capillary tube, a thin cover glass, or a small hole on a glass surface covered with a film, and can also be used for real-time analysis. With these characteristics and further improvements, it is clear that the amplification process can be easily monitored (real-time monitoring) and an automatic detection system (robotics). We conclude that the FA-PCR quantification method based on FCS has advantages in sensitivity, quantification accuracy and simplicity. This method is expected to open a new era of molecular diagnostics as well as rapid screening.
[0062]
【The invention's effect】
According to the present invention, the labeling molecule is labeled on the substrate molecule to perform the amplification reaction by PCR, and the ease of movement of the labeling molecule is measured, so that the abundance of the target nucleic acid in the sample can be reduced with a simple configuration. It can be detected accurately and quantitatively.
[Brief description of the drawings]
FIG. 1 is a schematic diagram showing an example of an apparatus for carrying out the method of the present invention.
FIG. 2 is a partially enlarged view of the apparatus shown in FIG.
FIG. 3 is a copy showing the characteristics of the amplified product 4 kb in agarose electrophoresis.
FIG. 4 is a graph showing the relationship between the label concentration and the amount of product obtained as a function of the% ratio of Flu-dUTP in PCR.
FIG. 5 is a graph showing the transition of the autocorrelation function during the PCR reaction.
FIG. 6 is a graph in which each autocorrelation function is normalized at a zero time delay.
FIG. 7 is a graph showing the number of amplified DNA molecules in PCR reaction solution (5 μl) as a function of PCR cycle number.
FIG. 8 is a graph showing the number of DNA molecules after 20 cycles as a function of the initial number of templates.
[Explanation of symbols]
1 ... fluorescence microscope,
2 ... Photomultiplier,
3 ... Data processing device,
4 ... display device,
11 ... Sample-containing liquid,
12 ... Sample stage,
13 ... slide glass,
14 ... lid,
15 ... Objective lens,
16: Laser generator.

Claims (5)

A method for analyzing a target nucleic acid using a nucleic acid amplification reaction by a test solution containing a primer, a substrate molecule, a nucleic acid synthase, and a target nucleic acid molecule,
Performing the nucleic acid amplification reaction using a test solution containing a substrate molecule labeled with a labeled molecule capable of generating a measurable signal;
For the nucleic acid-amplified test solution, the speed of movement of the labeled molecules entering and exiting the minute measurement visual field is measured using the increase or decrease or disappearance of the output intensity measured from one or more individual labeled molecules in a predetermined space as an index. Measuring the fluctuation motion of the labeled nucleic acid molecule in the liquid;
Based on the measured fluctuations motion, the step of evaluating the ease of movement of in said test solution of the labeled molecule,
A method for analyzing a target nucleic acid, comprising a step of quantifying an amplified nucleic acid fragment labeled with the labeling molecule based on the evaluation result. The method according to claim 1, wherein the labeling molecule is a fluorescent substance or a luminescent substance . The method according to claim 1, wherein the measuring step includes a step of measuring an abundance of a labeled molecule in a predetermined measurement region. The method of claim 1, wherein the step of evaluating includes performing an operation with an autocorrelation function. The step of performing the amplification reaction and the step of measuring further include a step of removing the labeled substrate molecule that was not involved in the nucleic acid amplification reaction, and the step of measuring is in a state that is not in contact with the test solution The method according to any one of claims 1 to 4 , wherein the measurement is performed by the method.

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