JP2004512027A - Methods for identifying low abundance polynucleotides and related compositions - Google Patents

Abstract

The present invention provides a novel method for producing a plurality of polynucleotides produced from a polynucleotide sample and a plurality of polynucleotides produced thereby. In one embodiment, the plurality of polynucleotides are produced by subtractive hybridization between a test and a reference polynucleotide sample and are not present in the reference polynucleotide sample or substantially more than present in the test polynucleotide sample. The sequence present in the reference polynucleotide sample at a lower concentration is substantially enriched. The plurality of polynucleotides are also enriched in substantially lower abundance sequences as compared to the test polynucleotide sample. The method also provides a kit for using the polynucleotides produced in and by the method. Polynucleotides are useful in a wide variety of applications, such as cloning, expression and hybridization studies.

Description

（太字で示される３’“ＧＧＧ” はリボヌクレオチドである；シーケンスＩＤ　ＮＯ：３）、及び２μｌの脱イオン化Ｈ_２ Ｏを含む５μｌのｃＤＮＡ合成混合物であった。該混合物を７２℃で２分間及びそれから３７℃で２分間インキュベートした。それから体積を以下の試薬を用いて１０μｌに調節した：２μｌの５Ｘ第一鎖合成バッファー（２５０ｍＭのＴｒｉｓ−ＨＣｌ、ｐＨ８．３；３０ｍＭのＭｇＣｌ_２ ；及び３７５ｍＭのＫＣｌ）、０．５μｌのジチオトレイトール（ＤＴＴ；５０ｍＭ）、１μｌのｄＮＴＰ混合物（１０ｍＭの各ｄＡＴＰ、ｄＣＴＰ、ｄＧＴＰ、ｄＴＴＰ）、０．５μｌのＲｎａｓｉｎ（２０単位、プロメガ）、１μｌ（２００単位）のＭＭＬＶ逆転写酵素（ＳｕｐｅｒｓｃｒｉｐｔＩＩ（登録商標）、Ｌｉｆｅ　Ｔｅｃｈｎｏｌｏｇｉｅｓ）。逆転写を４２℃にて９０分間行った。反応は氷上にチューブを静置することにより終了させた。
【０２１８】
第一鎖ｃＤＮＡを約１ｐｇ／μｌ（約１×１０^−６）まで希釈し、希有の転写物を除去し、続けてＰＣＲを、４０ｍＭ　Ｔｒｉｃｉｎｅ−ＫＯＨ、ｐＨ９．２；１５ｍＭ　ＫＯＡｃ；３．５ｍＭ　Ｍｇ（ＯＡｃ）_２；及び０．２μＭの５’ −アンカープライマー（５’ −ＴＧＣ−ＴＧＣ−ＧＡＧ−ＡＡＧ−ＡＣＧ−ＡＣＡ−ＧＡＡ−３’ ）；０．２μＭのランダム−Ｔ７プライマー；０．２ｍＭの各ｄＡＴＰ、ｄＧＴＰ、ｄＣＴＰ、ｄＴＴＰ；及び２μｌのＡｄｖａｎｔａｇｅ（登録商標）ｃＤＮＡポリメラーゼ混合物（５０Ｘ；ＫｌｅｎＴａｑ−１及びＤｅｅｐ　Ｖｅｎｔポリメラーゼ；Ｃｌｏｎｔｅｃｈを含む）を含む１００μｌ反応中で行った。ＰＣＲを以下の循環条件を使用して、ＤＮＡ　Ｔｈｅｒｍａｌ　Ｃｙｃｌｅｒ　４８０（ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ）中で行った：９５℃で１分間；９５℃で１５秒間、３０−３５サイクル；６２℃で３０秒；及び７２℃で６分間。
【０２１９】
ＰＣＲ後、二本鎖ｃＤＮＡをＳ−２００スピンカラム（Ｃｌｏｎｔｅｃｈ）を通すことにより精製した。アンチセンスＲＮＡドライバー合成の前に、二本鎖ｃＤＮＡを、グルコース３−ホスフェート脱水素酵素（Ｇ３ＰＤＨ）及びｃ−ｍｙｃ遺伝子の増幅について試験した。ＰＣＲ増幅の２５サイクル後、Ｇ３ＰＤＨをエチジウムブロマイドを用いて染色された１．１％アガロースゲル上の濃い色のバンドとして視覚化した。ｃ−ｍｙｃは増幅の３５サイクル後でさえ、エチジウムブロマイド染色ゲル上で視覚化されなかった。
【０２２０】
２．　ホモポリマー配列化により加えられるユニバーサルＰＣＲアンカー
第一鎖ｃＤＮＡを、方法１のランダム−Ｔ７プライマーを使用してヒト胎盤ポリＡ＋ＲＮＡ上で合成した。短く言えば、１μｌのヒト胎盤ポリＡ＋ＲＮＡ（１μｇ）、１μｌのランダム−Ｔ７プライマー（２０μＭ）、及び３μｌの脱イオン化Ｈ_２ Ｏを含む５μｌのｃＤＮＡ合成混合物を、７２℃で２分間及びそれから３７℃で２分間インキュベートした。体積を以下の試薬を用いて１０μｌに調節した：２μｌの５Ｘ第一鎖合成バッファー（２５０ｍＭのＴｒｉｓ−ＨＣｌ、ｐＨ８．３；３０ｍＭのＭｇＣｌ_２ ；及び３７５ｍＭのＫＣｌ）、０．５μｌのＤＴＴ（５０ｍＭ）、１μｌのｄＮＴＰ混合物（１０ｍＭの各ｄＡＴＰ、ｄＣＴＰ、ｄＧＴＰ、ｄＴＴＰ）、０．５μｌのＲｎａｓｉｎ（２０単位、プロメガ）、１μｌ（２００単位）のＭＭＬＶ逆転写酵素（ＳｕｐｅｒｓｃｒｉｐｔＩＩ（登録商標）、Ｌｉｆｅ　Ｔｅｃｈｎｏｌｏｇｉｅｓ）。逆転写を４２℃にて９０分間行った。反応を氷上にチューブを静置することにより終了させた。
【０２２１】
ホモポリマー配列化は以下の改変を伴って、先に報告されたように（Ｄ．Ｊ．Ｂｅｒｔｉｏｌｉ等．，　ＢｉｏＴｅｃｈｎｏｌｏｇｙ，１９９４）行った。２０μｌの脱イオン化Ｈ_２ Ｏを第一鎖ｃＤＮＡ混合物へ添加した。希釈した第一鎖混合物を、ＣＨＲＯＭＡ　ＳＰＩＮ−１００カラム（Ｃｌｏｎｔｅｃｈ）を使用して精製し、小さなｃＤＮＡフラグメント同様にプライマー及びｄＮＴＰを除去した。オリゴ−ｄＣ配列化を以下を混合することにより行った：３０μｌの精製第一鎖ｃＤＮＡ；１μｌの５Ｘ配列化バッファー（０．５Ｍカリウムカコジレート、ｐＨ７．２、１０ｍＭ　ＣｏＣｌ_２ 、１ｍＭ　ＤＴＴ）、１μｌの１ｍＭ　ｄＣＴＰ、１μｌの末端トランスフェラーゼ（１５単位／μｌ；Ｌｉｆｅ　Ｔｅｃｈｎｏｌｉｇｉｅｓ）、８μｌの脱イオン化Ｈ_２ Ｏ（最終体積は５０μｌ）。反応混合物を３７℃で１時間インキュベートし、続けてフェノール／クロロホルム抽出及び析出を行った。
【０２２２】
オリゴ−ｄＣ−配列化ｃＤＮＡを約１０ｐｇ／μｌ（約１×１０^−５）まで希釈し、希有な転写物を除去し、続けてＰＣＲを、４０ｍＭ　Ｔｒｉｃｉｎｅ−ＫＯＨ、ｐＨ９．２；１５ｍＭ　ＫＯＡｃ；３．５ｍＭ　Ｍｇ（ＯＡｃ）_２；及び０．２μＭの５’ −ｄＧ_１２プライマー（５’ −ｄ（Ｇ）_１２ −ＶＮ−３’ 、Ｎ＝Ａ、Ｇ，Ｃ又はＴ；Ｖ＝Ａ、Ｇ又はＣ）；０．２μＭのランダム−Ｔ７プライマー；０．２ｍＭの各ｄＡＴＰ、ｄＧＴＰ、ｄＣＴＰ、ｄＴＴＰ；２μｌのＡｄｖａｎｔａｇｅ（登録商標）ｃＤＮＡポリメラーゼ混合物（５０Ｘ；ＫｌｅｎＴａｑ−１及びＤｅｅｐ　Ｖｅｎｔポリメラーゼ；Ｃｌｏｎｔｅｃｈ）を含む１００μｌ反応中で行った。ＰＣＲを以下の循環条件を使用して、ＤＮＡ　Ｔｈｅｒｍａｌ　Ｃｙｃｌｅｒ　４８０（ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ）中で行った：９５℃で１分間；９５℃で１５秒間　３０−３５サイクル；５２℃で３０秒；及び７２℃で６分間。
【０２２３】
ＰＣＲ後、二本鎖ｃＤＮＡをＳ−２００スピンカラム（Ｃｌｏｎｔｅｃｈ）を通すことにより精製した。アンチセンスＲＮＡドライバー合成の前に、二本鎖ｃＤＮＡを（Ｇ３ＰＤＨ）及びｃ−ｍｙｃ遺伝子の増幅について試験した。ＰＣＲ増幅の２５サイクル後、Ｇ３ＰＤＨをエチジウムブロマイドを用いて染色された１．１％アガロースゲル上の濃い色のバンドとして視覚化した。ｃ−ｍｙｃは増幅の３５サイクル後でさえ、エチジウムブロマイド染色ゲル上で視覚化できなかった。
【０２２４】
Ｂ．　アンチセンスＲＮＡ合成
アンチセンスＲＮＡを以下を混合することにより合成した：上記二本鎖ｃＤＮＡ（１μｇ）；５ＸＲＮＡ翻訳バッファー（２００ｍＭのＴｒｉｓ−ＨＣｌ、ｐＨ８．５；６０ｍＭのＭｇＣｌ_２ ；及び３５０ｍＭのＫＣｌ；２５ｍＭのＤＴＴ；２．５ｍＭのＩＴＰ；７．５ｍＭのＧＴＰ；１０ｍＭのＡＴＰ；１０ｍＭのＵＴＰ；１０ｍＭのＣＴＰ）そして、それから４０単位のＲＮａｓｉｎ（プロメガ）及び８０単位のＴ７　ＲＮＡポリメラーゼ（Ｂｏｅｈｒｉｎｇｅｒ　Ｍａｎｎｈｅｉｍ）を加えた。Ｈ_２ Ｏを体積５０μｌの最終反応物に加えた。混合物を、４１℃で９０分間インキュベートした。それから１０単位のＤＮａｓｅ　Ｉ（ＲＮａｓｅフリー；Ｂｏｅｈｒｉｎｇｅｒ　Ｍａｎｎｈｅｉｍ）を加えた。この反応物を３７℃で１５分間インキュベートし、続けてフェノール／クロロホルム抽出及び析出を行った。
【０２２５】
Ｃ．　刪減ハイブリダイゼーション
刪減ハイブリダイゼーションを（高存在量シーケンスに富む）ヒト胎盤アンチセンスＲＮＡをドライバーとして使用して、並びに正常なヒト胎盤ｍＲＮＡ（アンチセンスＲＮＡと同起源）を“テスター”として使用して行った、第１表に、各混合物中のアンチセンスＲＮＡと胎盤ｍＲＮＡの種々の量を列挙する。
【０２２６】

混合後、チューブをＰＥ９６００　Ｔｈｅｒｍａｌ　Ｃｙｃｌｅｒ中で６８℃で２０分インキュベートした。それから温度を１６ｈインキュベーション中に４５℃まで上昇させた。
【０２２７】
Ｄ．　ｃＤＮＡ合成
（上記）試料Ａ、Ｂ、Ｃ及びＤを、オリゴｄ（Ｔ）_３０ プライマー（５’ −ｄ（Ｔ）_３０ −ＶＮ−３’ 、Ｎ＝Ａ、Ｇ、Ｃ又はＴ；Ｖ＝Ａ、Ｇ又はＣ）を第一鎖ｃＤＮＡ合成用の鋳型として使用した。短く言えば、２μｌの１０ｍＭ　オリゴｄ（Ｔ）_３０ を、各試料に加え、続けて７２℃で２分間インキュベートした。それから反応混合物を即座に氷浴に２分間移した。体積を以下の試薬を用いて１０μｌに調節した：２μｌの５Ｘ第一鎖合成バッファー（２５０ｍＭのＴｒｉｓ−ＨＣｌ、ｐＨ８．３；３０ｍＭのＭｇＣｌ_２ ；及び３７５ｍＭのＫＣｌ）、０．５μｌのＤＴＴ（５０ｍＭ）、１μｌのｄＮＴＰ混合物（１０ｍＭの各ｄＡＴＰ、ｄＣＴＰ、ｄＧＴＰ、ｄＴＴＰ）、０．５μｌのＲｎａｓｉｎ（２０単位、プロメガ）、１μｌ（２００単位）のＭＭＬＶ逆転写酵素（ＳｕｐｅｒｓｃｒｉｐｔＩＩ（登録商標）、Ｌｉｆｅ　Ｔｅｃｈｎｏｌｏｇｉｅｓ）。逆転写を４２℃にて９０分間行った。反応を、氷上にチューブを静置することにより終了させた。
【０２２８】
Ｅ．　オリゴ−ｄＧ配列化
第一鎖合成後、ＲＮＡを１００ｍＭ　１μｌのＮａＯＨを各第一鎖合成混合物に加え及び６８℃で３０分間インキュベートすることにより分解した。それから１９μｌの脱イオン化Ｈ_２ Ｏを各第一鎖混合物へ加え、そしてＤＮＡをＣｈｒｏｍａ　Ｓｐｉｎ　１００（登録商標）カラム（Ｃｌｏｎｔｅｃｈ）を使用して精製した。オリゴ−ｄＧ配列化を以下のものを混合することにより行った：３０μｌの精製第一鎖ｃＤＮＡ；１μｌの５Ｘ配列化バッファー（０．５Ｍカリウムカコジレート、ｐＨ７．２、１０ｍＭ　ＣｏＣｌ_２ 、１ｍＭ　ＤＴＴ）、１μｌの１ｍＭ　ｄＧＴＰ、１μｌの末端トランスフェラーゼ（１５単位／μｌ；Ｌｉｆｅ　Ｔｅｃｈｎｏｌｉｇｉｅｓ）、８μｌの脱イオン化Ｈ_２ Ｏ（最終体積は５０μｌ）。反応混合物を３７℃で１時間インキュベートし、続けてフェノール／クロロホルム抽出及び析出を行った。
【０２２９】
Ｆ．　低サイクルＰＣＲによる二本鎖ｃＤＮＡ合成及びＰＣＲ生成物の分析
１．　０．９ｋｂ胎盤転写物：
試料Ａ−Ｄにおける低存在量シーケンスの刪減を、０．９ｋｂ胎盤“ハウスキーピング” 転写物に相当するｃＤＮＡのレベルを検査することにより行った。低サイクルＰＣＲ増幅を、先の段階で得られたペレットを、４０ｍＭ　Ｔｒｉｃｉｎｃ−ＫＯＨ、ｐＨ９．２；１５ｍＭ　ＫＯＡｃ；３．５ｍＭ　Ｍｇ（ＯＡｃ）_２；及び０．２μＭの５’ −ｄＣ_１２プライマー（５’ −ｄ（Ｃ）_１２ −ＶＮ−３’ 、Ｎ＝Ａ、Ｇ，Ｃ又はＴ；Ｖ＝Ａ、Ｇ又はＣ）；０．２μＭのオリゴｄ（Ｔ）_３０ プライマー；０．２ｍＭの各ｄＡＴＰ、ｄＧＴＰ、ｄＣＴＰ、ｄＴＴＰ；２μｌのＡｄｖａｎｔａｇｅ（登録商標）ｃＤＮＡポリメラーゼ混合物（５０Ｘ；ＫｌｅｎＴａｑ−１及びＤｅｅｐ　Ｖｅｎｔポリメラーゼ；Ｃｌｏｎｔｅｃｈを含む）を含む１００μｌ反応物中に溶解させることにより行った。ＰＣＲを以下の循環条件を使用して、ＤＮＡ　Ｔｈｅｒｍａｌ　Ｃｙｃｌｅｒ　４８０（ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ）中で行った：９５℃で１分間；９５℃で１５秒間　８サイクル；５２℃で３０秒；及び７２℃で６分間。
【０２３０】
試料Ａ、Ｂ、Ｃ及びＤからのＰＣＲ生成物を、２００ｎｇ　１ｋｂ　ＤＮＡラダー（Ｌｉｆｅ　Ｔｅｃｈｎｏｌｏｇｉｅｓ）とともに、１．１％アガロースゲル（５μｌ／ウェル）上に充填した。エチジウムブロマイドを用いた染色は、胎盤ｍＲＮＡに対するアンチセンスｍＲＮＡドライバーの比とともにバンディングプロファイル（ｂａｎｄｉｎｇ　ｐｒｏｆｉｌｅ）が変化したことを表した。特に、ヒト胎盤ハウスキーピング遺伝子に相当する０．９ｋｂにおけるバンドの強度は、アンチセンスＲＮＡ／ｍＲＮＡの比の増加とともに減少し、試料Ｄにおいてはほとんど検出できなかった、それはアンチセンスＲＮＡドライバーが高存在量ＲＮＡシーケンスに対してハイブリダイズし、そしてそれらを刪減することを示す。
【０２３１】
２．　グルコース３−ホスフェート脱水素酵素（Ｇ３ＰＨＤ）：
試料Ａ−Ｄ中の高存在量シーケンスの刪減も、ハウスキーピング遺伝子Ｇ３ＰＨＤに相当するｃＤＮＡのレベルを検査することにより行った。試料Ａ、Ｂ、Ｃ及びＤを、４０ｍＭ　Ｔｒｉｃｉｎｅ−ＫＯＨ、ｐＨ９．２；１５ｍＭ　ＫＯＡｃ；３．５ｍＭ　Ｍｇ（ＯＡｃ）_２；及び０．２μＭの５’ 及び３’ Ｇ３ＰＨＤ　Ａｍｐｌｉｍｅｒセット（Ｃｌｏｎｔｅｃｈ）；０．２ｍＭの各ｄＡＴＰ、ｄＧＴＰ、ｄＣＴＰ、ｄＴＴＰ；２μｌのＡｄｖａｎｔａｇｅ（登録商標）ｃＤＮＡポリメラーゼ混合物（５０Ｘ；ＫｌｅｎＴａｑ−１及びＤｅｅｐ　Ｖｅｎｔポリメラーゼ；Ｃｌｏｎｔｅｃｈを含む）を含む５０μｌの反応混合物中でＰＣＲ増幅した。ＰＣＲを以下の循環条件を使用して、ＤＮＡ　Ｔｈｅｒｍａｌ　Ｃｙｃｌｅｒ　４８０（ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ）中で行った：９５℃で１分間；９５℃で１５秒間　２５サイクル；６８℃で２分間。
【０２３２】
試料Ａ、Ｂ、Ｃ及びＤからのＰＣＲ生成物を、２００ｎｇ　１ｋｂ　ＤＮＡラダー（Ｌｉｆｅ　Ｔｅｃｈｎｏｌｏｇｉｅｓ）とともに、１．１％アガロースゲル（５μｌ／ウェル）上に充填した。エチジウムブロマイドを用いた染色は、ハウスキーピング遺伝子Ｇ３ＰＨＤに相当するバンドの濃い色がアンチセンスＲＮＡ／ｍＲＮＡの比の増加とともに減少し、試料Ｄにおいてはほとんど検出できず、それはアンチセンスＲＮＡが高存在量ｍＲＮＡシーケンスを刪減することが確認される。
【０２３３】
３．　ｃ−ｍｙｃ：
ｃ−ｍｙｃ遺伝子をマーカーとして使用し、試料Ａ−Ｄにおける低存在量シーケンスの富化をアッセイした。各試料をＧ３ＰＨＤの研究で記載したようにＰＣＲ増幅したが、５’ 及び３’ ｃ−ｍｙｃ　Ａｍｐｌｉｍｅｒセット（Ｃｌｏｎｔｅｃｈ）をプライマーとして使用し、そして以下の循環条件を使用してＰＣＲを行った：９５℃で１分間；９５℃で１５秒間　３０サイクル；６８℃で２分間。
【０２３４】
試料Ａ、Ｂ、Ｃ及びＤからのＰＣＲ生成物を、２００ｎｇ　１ｋｂ　ＤＮＡラダー（Ｌｉｆｅ　Ｔｅｃｈｎｏｌｏｇｉｅｓ）とともに、１．１％アガロースゲル（５μｌ／ウェル）上に充填した。エチジウムブロマイドを用いた染色は、ｃ−ｍｙｃ表示の濃い色がアンチセンスＲＮＡ／ｍＲＮＡの比の増加とともに減少することを表した。この結果はｃ−ｍｙｃが正常なヒト胎盤ＲＮＡ中に存在するが、アンチセンスＲＮＡドライバー中には存在しないことを示す、そして刪減ハイブリダイゼーション、後に続く増幅はｃ−ｍｙｃのような低存在量シーケンスに富むポリヌクレオチドプールを生成する。
【０２３５】
例２
種々の組織から製造されるｍＲＮＡからの低存在量シーケンスに富むｃＤＮＡの製造
本発明の方法の再現性を試験するために、アンチセンスＲＮＡドライバーを、ヒト脳、胎児脳、肝臓及び腎臓ｍＲＮＡから、例１に記載されるように生成させた。各組織からのｍＲＮＡ試料は、同じ組織（Ａ群）から製造されるアンチセンスＲＮＡドライバーを用いた刪減ハイブリダイゼーションを受けた。ネガティブコントロール（すなわちアンチセンスＲＮＡで処理されない）が各試料（Ｂ群）について含まれた。刪減後、低サイクルＰＣＲを行い、ハイブリダイズしていないｍＲＮＡシーケンスに相当する二本鎖ｃＤＮＡが発生させた。
【０２３６】
Ｐ５０遺伝子をマーカーとして使用し、低存在量シーケンスの富化をアッセイした。各試料を４０ｍＭ　Ｔｒｉｃｉｎｅ−ＫＯＨ、ｐＨ９．２；１５ｍＭ　ＫＯＡｃ；３．５ｍＭ　Ｍｇ（ＯＡｃ）_２；及び０．２μＭの５’ 及び３’ Ｐ５０　Ａｍｐｌｉｍｅｒセット（Ｃｌｏｎｔｅｃｈ）；０．２ｍＭの各ｄＡＴＰ、ｄＧＴＰ、ｄＣＴＰ、ｄＴＴＰ；２μｌのＡｄｖａｎｔａｇｅ（登録商標）ｃＤＮＡポリメラーゼ混合物（５０Ｘ；ＫｌｅｎＴａｑ−１及びＤｅｅｐ　Ｖｅｎｔポリメラーゼ；Ｃｌｏｎｔｅｃｈを含む）を含む５０μｌの反応物中で増幅した。ＰＣＲを以下の循環条件を使用して、ＤＮＡ　Ｔｈｅｒｍａｌ　Ｃｙｃｌｅｒ　４８０（ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ）中で行った：９５℃で１分間；９５℃で１５秒間　３０サイクル；６８℃で２分間。
【０２３７】
ＰＣＲ生成物の分析を、１．１％アガロースゲル（５μｌ／ウェル）上における電気泳動により行い、続けてエチジウムブロマイドを用いた染色によりＡ群試料（低存在量シーケンスに富む）がＰ５０シーケンスを含むことを表したが、Ｂ群試料（ネガティブコントロール）は検出可能なＰ５０シーケンスを示さなかった。結果は本発明の方法が組織の型にかかわらず選択されたポリヌクレオチドプールを製造するために効果的であることを示す。
【０２３８】
例３
ヒト睾丸ｍＲＮＡから製造される変化する長さの低存在量シーケンスに富むｃＤＮＡの生成
変化する長さの低存在量シーケンスの富化について試験するために、アンチセンスＲＮＡドライバーをヒト睾丸ｍＲＮＡから例１に記載されるように生成させた。ヒト睾丸ｍＲＮＡ試料は、アンチセンスＲＮＡドライバーを用いた刪減ハイブリダイゼーションを受け（Ａ群試料を発生させ）た。ネガティブコントロール（すなわちアンチセンスＲＮＡで処理されない）を含んだ（Ｂ群試料を発生させた）。刪減後、低サイクルＰＣＲを行い、ハイブリダイズしていないｍＲＮＡシーケンスに相当する二本鎖ｃＤＮＡを生成した。
【０２３９】
インターロイキン−６（ＩＬ６、０．６ｋｂ　ＯＲＦフラグメント）、Ｐ５０（２．８ｋｂ　ＯＲＦ）及びインスリン成長因子レセプター（ＩＧＦＲ；４．８ｋｂ　ＯＲＦフラグメント）をマーカーとして使用し、低存在量遺伝子富化及びサイズ表示をチェックした。Ａ及びＢ試料を、４０ｍＭ　Ｔｒｉｃｉｎｅ−ＫＯＨ、ｐＨ９．２；１５ｍＭ　ＫＯＡｃ；３．５ｍＭ　Ｍｇ（ＯＡｃ）_２；及び０．２μＭの５’ 及び３’ ＩＬ６−，Ｐ５０又はＩＧＦＲ特異的プライマーセット（Ｃｌｏｎｔｅｃｈ）；０．２ｍＭの各ｄＡＴＰ、ｄＧＴＰ、ｄＣＴＰ、ｄＴＴＰ；２μｌのＡｄｖａｎｔａｇｅ（登録商標）ｃＤＮＡポリメラーゼ混合物（５０Ｘ；ＫｌｅｎＴａｑ−Ｉ及びＤｅｅｐ　Ｖｅｎｔポリメラーゼ；Ｃｌｏｎｔｅｃｈを含む）を含む５０μｌの反応物中でＰＣＲ増幅した。ＰＣＲを以下の循環条件を使用して、ＤＮＡ　Ｔｈｅｒｍａｌ　Ｃｙｃｌｅｒ　４８０（ＰＥ　Ｂｉｏｓｙｓｔｅｍｓ）中で行った：９５℃で１分間；９５℃で１５秒間　３０サイクル；６８℃で５分間。
【０２４０】
ＰＣＲ生成物の分析を、１．１％アガロースゲル（５μｌ／ウェル）上で電気泳動させることにより行い、続けてエチジウムブロマイドを用いた染色によりＡ群試料（低存在量シーケンスに富む）がＩＬ６、Ｐ５０又はＩＧＦＲシーケンスに相当するバンドを含むことを表したが、Ｂ群試料（ネガティブコントロール）はこれらのシーケンスに相当する検出可能なバンドを示さなかった。結果は本発明の方法が変化するサイズの低存在量転写物について富化することをを示す。
【０２４１】
例４
試験及び参照試料間で異なる低存在量ポリヌクレオチドを同定するための好適な刪減ハイブリダイゼーション方法
この例は、本発明の一般的な刪減ハイブリダイゼーション方法の好適な具体例についてのプロトコルを提供する。この具体例は、高存在量富化試験ポリヌクレオチドプール及び低存在量富化参照ポリヌクレオチドプールを使用し、それらは本発明の例示的な選択方法により生成される。
【０２４２】
Ａ．　高存在量シーケンスに富むアンチセンス試験ＲＮＡプールの製造
高存在量富化アンチセンスＲＮＡプールを、図１に示し及び以下に記載するように試験ＲＮＡ試料から製造する。
１．　第一鎖（アンチセンス）試験ｃＤＮＡを、逆転写酵素及びアンチセンスプライマー複合物の混合物を使用して試験ＲＮＡ試料から合成する。各アンチセンスプライマー複合物はランダムシーケンスを含み、それはアンチセンスプライマーとして働く。Ｔ７　ＲＮＡプロモーターシーケンスをランダムシーケンスの５’ 末端にリンクさせる。
２．　ユニバーサルプライマーサイトをオリゴヌクレオチド配列化によりアンチセンス試験ｃＤＮＡ鎖の３’ 末端に加える。この例において、ｄＧ配列化をポリ−Ｇ配列を製造するために使用する。
３．　段階２の反応混合物を連続的に希釈し、低存在量シーケンスを除去する。このようなシーケンスの除去を上述の例のようにＰＣＲによりモニターする。
４．　それから希釈した反応混合物を、新たに合成されるアンチセンス試験ｃＤＮＡ鎖の３’ ユニバーサルプライマーサイトに対してハイブリダイズする５’ ユニバーサルプライマーを使用して、それからＰＣＲを受けさせる。図１Ｂ（４）に表されるように、３’ プライマーはアンチセンス試験ｃＤＮＡ鎖の５’ 末端においてＴ７　ＲＮＡプロモーターシーケンスに対してハイブリダイズするものである。ＰＣＲは高存在量シーケンスに富む二本鎖試験ｃＤＮＡ分子のプールを生成する。
５．　アンチセンス試験ＲＮＡを、Ｔ７　ＲＮＡポリメラーゼを使用して高存在量シーケンスに富むｃＤＮＡ分子から合成し、手順Ｃ（段階Ｃ．１、以下）の第一刪減ハイブリダイゼーションにおける使用のためのドライバーを生成させる。このドライバーは高存在量試験ＲＮＡシーケンスに富む。
【０２４３】
Ｂ．　低存在量シーケンスに富むセンス参照ｃＤＮＡ鎖のプールの製造
アンチセンス参照ＲＮＡを、Ａ．５（上記）においてアンチセンス試験ＲＮＡについて記載されたように製造する。高存在量シーケンスに富むアンチセンス参照ＲＮＡは、図２に示し及び以下説明するように、センス参照ｃＤＮＡ鎖の低存在量富化プールを生成することに使用される。
１．　アンチセンス参照ＲＮＡを、参照ｍＲＮＡを用いる刪減ハイブリダイゼーション反応におけるドライバーとして使用し、高存在量ｍＲＮＡをブロックし、低存在量参照ｍＲＮＡ分子をフリーなままにし、第一鎖ｃＤＮＡ合成のための鋳型として働かせる。第一鎖（アンチセンス）参照ｃＤＮＡは、逆転写酵素及び５’ 末端においてＳＰ６　ＲＮＡプロモーターシーケンスに対してリンクするオリゴ−ｄＴシーケンスを含むアンチセンスプライマー複合物を使用して合成する。（これらのプライマー及びプロモーターシーケンスは上記Ａ．１．において使用されるものと異ならなければならない。）
２．　ユニバーサルプライマーサイトをオリゴヌクレオチド配列化によりアンチセンス参照ｃＤＮＡ鎖の３’ 末端に加える。ユニバーサルプライマーサイトは上記Ａ．２．にてアンチセンス試験ｃＤＮＡ鎖に加えられるものとは異ならなくてはならない、及びそれゆえｄＣ配列化はポリｄＣ配列を生成するために使用される。
３．　それからＢ．２．の反応混合物を、１００ｍＭのＮａＯＨを用いて６８℃、６０分間処理し、参照ｍＲＮＡを分解する。低存在量富化アンチセンス参照ｃＤＮＡ鎖を残す。
４．　それからアンチセンス参照ｃＤＮＡ鎖を、３’ ユニバーサルプライマーサイトに対してハイブリダイズする５’ ユニバーサルプライマー（ここではポリ−Ｇ）、並びにＳＰ６プロモーターシーケンスにリンクするアンチセンス参照ｃＤＮＡ（ここではポリ−Ａ）の５’ アンチセンスプライマーサイトに対してハイブリダイズする３’ アンチセンスプライマーを使用してＰＣＲを受けさせる。ＰＣＲは、低存在量シーケンスに富む二本鎖参照ｃＤＮＡ分子のプールを生成する。
５．　アンチセンス参照ＲＮＡを、低存在量富化参照ｃＤＮＡ分子から、ＳＰ６ポリメラーゼを使用して合成する。
６．　センス参照ｃＤＮＡ鎖を、アンチセンス参照ＲＮＡから、逆転写酵素及び５’ 末端に制限サイト（例えばポリ−ＧにリンクするＡｌｕ　Ｉ）を含む５’ ユニバーサルプライマーを使用して合成する。この制限サイトは、二本鎖ＤＮＡに特異的な酵素により開裂されるものである。この態様は（以下の）Ｃの一般的ハイブリダイゼーション方法の第二ハイブリダイゼーション反応後にハイブリダイズされたシーケンスの選択的な開裂を容易化する。
７．　それからＢ．６の反応混合物を１００ｍＭのＮａＯＨを使用して、６８℃で６０分間処理し、アンチセンス参照ＲＮＡを分解し、センス参照ｃＤＮＡを残す。
８．　結果として生じる参照ｃＤＮＡ鎖は低存在量シーケンスに富み、Ｃの一般的な刪減ハイブリダイゼーション方法の第二刪減ハイブリダイゼーションにて使用できる。ポリ−Ａ及びポリ−Ｔシーケンス間の非特異的ハイブリダイゼーションを減少させるために、センス参照ｃＤＮＡ鎖のポリ−Ａ配列を、本発明の第二刪減ハイブリダイゼーション反応にて使用する前に、Ｅ．ｃｏｌｉエキソヌクレアーゼＩを用いた部分消化により除去する。好適な反応条件を、（例えば約５〜約３分の複数の反応を設定し、そしてオリゴヌクレオチド−ｄＴプライマーを使用して増幅することによりポリ−Ａ除去をモニタリングすることを使用することによって）経験的に決定する。
【０２４４】
Ｃ．　試験及び参照ポリヌクレオチド間の一般的な刪減ハイブリダイゼーション
１．　試験ｍＲＮＡを用いた第一刪減ハイブリダイゼーション反応において、Ａ．５．のアンチセンス試験ＲＮＡをドライバーとして使用し、高存在量試験ｍＲＮＡ分子をブロックし、第一鎖ｃＤＮＡ合成用の鋳型として働かせるために、低存在量試験ｍＲＮＡ分子をフリーなままにしておく。低存在量富化第一鎖（アンチセンス）試験ｃＤＮＡを、ハイブリダイズしていない試験ｍＲＮＡ分子から、逆転写酵素並びにオリゴｄＴシーケンスとオリゴｄＴシーケンスの２種の制限サイト５’ とを含むアンチセンスプライマーを使用して、合成する（プライマーシーケンスはＢ．２．にて使用されるものとは異なるものでなければならないが、Ａ．１．にて使用されるものとは同じか又は異なるものであることができる）。１つの制限サイト（例えばＡｌｕ　Ｉ）は二本鎖ＤＮＡについて特異的な酵素により開裂される。この態様は、（下記）方法Ｃの一般的なハイブリダイゼーションの第二ハイブリダイゼーション反応後に、ハイブリダイズされるシーケンスの選択的な開裂を容易化するために使用する。他の制限サイトは、試験特異的ポリヌクレオチドを第二ハイブリダイゼーション反応後にクローニングベクター内へクローン化するために使用する。
２．　それからＣ．２．の反応混合物を、１００ｍＭのＮａＯＨを用いて６８℃、６０分間処理し、試験ｍＲＮＡを分解する。低存在量富化アンチセンス試験ｃＤＮＡ鎖を残す。
３．　Ｂ．７．のセンス参照ｃＤＮＡを、段階３のアンチセンス試験ｃＤＮＡ鎖を用いた刪減ハイブリダイゼーション反応におけるドライバーとして使用し、試験及び参照試料の両方において存在するポリヌクレオチドシーケンスに相当するアンチセンス試験ｃＤＮＡ鎖をブロックする。この第二ハイブリダイゼーション反応は、二本鎖ｃＤＮＡ分子、ハイブリダイズしていない低存在量富化アンチセンス試験ｃＤＮＡ鎖及びハイブリダイズしていない低存在量富化センス参照ｃＤＮＡ鎖（すなわちドライバー）を生成する。ハイブリダイズしていないアンチセンス試験ｃＤＮＡ鎖は試験特異的なシーケンスに相当するが、ハイブリッド二本鎖は、試験及び参照試料の両方において存在するシーケンスに相当する。
４．　ハイブリッド二本鎖の“粘着末端” を、Ｔａｑポリメラーゼを使用して充填し、二本鎖Ａｌｕ　Ｉ制限サイトを発生させる。
５．　オリゴヌクレオチド配列化をｄＣを使用して実行し、反応混合物中の全ての分子にポリ−Ｃ配列を加える。
６．　ハイブリッド二本鎖をＡｌｕ　Ｉを用いて消化し、それは二本鎖の両方の末端において３’ ポリ−Ｃ配列を開裂する。この反応はハイブリダイズしていない低存在量富化アンチセンス試験ｃＤＮＡ鎖、及びハイブリダイズしていない低存在量富化センス参照ｃＤＮＡ鎖（すなわちドライバー）を完全なまま残す。しかし、ドライバーは自己アニールする５’ ポリ−Ｇ及び３’ ポリ−Ｃシーケンスを有し、増幅を妨げる。
７．　低存在量富化アンチセンス試験ｃＤＮＡ鎖を、好適なプライマーを使用する選択的なＰＣＲにより二本鎖試験ｃＤＮＡ分子に変換する。この例において、５’ プライマーはポリ−Ｇを含み、３’ プライマーはアンチセンス試験ｃＤＮＡ鎖の５’ 末端にてＳｆｉ　Ｉシーケンスと結合するＳｆｉ　Ｉシーケンスを含む。５’ プライマーも５’ 末端に制限サイトを含む（ここではＮｏｔ　Ｉ）。ＰＣＲは低存在量試験シーケンス並びに出発試験及び参照ＲＮＡ試料間の異なるシーケンスに富む刪減ｃＤＮＡ分子のプールを生成する。各二本鎖分子は５’ 末端においてＮｏｔ　Ｉ制限サイトを有し、そして３’ 末端においてＳｆｉ　Ｉ制限サイトを有する。
８．　７の混合物をＳｆｉ　Ｉ及びＮｏｔ　Ｉを使用して消化させ、好適なベクター内へライゲートし、低存在量富化の試験特異的なクローンのライブラリーを生成する。Ｓｆｉ　Ｉ及びＮｏｔ　Ｉは“インフリークエントカッター（ｉｎｆｒｅｑｕｅｎｔ　ｃｕｔｔｅ）” であり、ほとんど全ての試験特異的なシーケンスを完全なままにし、それゆえ完全長ｃＤＮＡの回収を高める。この手順における２種の異なる制限サイトの使用は、発現ベクターへ指向的なクローニングを容易化し、続けて発現された遺伝子の迅速な分析を容易化する。
【図面の簡単な説明】
【図１Ａ】ポリヌクレオチド試料に比べて高存在量シーケンスに富むポリヌクレオチドのプールを製造するために有用である本発明の選択方法の好適な具体例の概略図である。
【図１Ｂ】ポリヌクレオチド試料に比べて高存在量シーケンスに富むポリヌクレオチドのプールを製造するために有用である本発明の選択方法の好適な具体例の概略図である。
【図２Ａ】本発明の第二の選択方法の好適な具体例の概略図である。
【図２Ｂ】本発明の第二の選択方法の好適な具体例の概略図である。
【図２Ｃ】本発明の第二の選択方法の好適な具体例の概略図である。
【図３Ａ】本発明の一般的刪減ハイブリダイゼーション方法の好適な具体例の概略図である。
【図３Ｂ】本発明の一般的刪減ハイブリダイゼーション方法の好適な具体例の概略図である。
【図３Ｃ】本発明の一般的刪減ハイブリダイゼーション方法の好適な具体例の概略図である。
【図３Ｄ】本発明の一般的刪減ハイブリダイゼーション方法の好適な具体例の概略図である。[0001]
Related application information
This application is a continuation-in-part of application Ser. No. 09 / 632,898 (filed on Aug. 7, 2000) and is a continuation-in-part of application No. 06 / 288,777 (filed on May 4, 2001). Applications, both of which are fully incorporated herein by reference.
[0002]
Field of the invention
The present invention relates to a method and a kit for selecting a polynucleotide pool from a sample, and a selected polynucleotide pool produced thereby. In particular, the present invention provides methods for producing polynucleotide pools that are rich in high-abundance sequences as compared to samples, and that are rich in low-abundance sequences using such polynucleotide pools. The present invention provides a subtractive hybridization reaction for producing a polynucleotide pool. The present invention further provides a general method of reducing hybridization in which a reduced polynucleotide pool is prepared from test and reference polynucleotide samples.
[0003]
Background of the Invention
Biologically active proteins are the subject of intense investigation as candidates for therapeutic, diagnostic and other applications. The first step in these attempts is typically the cloning of a gene encoding a protein from messenger RNA (mRNA). The mRNAs of human and other mammalian cells have three frequency classes: (1) high abundance sequences representing about 10-20% of the total mRNA population; (2) about 40-45% of mRNA. (3) can be divided into low abundance sequences representing the other 40-45% of the mRNA. Genes encoding proteins with important regulatory functions, such as hormones and their receptors, are expressed at low levels, and the corresponding transcripts fall into the low abundance class of the sequence.
[0004]
Attempts to clone low abundance sequences have used standardized cDNA libraries where the frequency of all clones in the library is within a very narrow range. However, this approach does not address the loss of low abundance sequences in the process of generating a cDNA library, which preferentially clones high and medium abundance sequences as well as shorter sequences. A method that facilitates the selection of pooled low abundance polynucleotides from mRNA distribution without cloning losses and a method that provides a means to provide large amounts of such sequences will be useful in identifying key regulatory proteins. Greatly facilitates the intended search.
[0005]
Of particular interest are methods that facilitate the identification of low abundance polynucleotides that are differentially expressed between two samples. Attempts to identify such polynucleotides have generally used several variations of exclusion hybridization. In this technique, the two samples are typically two mRNA samples, a sample designated as a "test" sample containing important transcripts and a "reference" sample or " And a sample called “driver”. For example, the test sample consists of mRNA from tissue cells and the reference sample consists of mRNA from normal cells. Both samples are first converted to cDNA and then hybridized. The polynucleotide common to both samples forms a hybrid, which is removed in several ways, leaving a single stranded polynucleotide that differs between the two samples. Although traditional lip-reduction hybridization techniques have been successful in several cases, two major drawbacks of these techniques are that they are not suitable for identifying rare mRNAs, and that they are not suitable for full-length transcripts. That is not to produce. Full-length transcripts are essential for expressing the encoded protein and performing it in functional studies.
[0006]
In a traditional hybridization technique, mRNA samples are converted to cDNA and then digested with restriction enzymes that cut frequently to ensure that each cDNA molecule is "sticky ended". I do. The adapter is ligated onto the sticky end and the fragment is cloned into the vector. Digestion with frequently cleaving restriction enzymes needs to ensure that the resulting cDNA library represents as much of the original mRNA as possible, but it is desirable to generate truncated cDNA clones. Has no effect. Such clones can be used to probe additional libraries in an attempt to obtain full-length clones, and this is at best difficult and, typically, in all applicable libraries It is often wasteful in the case of low abundance transcripts that are poorly displayed or absent.
[0007]
Ideally, methods specifically intended to identify low abundance polynucleotides that differ between samples will replicate a wide range of copies without prior cloning into a vector and without the need for sequencing information. can do. Preferably, the low abundance polynucleotide provided by such a method represents a full-length copy (eg, a full-length cDNA clone). Finally, methods of facilitating the identification of low abundance polynucleotides that differ between the two samples are of particular interest.
[0008]
Summary of the Invention
The invention provides reduced hybridization to identify one or more polynucleotides in a test sample that are absent or low abundant in a reference sample. The method is particularly useful for identifying low abundance sequences that are differentially expressed between different samples. In a preferred embodiment, the test and reference polynucleotide samples are selected from one of the following:
(A) ｍ mRNA from a first cell or tissue and mRNA from a second different cell or tissue;
(B) mRNA in the first stage of differentiation or growth and mRNA in the second different stage of differentiation or growth,
(C) mRNA from cells or tissues treated with an active reagent and mRNA not treated or treated with a second different active reagent; and
(D) ｍ mRNA from normal cells or tissues and mRNA from diseased cells or tissues, for example, infection or cancer.
[0009]
The present method is used as a “driver” in a first subtraction hybridization reaction to remove high abundance sequences from a hybridization mixture, as a test enriched in high abundance polynucleotide sequences as compared to a test or reference polynucleotide sample. Alternatively, a high abundance-enriched polynucleotide chain from or produced from a pool of reference polynucleotides is used. In one embodiment, the high abundance-enriched polynucleotide strand is that of or produced from a pool of test polynucleotides. Preferably, the high abundance-enriched polynucleotide strand is a high abundance-enriched antisense polynucleotide chain. For example, if the test or reference polynucleotide sample is an mRNA sample, the high abundance-enriched antisense polynucleotide strand is preferably an antisense RNA molecule.
[0010]
This first reduction hybridization involves contacting the high abundance-enriched polynucleotide chain with a test polynucleotide sample or a test polynucleotide chain produced therefrom under conditions to form a first hybridization mixture. Need. The test polynucleotide strand is preferably a sense test polynucleotide strand, such as an mRNA molecule. This reaction produces an unhybridized test polynucleotide that is rich in low abundance polynucleotide sequences as compared to the test polynucleotide sample.
[0011]
Polynucleotide strands for use in the second deletion hybridization reaction are then synthesized from unhybridized test polynucleotide strands, thereby producing a low abundance-enriched test polynucleotide strand. These low abundance enriched test polynucleotide strands are preferably antisense test polynucleotide strands, such as antisense cDNA strands. The driver for this second subtraction hybridization reaction is a reference pool of polynucleotides rich in the low abundance polynucleotide sequence compared to the reference polynucleotide sample, or a low abundance-enriched reference polynucleotide produced therefrom. Is a chain. The low abundance-enriched reference polynucleotide strand is preferably a sense reference polynucleotide strand, eg, a sense cDNA strand.
[0012]
This second hybridization reaction removes "sequences" that are present (in approximately the same amount) in both samples, leaving unhybridized sequences that are differentially expressed. The reaction is performed by contacting the low abundance-enriched reference polynucleotide chain with the low abundance-enriched test polynucleotide chain under hybridization conditions to form a second hybridization mixture, thereby comprising: A hybrid duplex, an unhybridized low abundance-enriched reference polynucleotide chain and a low abundance-enriched test polynucleotide chain are generated.
[0013]
Hybrid duplexes representing a consensus sequence are removed or digested, and "test-specific duplexes" are generated from unhybridized, low abundance-enriched test polynucleotide strands. Preferably, the hybrid duplex is digested with at least one enzyme, such as a restriction endonuclease, and the test-specific duplex is produced by amplification from an unhybridized antisense test polynucleotide strand. .
[0014]
In a preferred embodiment, the antisense test polynucleotide strand is synthesized from the unhybridized sense test polynucleotide strand using a first antisense primer or first antisense primer complex. In a variation of this embodiment, the first antisense primer or first antisense primer complex is: (a) {a sequence that binds to a primer site in the unhybridized sense test polynucleotide chain; (b)} (a) A first restriction site 5 'of the sequence, wherein the first restriction site is cleaved by a restriction endonuclease that cleaves the double-stranded polynucleotide but leaves the single-stranded polynucleotide substantially intact; And (c) the first universal primer site 5 'of the restriction site of (b). In this variation of the invention, the antisense test polynucleotide strand preferably comprises a first universal primer site of (c) comprising a first antisense primer and a second restriction site from an unhybridized sense test polynucleotide strand. Wherein the second restriction site is different from the first restriction site. Further, the sense reference polynucleotide strand preferably includes a third restriction site previously added to the 5 'end of the sense reference polynucleotide strand, wherein the third restriction site cleaves the double-stranded polynucleotide but does It is cleaved by a restriction endonuclease that leaves the single-stranded polynucleotide substantially intact.
[0015]
After introducing a suitable restriction site within the polynucleotide strand that forms the hybrid duplex, digestion of the hybrid duplex,
(A) treating the second hybridization mixture with an enzyme that double-strands the single-stranded portion of the hybrid duplex, such that the double-stranded first, second and Generate three restriction sites;
(B) adding a second universal primer site to the 3 'end of the polynucleotide strand in the second hybridization mixture; and
(C) treating the second hybridization mixture with one or more restriction endonucleases, wherein the restriction endonuclease cleaves the hybrid duplex at the duplex first and third restriction sites. Leaving the antisense test polynucleotide strand and the sense reference polynucleotide strand substantially intact,
This is conveniently achieved by:
[0016]
The method of the invention generally involves cloning one or more test-specific duplexes into a vector. In particular, such methods provide for the construction of "test-specific" cDNA libraries. This library is superior to libraries created by other subtractive hybridization techniques in that it contains low abundance sequences that are usually lost using conventional techniques. In a preferred variation of this embodiment, the cloned test-specific duplex encodes a polypeptide, and the vector is an expression vector. The method of the invention further comprises introducing the expression vector into a host cell and expressing the protein encoded by the test-specific duplex.
[0017]
In one embodiment, the test-specific duplex is synthesized from an unhybridized antisense test polynucleotide strand using a first antisense primer conjugate, and the method further comprises testing the test-specific duplex. And synthesizing one or more antisense RNA molecules from the main strand. Antisense RNA molecules produced in this manner can be introduced into cells for research or therapeutic purposes.
[0018]
Test-specific duplexes produced according to the methods of the invention, or polynucleotides produced directly or indirectly therefrom, can also be used in hybridization reactions. Such polynucleotides can be labeled with a detectable label and / or can be added to a substrate to produce a polynucleotide array. If desired, one or more test-specific duplexes can be amplified. In a preferred embodiment, the amplification is performed using one or more gene-specific primers. Accordingly, the methods of the present invention include each of these applications of a test-specific duplex.
[0019]
In another embodiment, the invention provides a method for functionally isolating a single-stranded polynucleotide in a mixture of single- and double-stranded polynucleotides. The method requires contacting the mixture of single- and double-stranded polynucleotides with one or more restriction endonucleases under conditions sufficient to digest the double-stranded polynucleotide; For a nucleotide synthesis reaction using a single-stranded polynucleotide as a template, one that cannot be used as a template is formed. Preferably, the restriction endonuclease cleaves the primer site from the double-stranded polynucleotide in the mixture. After restriction digestion, the uncleaved single-stranded polynucleotides in the mixture are converted to double-stranded polynucleotides, preferably by amplification.
[0020]
Another aspect of the invention is a plurality of polynucleotides produced by subtractive hybridization between a test and a reference polynucleotide sample, wherein the plurality of polynucleotides comprises at least 10 polynucleotides.³ Includes different types of polynucleotides. The plurality of polynucleotides is substantially:
(A) either not present in the reference polynucleotide sample or present at a substantially lower concentration in the reference polynucleotide sample than in the test polynucleotide sample;
(B) low abundance compared to the test polynucleotide sample;
Rich in sequence. Each polynucleotide in the plurality of polynucleotides can include an RNA promoter sequence and a universal primer site. In a preferred embodiment, these polynucleotides are double-stranded cDNA or antisense RNA.
[0021]
The invention also provides kits that are useful for performing the methods of the invention and / or using the plurality of polynucleotides of the invention. The first kit includes the antisense primer or antisense primer complex and instructions for performing the general deletion hybridization method of the present invention. The antisense primer or antisense primer complex comprises:
(A) a sequence that binds to the primer site;
(B) the first restriction site 5 'of the sequence of (a), wherein the first restriction site cleaves the double-stranded polynucleotide but keeps the single-stranded polynucleotide substantially intact; Cleaved by; and
(C) the first universal primer site 5 'of the restriction site of (b)
including.
[0022]
A second kit includes: a plurality of polynucleotides of the invention; an antisense primer complex comprising an antisense primer operably linked to the RNA promoter sequence, wherein the RNA promoter sequence is 5 'of the antisense primer; Includes sense primer. A third kit comprises: a plurality of polynucleotides of the invention and an RNA polymerase capable of transcribing antisense RNA from the plurality of polynucleotides.
[0023]
The invention also provides a method of producing a selected polynucleotide pool from a polynucleotide sample. In a preferred embodiment, the selected polynucleotide pool is enriched in one or more abundant polynucleotides as compared to a polynucleotide sample. The method requires that a first antisense polynucleotide strand be synthesized from a polynucleotide sample or from a sense polynucleotide produced therefrom using an antisense primer conjugate. The antisense primer complex includes an antisense primer operably linked to an RNA promoter sequence, wherein the RNA promoter sequence is 5 'of the antisense primer. Next, a universal primer site is added to the 3 'end of the first antisense polynucleotide strand. The resulting first antisense polynucleotide strand is then diluted to substantially remove at least some low abundance first antisense polynucleotide strand. After dilution, a first double stranded polynucleotide is formed from the remaining first antisense polynucleotide strand. The first double-stranded polynucleotide is enriched in high abundance polynucleotide sequences as compared to a polynucleotide sample.
[0024]
In a preferred embodiment of the method, the polynucleotide sample is an mRNA sample, the first antisense polynucleotide strand is a first antisense cDNA strand, and the first double-stranded polynucleotide is a first double-stranded polynucleotide. Chain cDNA molecule. Synthesis of the first antisense cDNA strand can be primed using random primers or oligonucleotide-dT primers. Universal primer sites can be added to the 3 'end of the first antisense cDNA strand by template exchange, oligonucleotide sequencing or ligation. The RNA promoter sequence is one that is conveniently recognized by a bacteriophage RNA polymerase such as T7, T3 or SP6 polymerase.
[0025]
Preferably, the first double-stranded polynucleotide is produced by amplifying the first antisense polynucleotide strand remaining after dilution, and the amplification is performed by a universal primer that hybridizes as a 5 'primer to a universal primer site. And using an antisense primer conjugate as the 3 'primer. Most preferably, amplification is performed by an enhanced polymerase chain reaction. This reaction produces a pool of double-stranded polynucleotides that is rich in high abundance sequences compared to the original polynucleotide sample. The method optionally comprises synthesizing a first antisense RNA molecule from the first double-stranded polynucleotide. The pool of antisense RNA molecules is rich in high abundance sequences and can therefore be used as a "driver" in subtractive hybridization.
[0026]
The present invention also provides a method of using an antisense polynucleotide strand, preferably a high abundance-enriched antisense RNA molecule produced as described above, to generate a selected polynucleotide pool from a polynucleotide sample. In a preferred embodiment, the selected polynucleotide pool is enriched in one or more low abundance polynucleotides as compared to a polynucleotide sample. The method requires that the first antisense polynucleotide strand hybridize under hybridization conditions to a polynucleotide sample or a sense polynucleotide strand produced therefrom. Preferably, the molar ratio of the first antisense polynucleotide strand to the other polynucleotides in the hybridization mixture is from about 1 to about 100: 1.
[0027]
The resulting hybridization mixture contains unhybridized sense polynucleotide strands that are rich in low abundance polynucleotide sequences as compared to the polynucleotide sample. A second antisense polynucleotide strand is synthesized from the unhybridized sense polynucleotide strand using an antisense primer or an antisense primer complex. The antisense primer complex includes an antisense primer operably linked to an RNA promoter sequence, wherein the RNA promoter sequence is 5 'of the antisense primer. Antisense primer complexes are used when it is desired to generate a pool of selected polynucleotides, each containing an RNA promoter, and to facilitate the synthesis of antisense RNA from the selected polynucleotides. Is preferred.
[0028]
In addition, a universal primer site is added to the 3 'end of the second antisense polynucleotide strand. A second double stranded polynucleotide is then generated from the second antisense polynucleotide strand. This pool of polynucleotides is rich in low abundance polynucleotide sequences as compared to a polynucleotide sample.
[0029]
In a preferred embodiment of the method, the polynucleotide sample is an mRNA sample, the sense polynucleotide strand is an mRNA molecule, the second antisense polynucleotide strand is a second antisense cDNA strand, and the second A single-stranded polynucleotide is a second double-stranded cDNA molecule. Synthesis of the second antisense cDNA strand can be primed using an oligonucleotide-dT primer. Universal primer sites can be added to the 3 'end of the second antisense cDNA strand by template exchange, oligonucleotide sequencing or ligation. If an antisense primer complex is used, the RNA promoter sequence is one that is conveniently recognized by a bacteriophage RNA polymerase such as T7, T3 or SP6 polymerase.
[0030]
Preferably, the second double stranded polynucleotide is produced by amplifying a second antisense polynucleotide strand, and the amplification uses a universal primer that hybridizes to a universal primer site as a 5 'primer. And by using an antisense primer or an antisense primer complex as the 3 'primer. Most preferably, the amplification is performed by an enhanced polymerase chain reaction. This reaction produces a low abundance sequence rich pool of double-stranded polynucleotides compared to the original polynucleotide sample. If an antisense primer complex is used to generate second double-stranded polynucleotides, these polynucleotides will include an RNA promoter. In this case, the method is optional. Synthesizing an antisense RNA molecule from the second double-stranded polynucleotide can be included.
[0031]
In a preferred embodiment of the method of the present invention, the universal primer and / or the antisense primer or antisense primer complex each contain a restriction site. The method of the invention can optionally include cloning one or more second double-stranded (low abundance-enriched) polynucleotides into a vector. In particular, such methods allow for the construction of "standardized" cDNA libraries. This library is superior to standardized libraries produced by other techniques in that the number of copies of the cDNA in the library varies much less than that of the original polynucleotide sample; for example, cDNA replication. Highly representative cDNA libraries are produced whose numbers vary by no more than an order of magnitude. In a preferred variation of this embodiment, the cloned double-stranded polynucleotide encodes a polypeptide and the vector is an expression vector. The method of the invention further comprises introducing the expression vector into a host cell and expressing the protein encoded by the cloned double-stranded polynucleotide.
[0032]
Double-stranded polynucleotides produced according to the methods of the present invention, or polynucleotides produced directly or indirectly therefrom, can also be used in hybridization reactions. Such polynucleotides can be labeled with a detectable label and / or can be attached to a substrate, producing a polynucleotide array. If desired, one or more second double-stranded polynucleotides can be amplified. In a preferred embodiment, the amplification is performed using one or more gene-specific primers. Thus, the method of the invention encompasses each of these applications of these polynucleotides.
[0033]
In an alternative embodiment, the method of producing a selected polynucleotide pool from a polynucleotide sample comprises synthesizing a first antisense polynucleotide strand from a polynucleotide sample or a sense polynucleotide produced therefrom, and This is done by diluting the sense polynucleotide strand and substantially removing at least some of the low abundance first antisense polynucleotide strand. A first double-stranded polynucleotide is then generated from the remaining first antisense polynucleotide strand. These first double-stranded polynucleotides are rich in high abundance polynucleotide sequences as compared to polynucleotide samples. They are used to produce a second antisense polynucleotide strand, which is contacted under hybridization conditions with a polynucleotide sample therefrom or a sense polynucleotide produced therefrom. The resulting hybridization mixture contains unhybridized sense polynucleotide strands that are rich in low abundance polynucleotide sequences relative to the polynucleotide sample. A third antisense polynucleotide strand is synthesized from the unhybridized sense polynucleotide strand, and a second double-stranded polynucleotide is generated from the third antisense polynucleotide strand. These second double stranded polynucleotides utilize a selected pool of polynucleotides that is rich in low abundance polynucleotide sequences.
[0034]
Another aspect of the invention is a plurality of polynucleotides produced from a polynucleotide sample, wherein the plurality of polynucleotides is at least 10%.³ It includes a variety of different polynucleotides, each of which is substantially rich in high abundance polynucleotide sequences as compared to a polynucleotide sample, or rich in low abundance polynucleotide sequences as compared to a polynucleotide sample. Each polynucleotide in the plurality of polynucleotides preferably comprises an RNA promoter sequence and a universal primer site. In a preferred embodiment, these polynucleotides are double-stranded cDNA or antisense RNA.
[0035]
The present invention provides kits useful for performing the methods of the invention and / or kits for using the plurality of polynucleotides of the invention. The first kit is: an antisense primer conjugate comprising an antisense primer operably linked to an RNA promoter sequence, wherein the RNA promoter sequence is 5 'of the antisense primer; a sense primer; Instructions for performing at least one of the above methods of the invention. A second kit is: an antisense primer complex comprising a plurality of polynucleotides of the invention; an antisense primer operably linked to an RNA promoter sequence, wherein the RNA promoter sequence is 5 'of the antisense primer. The complex; and a sense primer. A third kit comprises: a plurality of polynucleotides of the invention and an RNA polymerase capable of transcribing antisense RNA from the plurality of polynucleotides.
[0036]
BRIEF DESCRIPTION OF THE FIGURES
1A-1B show schematics of preferred embodiments of the selection methods of the invention that are useful for producing a pool of polynucleotides that are enriched in high abundance sequences relative to a polynucleotide sample. This embodiment, described in detail in Example 4, demonstrates the production of a high abundance-enriched antisense RNA driver for use in the first reduction hybridization reaction of the general reduction hybridization method of the present invention. Can be used for
[0037]
2A-2C show schematics of a preferred embodiment of the second selection method of the present invention, which is useful for producing pools of polynucleotides that are rich in low abundance sequences as compared to polynucleotide samples. This embodiment, described in detail in Example 4, is used to produce a low abundance-enriched sense cDNA driver for use in the second deletion hybridization reaction of the general deletion hybridization method of the present invention. Can be used for
[0038]
3A to 3D are schematic diagrams of preferred embodiments of the general curtailing hybridization method of the present invention. The steps of this embodiment are described in detail in Example 4C.
[0039]
Detailed description
SUMMARY OF THE INVENTION The present invention provides novel novel high abundance polynucleotides that can identify low abundance polynucleotides present in one polynucleotide sample but not present (or substantially reduced) in a second polynucleotide sample. Hybridization methods. This method relies on two types of reduced hybridization reactions. First, an excess abundant polynucleotide strand from a test or reference polynucleotide sample is contacted with a polynucleotide strand from a test sample, thereby removing the abundant polynucleotide sequence. The resulting mixture is used to generate a low abundance-enriched polynucleotide chain corresponding to the test sample. In a second hybridization reaction, the low abundance-enriched polynucleotide strands derived from the test sample are contacted with excess low abundance polynucleotide strands from the reference sample and these low abundance polynucleotide strands present in both samples Block the polynucleotide sequence. The residual single-stranded abundance polynucleotide that is unique to the test sample can then be used for subsequent analysis or manipulation, after recreating the duplex and cloning, typically into a vector. This method is particularly useful in studies intended to identify low abundance disease-related genes, for example, which are difficult to identify using conventional subtractive hybridization techniques.
[0040]
This method relies on the availability of a pool of polynucleotides that are rich in high and low abundance polynucleotide sequences relative to the polynucleotide sample to be derived (ie, compared to the “starting polynucleotide sample”). Preferred embodiments use a novel method for generating such a polynucleotide pool. In particular, high abundance polynucleotides are selected by taking advantage of the loss of low abundance polynucleotides that occur on dilution. Low abundance polynucleotides can then be selected by reduced hybridization between the high abundance polynucleotide-enriched pool and the sample polynucleotide.
[0041]
The methods described herein can be used to replicate a wide range of polynucleotides without prior cloning into a vector and without sequence information. If desired, a polynucleotide representing a full-length mRNA transcript can be generated. Polynucleotides produced by this method are useful in a wide variety of applications, such as cloning, expression and hybridization studies.
[0042]
I. Definition
The term "polynucleotide" means a deoxyribonucleotide or ribonucleotide polymer, and, unless otherwise limited, includes known natural nucleotide analogs that can function in a manner similar to naturally occurring nucleotides.
[0043]
The term "polynucleotide" refers to, for example, genomic DNA; complementary strand DNA (cDNA), a DNA representation of mRNA, usually obtained by reverse transcription or amplification of messenger RNA (mRNA); DNA produced synthetically or by amplification And any form of DNA or RNA, including molecules; and mRNA.
[0044]
The term "polynucleotide" includes double-stranded polynucleotides as well as single-stranded molecules. Double-stranded polynucleotides that encode proteins include "sense" polynucleotide strands that hydrogen bond to "antisense" polynucleotide strands. A sense polynucleotide chain is a chain that, when translated, provides the amino acid sequence of the protein whose nucleotide sequence is encoded. The term "sense polynucleotide strand" refers to the sense strand of a double-stranded DNA molecule, for example, as well as mRNA. The antisense polynucleotide strand is complementary to a sense polynucleotide strand. Examples of antisense polynucleotide strands include the antisense strand of a double-stranded DNA molecule (eg, an antisense cDNA strand) and an antisense RNA molecule. In double-stranded polynucleotides, the polynucleotide strands need not be coextensive (ie, the double-stranded polynucleotide need not be double-stranded along the entire length of both strands). Absent).
[0045]
As used herein, the term “double-stranded” refers to double-stranded nucleotides. A "hybrid duplex" is a duplex formed between two polynucleotides in a hybridization hybridization reaction, each strand being derived from a different polynucleotide sample.
[0046]
As used herein, the term "complementary" refers to the ability to pair exactly between two nucleotides. That is, a nucleotide at a given position in a polynucleotide can hydrogen bond with another polynucleotide, and the two polynucleotides are then found to be complementary to each other at that position. The term "substantially complementary" describes a sequence that is sufficiently complementary to one another to cause a particular hybridization under stringent hybridization conditions.
[0047]
The phrase "stringent hybridization conditions" generally refers to the melting point (T.sub.T) for a particular sequence at a defined ionic strength and pH._m ) Means about 5 ° C. lower. An example of a stringent condition that is suitable to achieve particular hybridization for most sequences is a salt concentration of about 0.2 molar at a temperature of at least about 60 degrees and a pH of 7.
[0048]
"Specific hybridization" refers to the binding of a polynucleotide to a target nucleotide sequence under defined stringent conditions in the absence of substantial binding to other nucleotide sequences present in the hybridization mixture. I do. One skilled in the art will appreciate that reducing the stringency of hybridization conditions is tolerating sequence mismatches.
[0049]
Hybridization converts a single-stranded polynucleotide to a double-stranded polynucleotide, which can prevent the single-stranded polynucleotide from first serving as a target for hybridization or as a template for further polynucleotide strand synthesis. . Thus, hybridization is said to "block" a single-stranded polynucleotide in advance.
[0050]
As used with respect to polynucleotide strands, the term "unhybridized" refers to the presence of one or more polynucleotide strands after hybridization reaction under conditions that hybridize and form double-stranded polynucleotides. A polynucleotide that remains single-stranded is meant.
[0051]
As used herein, the term "driver" refers to a special type of polynucleotide strand that is added to a subtractive hybridization reaction and blocks complementary sequences present in the reaction mixture. Usually, a driver is added in molar excess to the reaction mixture to drive hybridization.
[0052]
The term "oligonucleotide" is used to mean a polynucleotide that is relatively short, usually shorter than 200 nucleotides, especially shorter than 100 nucleotides, especially shorter than 50 nucleotides. Typically, the oligonucleotide is a single-stranded DNA molecule.
[0053]
The term “selected polynucleotide pool” is used to describe a collection of polynucleotides that represent a subset of the polynucleotides present in a polynucleotide sample used to generate the selected polynucleotide pool. You. As used herein, this term describes the high- and low-abundance pools produced by dilution and post-dilution reduction hybridization, respectively.
[0054]
The term “reduced polynucleotide pool” as used herein refers to a reduced polynucleotide pool between polynucleotides derived from two different samples (eg, using the general reduced hybridization method of the present invention). Used to mean the pool of polynucleotides generated by hybridization. Thus, a "reduced polynucleotide pool" includes polynucleotides that are present in one sample and not present (or substantially reduced) in the other sample. If the reference polynucleotide is deleted from the test polynucleotide, the polynucleotides of the reduced polynucleotide pool are called "test-specific" and their presence in the test sample and the substantial Indicates absence. The reduced polynucleotide pool generated by the general reduced hybridization method of the present invention is typically used as a library of polynucleotides cloned into a vector (ie, a library of polynucleotide clones). Is generated.
[0055]
The term “primer” refers to an oligonucleotide that can hybridize with a polynucleotide (also called “annealing”) and can serve as a starting site for nucleotide (RNA or DNA) polymerization. .
[0056]
An “antisense primer” is a primer that hybridizes with a nucleotide sequence present in a sense polynucleotide and can act as a starting site for the synthesis of the antisense polynucleotide.
A "sense primer" is a primer that hybridizes with a nucleotide sequence present in an antisense polynucleotide and can act as a starting site for the synthesis of a sense polynucleotide. As used herein, a sense primer has a sequence that allows it to amplify one or more target polynucleotide sequences using an antisense primer or an antisense primer complex. .
[0057]
"Universal primers" are those that are capable of hybridizing to a nucleotide sequence present in substantially all polynucleotides intended to serve as template molecules for nucleotide polymerization.
[0058]
A “gene-specific primer” is one which hybridizes to a nucleotide sequence present in or flanking a unique expression sequence, and which essentially replaces the unique expression sequence or portions thereof with other sequences. Amplify without amplification.
[0059]
The term “primer site” refers to a region of a polynucleotide that hybridizes with a primer and can act as a starting site for nucleotide (RNA or DNA) polymerization.
[0060]
"Universal primer sites" are primer sites present in virtually all polynucleotides intended to act as template molecules for nucleotide polymerization.
[0061]
The term “antisense primer complex” is used herein to indicate an antisense primer operably linked to an oligonucleotide comprising an “RNA promoter sequence”. The latter sequence provides a promoter in the correct orientation and serves as a starting site for RNA polymerization.
[0062]
As used herein, the term "operably linked" means a functional linkage between a control sequence (typically a promoter) and a ligation sequence.
[0063]
The term "abundance" is used to describe the number of copies of a polynucleotide in a polynucleotide sample. For a sample polynucleotide, a polynucleotide that is present in a sample in a number greater than the medium number of replicates is referred to as a "high abundance" polynucleotide. Polynucleotides that are present in a sample in less than a moderate number are referred to as "low abundance polynucleotides". The absolute number of high and low abundance sequence replicas will vary depending on the polynucleotide sample. In mRNA samples, high abundance sequences include mRNA transcribed from so-called "housekeeping genes" and low abundance sequences include those encoding regulatory proteins such as hormones, receptors or other signals and control molecules. . The low abundance mRNA that can be selected by the method of the invention is typically less than about 1%, less than about 0.1%, less than about 0.01%, or less than about 0.001% of the RNA present in the cell. Occupy. The method of the present invention can also be used to select the most rare mRNAs that occupy only about 0.0000001% in cells. mRNA frequency is typically assessed by screening a cDNA library with a probe that specifically hybridizes to the mRNA of interest. The number of positive clones, divided by the total number of clones in the library and multiplied by 100%, gives an indication of the sequence in the library and produces an estimate of the frequency of mRNA in the cells in which the library is generated I do.
[0064]
Polynucleotides that are present in high abundance in a polynucleotide sample are said to be "represented" in the sample.
[0065]
A pool of polynucleotides is a polynucleotide of a given type compared to a polynucleotide sample if a given type of polynucleotide is present in the pool at a higher concentration than in the sample compared to the polynucleotide sample. Is said to be "rich." This term is used herein to describe the product of a hybridization hybridization reaction as follows. For example, if the subtractive hybridization reaction requires that a pool of high abundance-enriched polynucleotide strands selected from a sample be hybridized with (non-enriched) polynucleotide strands of the same sample, The quantity sequence hybridizes. Low abundance sequences present in the sample remain unhybridized. Thus, a curtailing hybridization reaction produces an unhybridized polynucleotide strand that is "rich" in the low abundance polynucleotide sequence compared to the polynucleotide sample. One skilled in the art understands that the concentration of low abundance polynucleotide sequence present in the reaction mixture is the same after the reaction as before the reaction. However, the concentration of the low abundance sequence is higher in the pool of unhybridized polynucleotide strands than in the sample polynucleotide strands.
[0066]
The phrase “high abundance-enriched polynucleotide chain” refers to a collection of polynucleotide chains that are rich in high abundance polynucleotide as compared to a polynucleotide sample.
[0067]
The phrase "low abundance-enriched polynucleotide chain" refers to a collection of polynucleotide chains that are rich in low abundance polynucleotide as compared to a polynucleotide sample.
[0068]
As used herein, "substantially enriched" means about 100-fold enrichment; that is, the concentration of a plurality of high or low abundance sequences is at least selected poly. Within the nucleotide pool, the selected polynucleotide pool is substantially rich in high or low abundance sequences when at least about 100-fold higher compared to the original polynucleotide sample from which the pool is derived. To this end, enrichment is assessed by hybridizing the labeled probe to a polynucleotide sample and a selected pool of polynucleotides and comparing the observed hybridization signal for each. For example, a Northern blot can be generated from a polynucleotide sample and a selected pool of polynucleotides, and hybridized with a radiolabeled probe, followed by radiography. Automated radiography can be scanned using a laser densitometer to quantify the hybridization signal. Other methods for measuring the strength of the hybridization signal, such as array-based methods, are known and can be used to assess the enrichment of the polynucleotide sequences of the present invention. "Hold enrichment" is calculated by dividing the hybridization signal observed for the selected polynucleotide pool by the hybridization signal observed for the polynucleotide sample from which the pool was selected.
[0069]
Certain types of polynucleotides (e.g., low abundance polynucleotides) can reduce the concentration of such polynucleotides in a pool of polynucleotides sufficiently for applications in which the presence of such polynucleotides is undesirable. "Substantially removed" when a pool of polynucleotides is available.
[0070]
A polynucleotide is "present in a reference sample at a substantially lower concentration than in a test polynucleotide sample" if the abundance of the polynucleotide in the reference sample is at least about 100 times less than that in the test sample. Say. The difference in abundance is about 10³ , About 10⁴ , About 10⁵ And about 10⁶ Or more orders may be used.
[0071]
The phrase “polynucleotide of a polynucleotide sample” refers to a sample polynucleotide. The phrase “polynucleotide produced from a polynucleotide sample” refers to a polynucleotide produced from a sample polynucleotide by RNA or DNA polymerization (eg, reverse transcription, amplification, synthesis of antisense RNA, etc.). Polynucleotides are "directly produced" from a sample polynucleotide if the sample polynucleotide serves as a template for RNA or DNA polymerization. If more than one polymerization step is performed, the polynucleotide is "indirectly produced" from the sample polynucleotide. Polynucleotides produced from or from a sample and used as a starting material in either the selection or subtraction methods of the present invention are referred to as "starting polynucleotides."
[0072]
As used herein, the terms “enhanced polymerase chain reaction” or “enhanced PCR” refers to a polymerase chain reaction that can amplify a polynucleotide sequence of at least 10 kilobases (kb) in length. I do.
[0073]
The term "vector" is used herein to describe a DNA structure containing a polynucleotide. Such vectors grow stably or transiently in host cells. This vector can be, for example, a plasmid, a viral vector, or simply a potential genomic insert. Once introduced into a suitable host, the vector can copy and function independently of the host genome, and in some instances can be integrated into the host genome.
[0074]
"Expression vector" refers to a DNA structure containing a polynucleotide molecule that is operably linked to a control sequence that can result in expression of the polynucleotide in a suitable host. Examples of control sequences include a promoter to effect transcription, an optional operator sequence to regulate transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence controlling transcription and translation termination.
[0075]
The term “host cell” refers to a cell that can temporarily or stably maintain a vector. Host cells of the present invention include, but are not limited to, bacterial cells, yeast cells, insect cells, plant cells, and mammalian cells. Other known or known host cells are suitable for use in the present invention.
[0076]
The term "array" refers to a collection of elements, where the elements are uniquely identifiable. For example, the term refers to a substrate that yields an array of elements such that each element has a different physical location on the substrate surface than the location of all other elements. In such an array, each element is simply identifiable by its orientation. Although the term "array" includes any arrangement of elements and can include elements arranged in a planar as well as non-planar manner, typical arrays of this type are arranged in a linear or two-dimensional matrix. Element. Non-planar arrays can be made by, for example, arranging beads, pins or fibers to form an array. The term "array" may include a collection of elements that have no fixed relationship to one another. For example, a collection of beads, with each bead identifying a property, can constitute an array.
[0077]
The elements of the array are called "target elements".
[0078]
As used herein with respect to a "target element", the term "different position" refers to a signal (e.g., a fluorescent signal) from every other target element from a labeled molecule that binds to the target element. Signal) is physically separated to uniquely contribute to the binding at its target element.
[0079]
"Microarray" means that the density of target elements on the substrate surface is at least about 100 / cm² Array.
[0080]
As used herein, the term "active reagent" is any reagent that, when added to a cell or tissue culture, for example, elicits a biological response in vivo or in vitro.
[0081]
"Normal" cells or tissues are those that are free of important specific physiological disorders. Thus, in one aspect, a normal cell may be "abnormal", but for the purposes of the present invention may still be a "normal" cell.
[0082]
"Disease" A cell or tissue is one that suffers from certain physiological disorders of interest.
[0083]
In a mixture comprising double-stranded and single-stranded polynucleotides, if single-stranded polynucleotides are suitably used as templates for the synthesis of further polynucleotide chains, single-stranded polynucleotides may be derived from double-stranded polynucleotides. It is said to be "functionally isolated." For example, a single-stranded polynucleotide can be functionally isolated from a double-stranded polynucleotide by suitably digesting the double-stranded polynucleotide with an enzyme.
[0084]
II. General elimination hybridization method
A. general
The present invention provides a general deletion hybridization method that facilitates the identification of one or more polynucleotides in a test polynucleotide sample that is absent or less abundant in a reference polynucleotide. This method requires a first trimming hybridization reaction to block high abundance polynucleotide sequences, leaving test polynucleotide chains rich for low abundance sequences. In a second subtractive hybridization reaction, the low abundance-enriched polynucleotide sequences present in both samples are removed and a test-specific duplex is generated.
[0085]
More specifically, the method uses a driver polynucleotide strand that is substantially rich in low abundance polynucleotide sequences as compared to either a test or reference polynucleotide. The method comprises forming a high abundance-enriched driver polynucleotide strand with a polynucleotide strand of a test sample or produced therefrom to form a first hybridization mixture, thereby blocking the high abundance polynucleotide sequence. Below, you need to make contact. This reaction produces unhybridized polynucleotide chains from the test sample that are rich in low abundance polynucleotide sequences as compared to the test sample.
[0086]
The unhybridized test polynucleotide strand serves as a template from which to synthesize a low abundance-enriched test polynucleotide strand. The latter are then contacted under hybridization conditions with a driver consisting of a reference polynucleotide chain that is rich in low abundance polynucleotide sequences compared to a reference sample. This second hybridization produces a hybrid duplex corresponding to the sequence separated by both samples, and an unhybridized low abundance-enriched polynucleotide chain corresponding to a sequence that differs between samples. The hybrid duplex is removed or digested and the non-hybridized, low abundance-enriched polynucleotide chain is functionally isolated, and the corresponding test sample is translated into a duplex. The resulting duplex is a low abundance polynucleotide sequence that is absent or substantially reduced in the reference sample. In this manner, test sample-specific or differentially expressed genes can be identified, even if their corresponding transcript is abundant in the test sample. An example of the protocol of the method is given in Example 4.
[0087]
As mentioned above, the driver for the first hybridization reaction can be derived from a test or reference sample. The method is described herein with reference to preferred embodiments in which the driver comprises a high abundance-enriched test polynucleotide chain. In a particularly preferred embodiment, the high abundance-enriched test polynucleotide strand is a high abundance-enriched antisense polynucleotide strand, which is used to sense the test polynucleotide strand in the first hybridization mixture. Hybridize. However, those skilled in the art can reverse the transcription of the polynucleotide component of such hybridization reactions, for example, abundance-enriched sense test polynucleotide strands can be hybridized using antisense test polynucleotide strands. Can be used as a driver.
[0088]
If the high abundance-enriched polynucleotide strand is an antisense polynucleotide strand, hybridization with the sense test polynucleotide strand will hybridize with a low abundance polynucleotide sequence compared to the test polynucleotide sample. Not generate a sense test polynucleotide strand. These unhybridized sense test polynucleotide strands then serve as templates for synthesizing low abundance-enriched antisense test polynucleotide strands. The latter is then contacted under hybridization conditions with a sense reference polynucleotide strand that is rich in low abundance polynucleotide sequences as compared to a reference sample. The resulting hybrid duplex is removed or digested to functionally isolate unhybridized, low abundance-enriched antisense polynucleotides, and those corresponding to these test samples are preferably selectively amplified. To make it double-stranded. This step produces low abundance "test-specific" duplexes that are generally cloned for further analysis.
[0089]
Starting polynucleotides useful in the general hybridization of the present invention can be obtained from any source. In particular, DNA or RNA useful in the present invention can be extracted and / or amplified from any source, including bacteria, yeast, viruses, organelles, as well as advanced organisms such as plants and animals. Preferably, mammals are used, and humans are most preferable. Starting polynucleotides can also be extracted or amplified from cells, fluids in the body (eg, blood) or tissue samples by various standard techniques. Starting polynucleotides useful in the present invention can also be derived from polynucleotide libraries, including cDNA, cosmid, YAC or BAC libraries, etc .; however, the use of such libraries is undesirable, because important Many low abundance sequences are not present in such libraries.
[0090]
The starting polynucleotide need not be in pure form, but must be sufficiently pure to effect the hybridization and synthesis reactions of the reduced hybridization to be performed.
[0091]
In a preferred embodiment, the test and reference polynucleotide strands are obtained from different polynucleotide samples. For example, test and reference polynucleotide samples include: a first cell or tissue and a second different cell or tissue; a cell or tissue in a first stage of differentiation or development and a cell or tissue of the same type in a second stage of differentiation or development. Derived from a cell or tissue treated with an active reagent or a cell or tissue not treated or treated with a second different active reagent, a normal cell or tissue or a diseased cell or tissue. Suitable examples of the latter include, but are not limited to, infectious diseases, inflammatory diseases, vascular diseases, cancer and / or genetic diseases.
[0092]
Although the hybridization method can be applied to any type of polynucleotide sample, the mRNA sample is preferably used to study differences in gene expression between two samples (ie, expression monitoring). As the skilled artisan will appreciate, mRNA can be converted to cDNA, which can optionally be cloned to generate a polynucleotide library. Similarly, an amplified DNA representation of the mRNA present in the cell is generated for use in the present invention. However, as noted above, such manipulations result in loss of polynucleotide sequences, especially low abundance sequences, and therefore, for this additional reason, the use of mRNA samples is preferred.
[0093]
B. First-reduction hybridization to block high abundance polynucleotides
In the first subtraction hybridization reaction of the general hybridization reaction of the present invention, the high abundance enrichment driver is hybridized to the test polynucleotide strand and blocks the high abundance polynucleotide sequence. The driver typically consists of a high abundance-enriched polynucleotide chain that is a pool of or produced from a test or reference polynucleotide that is each rich in the high abundance polynucleotide compared to the test or reference polynucleotide. When high abundance-enriched reference polynucleotide strands are used as drivers, the first trimming hybridization reaction blocks those polynucleotide sequences that are highly represented in the test or reference polynucleotide sample. When high abundance-enriched test polynucleotide strands are used as drivers, the first trimming hybridization reaction is performed on those polynucleotide sequences that are highly represented in the test polynucleotide sample (highly expressed in the reference polynucleotide sample). Is not exactly the same as what is represented). Preferably, the driver can be manufactured from the sample with the highest concentration of the high abundance sequence present in the test sample, which is often the test sample itself.
[0094]
In a preferred embodiment, the high abundance-enriched polynucleotide strand is an antisense polynucleotide strand. An antisense polynucleotide chain for use in the curative hybridization method of the present invention can be produced from a polynucleotide sample by any means known to those skilled in the art. For example, antisense polynucleotide strands that are enriched in high abundance polynucleotide sequences as compared to derived polynucleotide samples take advantage of the differences in reassociation kinetics between high and low abundance sequences. Can be manufactured. If the polynucleotide is denatured and recombined, sequences present in the sample at a high copy number will recombine before those at a low copy number. Therefore, sequences that become relatively double-stranded quickly (eg, Cot = 5.5 or less, where Co is moles of nucleotides per liter and t is seconds) are highly abundant poly Represents a nucleotide sequence. These double-stranded sequences can be recovered from the recombination mixture and used to produce antisense polynucleotides for use in general deletion hybridization of the present invention. In a preferred embodiment, the high abundance-enriched antisense polynucleotide strand is produced as described below in the section entitled "Method for Producing a Selected Polynucleotide Pool."
[0095]
For exclusion hybridization, the high abundance-enriched polynucleotide strand is contacted with the test polynucleotide strand under conditions where at least some of the polynucleotides specifically hybridize to each other. In a preferred embodiment, the high abundance-enriched antisense polynucleotide strand is contacted with a sense test polynucleotide strand. Preferably, the antisense polynucleotide strand is an antisense RNA molecule, and the sense test polynucleotide strand is an mRNA molecule.
[0096]
Although not required for this method, the antisense polynucleotide strand is usually added in excess to the hybridization reaction to drive hybridization (and is therefore called a driver). For most applications, the molar ratio of antisense polynucleotide to other polynucleotides in the reaction mixture will be from about 1: 1 to about 800: 1, preferably from about 1: 1 to about 200: 1, more preferably about 1: 1. : 1 to about 100: 1, but other ratios are possible.
[0097]
Hybridization reactions are performed at elevated temperatures, usually about 60-70 ° C., to achieve relatively specific hybridization. In addition, the buffers and salt concentrations used are adjusted to achieve the required stringency using techniques known to those skilled in the art. Typically, fairly high stringency is preferred. Acceptable methods for performing hybridization assays are known, and a general overview of the art is available at: Nucleic @ Acid. \\ Hybridization: A \ Practical \ Approach, \ Ed. {Hames, B .; D. And Higgins, S. J. , IRL Press, 1985; Hybridization of Nucleic Acid Immobilized on Solid Supports, Meinkoth, J.M. and \ Wahl, \ G. Analytic Cal Biochemistry, 238: 267-284, 1984. The tandem hybridization technique is also described in US Pat. No. 5,589,339 (December 31, 1996, Hampson et al.) And US Pat. No. 5,935,788 (August 10, 1999, Burmer et al.) ), And U.S. Patent No. 5,958,738 (issued September 28, 1999, Lindmann et al.).
[0098]
If the high abundance-enriched polynucleotide chain contains, for example, a poly-U or poly-T sequence and if the test polynucleotide chain contains, for example, a poly-A sequence, or vice versa, all high abundance-enriched polynucleotide chains The nucleotide strand is expected to hybridize to all test polynucleotide strands, and no clipping occurs. To prevent this type of "non-specific" hybridization, blocking oligonucleotides (ie, poly-A, poly-U and / or poly-T) are usually included in excess in the hybridization mixture. it can. Preferably, the blocking oligonucleotide is used in the hybridization mixture at a ratio of blocking oligonucleotide: polynucleotide of about 1: 1 to about 800: 1. More preferably, the ratio of blocking oligonucleotide to other polynucleotide is from about 1: 1 to about 200: 1, more preferably from about 1: 1 to about 100: 1, although other ratios are possible. Instead, the polynucleotide strands used in the hybridization hybridization reaction mixture are manufactured to contain no sequences that result in nonspecific hybridization, or such sequences are made using suitable enzymes. Either can be partially digested. For example, E. Partial digestion with E. coli exonuclease I can be used to remove the poly-A sequence from the 3 'end of the cDNA.
[0099]
Hybridization produces a double-stranded polynucleotide corresponding to the high abundance polynucleotide sequence, and an unhybridized test polynucleotide rich in the low abundance polynucleotide sequence as compared to the test polynucleotide sample. If the high abundance-enriched polynucleotide strand used for hybridization is derived from a reference sample rather than a test sample, the reaction mixture will generally also include an unhybridized high abundance-enriched reference polynucleotide strand. Including.
[0100]
C. Generation of low abundance enriched test polynucleotide chains
The next step of the general deletion hybridization method of the present invention is to use the unhybridized low abundance enriched test polynucleotide strand produced in the first hybridization reaction as a template, Abundance-enriched test polynucleotide chains.
[0101]
1. Synthesis of low abundance enriched test polynucleotide chains
In a preferred embodiment, the unhybridized low abundance test polynucleotide strand is used to synthesize an antisense polynucleotide test strand that is also rich in the low abundance polynucleotide sequence compared to the original test polynucleotide sample. Is a sense strand that serves as a template for Although any strand technique can be used for this purpose, in a particularly preferred embodiment, the synthesis of the antisense test polynucleotide strand is primed using an antisense primer.
[0102]
Antisense primers hybridize using antisense primer sites present in the sense test polynucleotide strand. This antisense primer site can be located at the 3 'end of the sense polynucleotide strand (e.g., the polyA sequence of the mRNA), which produces a full-length antisense polynucleotide strand. Alternatively, the antisense primer sites can be positioned such that the antisense test polynucleotide strand is synthesized for only a portion of the sense test polynucleotide strand. In the latter embodiment, at random sites in the polynucleotide strand, the random primer will prime, typically resulting in internal priming, producing a truncated polynucleotide strand.
[0103]
The synthesis of the antisense test polynucleotide strand is preferably primed using an antisense primer or, optionally, an antisense primer conjugate. The antisense primer complex has two components: (1) an antisense primer and (2) an RNA polymerase promoter sequence of specific origin. Antisense primer complexes are desirable, for example, to facilitate the production of antisense RNA from test-specific duplexes obtained using the general subtraction hybridization method of the present invention.
[0104]
Antisense primers produce polynucleotide synthesis, typically DNA replication, when placed under conditions suitable for primer extension, ie, in the presence of appropriate nucleotides and a replication reagent (eg, a DNA polymerase) under suitable reaction conditions. , Which are known in the art. The primer is preferably a single-stranded oligonucleotide, most preferably an oligodeoxynucleotide. The primer must be sufficiently long, and the sense test polynucleotide strand is used to form a sufficiently stable duplex, allowing synthesis of the extension product in the presence of a replication reagent. The exact length of the primers and the amount used will depend on many factors, including hybridization temperature, ionic conditions, degree of homology, and other requirements known to those skilled in the art. Primers designed to hybridize to a specific sequence motif typically contain about 10 to about 50 nucleotides, and preferably about 15 to about 25 or more nucleotides, although primers For example, fewer nucleotides can be included depending on the sequence motif. For other applications, oligonucleotide primers are not required, but are typically shorter, for example, from about 7 to about 15 nucleotides. As those skilled in the art have already appreciated, such short primer molecules generally require lower hybridization temperatures to form a sufficiently stable hybrid complex with the template polynucleotide. is there.
[0105]
Antisense primers are generated by any applicable method. Oligonucleotide primers are conveniently synthesized, for example, by known phosphotriester and phosphodiester methods, especially automated versions thereof. Standard automation methods use diethylphosphoramidites as a starting material, which are either commercially available or are described in Beaucage et al., Tetrahedron {Letters} 22: 1859-1962 (1981) or U.S. Pat. No. 4,458,066. Can be synthesized as described. Primers isolated from a biological source can also be used (eg, via restriction endonuclease digestion or amplification).
[0106]
Antisense primers useful in the methods of the invention are complementary to antisense primer sites on the sense polynucleotide strand. Therefore, a given antisense primer sequence need not be the exact complement of hybridizing antisense primer sites. If the primer sequence has sufficient complementarity with an antisense primer site to allow hybridization and polynucleotide extension, non-complementary bases or longer sequences can be present in the primer.
[0107]
As noted above, in a preferred embodiment, the antisense primer hybridizes to a low abundance-enriched sense test polynucleotide strand, such as an mRNA molecule. In this case, the antisense primer conveniently comprises a poly-T (also called oligonucleotide-dT or oligo-dT) sequence. This sequence usually contains about 5 to about 50, preferably about 5 to about 20, and more preferably about 10 to about 15 T residues, which are located at the 3 'end of each unhybridized mRNA molecule. Hybridizes with an existing poly-A sequence. Alternatively, if only RNAs that share a common nucleotide sequence motif are amplified, then the antisense primer is substantially complementary to this sequence motif.
[0108]
The second component of the antisense primer complex is an RNA promoter sequence. Such a sequence binds the RNA polymerase and can include a transcription start site. The RNA promoter sequence used in the antisense primer complex can be single-stranded or double-stranded. The promoter sequence is usually about 15 to about 250 nucleotides, preferably about 25 to about 60 nucleotides, from the naturally occurring RNA polymerase promoter, a consensus promoter sequence (Alberts et al., Molecular Biology of the Cell, 2nd ed. Edition, Garland, @NY (1989)), or modified versions thereof.
[0109]
Many types of promoters and polymerases that exhibit specificity for the same type of promoter are known. Generally, prokaryotic promoters are preferred over eukaryotic promoters, and phage or viral promoters are most preferred. Particularly preferred are the T3, T7 and SP6 phage promoter / polymerase systems. Probably the most studied is E.C. coli phage T7. T7 creates an entirely new polymerase highly specific for the 17-rate T7 promoter. E. FIG. The rate T7 promoter has a single highly-maintained sequence of -17 to +6 compared to the RNA starting site, rather than two separate highly-maintained regions such as the E. coli promoter. Salmonella phage SP6 is very similar to T7. Most RNA polymerases recognize double-stranded promoters, but E. coli. E. coli phage N4 creates an RNA polymerase that recognizes the N4 promoter early in native single-stranded N4 DNA. Transcription of the promoter and RNA synthesis on a DNA template are described in Watson et al., Molecular Biology of The Gene, 4th edition, verses 13-15, Benjamin / Cummings publishing Co. , Menlo Park, Calif. A preferred promoter sequence is a sequence from phage T7, which corresponds to its RNA site polymerase binding site (5'-AAT \ TCT \ AAT \ ACG \ ACT \ CAC \ TAT \ AGG \ G-3 '; sequence ID NO: 1).
[0110]
The RNA promoter sequence links with the antisense primer and facilitates transcription in the presence of ribonucleotides and RNA polymerase under suitable conditions. The primer and promoter components are ligated with the RNA promoter upstream (5 ') of the antisense primer in a direction that permits transcription of a polynucleotide strand complementary to the primer, ie, as described below. Linking is such that RNA transcription is generally in the same direction as primer extension. Any type of linkage that meets this criteria may be used, but nucleotide linkages are preferred. When present, the linker oligonucleotide between the components typically contains from about 5 to about 20 bases, but may be at least as large as desired.
[0111]
Furthermore, the antisense primer and the antisense primer complex preferably contain at least one (first) restriction site, and more preferably at least two restriction sites. The first restriction site is typically 5 'of the sequence in the antisense primer that binds to the template as well as an additional sequence 3' in the antisense primer that can serve as a primer site for amplification. Thus, the first restriction site can be used in a novel selective restriction cleavage method (described below), whereby the single-stranded polynucleotide remains intact, while the hybrid duplex remains the amplification primer site. Restriction digestion to remove will render amplification impossible. In this embodiment, the restriction site is one that is cleaved by a restriction enzyme that cuts double-stranded DNA instead of single-stranded. Examples are AluI, BbvI, DpnI, FnuDII, FokI, HpaII, HphI, MboI, MboII, MspI, Sau3AI, SfaNI and the like.
[0112]
If present, the second restriction site is usually the first 5 '. The second restriction site can be used to facilitate the cloning of test-specific duplexes generated by the general hybridization method of the present invention. Therefore, this site is preferably recognized by restriction enzymes that cut DNA relatively rarely and reduce the likelihood of unwanted internal cleavage within the test-specific duplex being cloned. Such enzymes are known in the art. As described in Example 4, the second restriction site may also serve as an amplification primer site. In this example, the antisense primer contains a (3′-5 ′) poly-T sequence, which hybridizes to the poly-A sequence of the mRNA linking to the AluΔI site (first restriction site), which Link to the site (second restricted site).
[0113]
When an antisense primer, optionally with an operably linked promoter region, hybridizes to a sense test polynucleotide, an antisense test polynucleotide chain is synthesized. If the sense polynucleotide is mRNA, the first strand of cDNA is conveniently generated through a reverse transcription process, where DNA is made from RNA and utilizes reverse transcription by standard techniques. This enzyme, present in all retroviruses (e.g., avian myeloblastoma virus), adds deoxyribonucleic acid to the 3 'end of the primer (Varmus, {Science} 240: 1427-1435} (1988)). Reverse transcription produces an antisense cDNA strand as a low abundance enriched test polynucleotide strand.
[0114]
2. Isolation of low abundance enriched test polynucleotide chains
Shortly after the synthesis of the low abundance enriched test polynucleotide strand, the reaction mixture further contained the template polynucleotide strand. Generally, it is desirable that the newly synthesized polynucleotide strand be separated from the template so that the newly synthesized strand can be used in the second trimming hybridization reaction of the general trimming hybridization reaction of the present invention. .
[0115]
Preferably, the newly synthesized strand is an antisense strand, more preferably an antisense cDNA strand, and the template consists of a sense polynucleotide strand, more preferably an mRNA strand. RNA is selectively degraded by any of a variety of conventional methods, and the DNA in the mixture can be left intact. As shown in the examples, treatment of the mixture with sodium hydroxide is one convenient way to remove RNA. The sodium hydroxide treatment is performed under such conditions that the DNA is not substantially decomposed and can be used for elimination hybridization. To this end, other methods of isolating the low abundance-enriched test polynucleotide strand can be readily devised by those skilled in the art for different applications of the general reduced hybridization of the present invention.
[0116]
D. Reduction hybridization to block low abundance polynucleotides present in test and reference samples
The newly synthesized low abundance enriched test polynucleotide strand is then used in a second deletion hybridization reaction. The second depletion hybridization reaction also includes a low abundance-enriched polynucleotide strand of, or produced from, a reference pool of polynucleotides that are enriched in the low abundance polynucleotide sequence compared to the reference polynucleotide sample. The low abundance reference polynucleotide chain acts as a driver, which blocks the low abundance polynucleotides present in both samples.
[0117]
If the newly synthesized polynucleotide strand is an antisense test polynucleotide strand, the low abundance-enriched reference polynucleotide strand is a sense reference polynucleotide strand. A low abundance-enriched sense reference polynucleotide chain can be produced from a polynucleotide sample by any means known to those of skill in the art. For example, a sense polynucleotide strand that is enriched in low abundance polynucleotide sequences as compared to a derived polynucleotide sample will exhibit differences in reassociation kinetics between the high and low abundance sequences as described above. It can be manufactured by utilizing. Sequences that become double stranded relatively quickly (eg, Cot = 5.5 or less, where Co is moles of nucleotides per liter and t is seconds) are those sequences that have high abundance polynucleotide sequences. Represent. These double-stranded sequences are removed from the reassembly mixture by methods known in the art (hydroxyapatite binding), leaving low abundance-enriched polynucleotides, which have corresponding counterparts for use in reduced hybridization. It can be used to produce a sense polynucleotide. In a preferred embodiment, the low abundance-enriched antisense polynucleotide strand is produced as described below in the section entitled "Methods for Producing Selected Polynucleotide Pools."
[0118]
For step hybridization, the low abundance-enriched reference polynucleotide chain is contacted with the low abundance-enriched test polynucleotide chain under conditions in which at least some of the polynucleotides specifically hybridize to each other. In a preferred embodiment, the low abundance sense reference polynucleotide strand is contacted with a low abundance antisense test polynucleotide strand. In a particularly preferred embodiment, the sense reference polynucleotide strand is a sense cDNA strand and the antisense test polynucleotide strand is an antisense test cDNA strand.
[0119]
The low abundance-enriched reference polynucleotide strand is usually added in excess to the hybridization reaction to drive hybridization, but this is not required for the present invention. For most applications, the molar ratio of the low abundance-enriched reference polynucleotide chain to other polynucleotides in the reaction mixture will be from about 1: 1 to about 800: 1, preferably from about 1: 1 to about 200: 1, More preferably from about 1: 1 to about 100: 1, other ratios are possible.
[0120]
Hybridization reactions are performed at elevated temperatures, usually 60-70 ° C, to achieve relatively specific hybridization. As described above for the first-reduction hybridization reaction, the buffer and salt concentration used can be adjusted to achieve the required stringency using techniques known to those skilled in the art. Typically, fairly high stringency is preferred.
[0121]
If the low abundance test polynucleotide strand contains, for example, a poly-U or poly-T sequence and the low abundance reference polynucleotide strand contains a poly-A sequence, or vice versa, all high abundance It is expected that the abundance-enriched polynucleotide strand will hybridize to all test polynucleotide strands and that no trimming will occur. If desired, blocking oligonucleotides may be added to the hybridization mixture, e.g., to allow hybridization between poly-A sequences within one set of polynucleotide chains to be poly-U or poly-U at another set of polynucleotide strands. Hybridization with the T sequence can be prevented.
[0122]
Alternatively, sequences that promote non-specific hybridization can be removed from at least one set of polynucleotide chains. For example, in a preferred embodiment, the low abundance test polynucleotide strand is an antisense cDNA and the low abundance reference polynucleotide strand is a sense cDNA. The latter contains a poly-A sequence, which can be removed, for example, by treating the sense cDNA strand with 3 'exonuclease under controlled conditions to remove the poly-A sequence. See Example 4, E.C. (commercially available from New England Biolabs). E. coli exonuclease I is used to remove poly-A sequences from the sense reference cDNA strand.
[0123]
Ultimately, at least one set of polynucleotide chains can be generated in a manner that eliminates sequences that promote non-specific hybridization. For example, in a preferred embodiment, a sense reference cDNA strand is generated from an antisense reference RNA for subtractive hybridization using an antisense test cDNA strand. If the antisense reference RNA comprises a poly-U sequence, the sense reference cDNA strand produced from this RNA contains the corresponding poly-A extension. The poly-U sequence is removed from the antisense reference RNA by heating (about 60 minutes at 72 ° C. or about 5 minutes at 90 ° C.) to degrade the poly-U sequence. This "poly-U-removed" antisense RNA is used to synthesize a sense reference cDNA strand lacking the corresponding poly-A extension.
[0124]
Hybridization includes the low abundance polynucleotide sequence present in both the test and reference samples, the unhybridized low abundance-enriched test polynucleotide strand, and the unhybridized low abundance-enriched reference polynucleotide strand. To generate a hybrid duplex corresponding to
[0125]
E. FIG. Removal of hybrid duplex
Since the general hybridization method of the invention is directed to isolating test-specific polynucleotides, hybrid duplexes representing a common sequence are preferably removed or digested. Methods for separating double-stranded and single-stranded polynucleotides are known, and any applicable method can be used in the present invention. Instead, the double-stranded polynucleotide is selectively degraded by nuclease digestion. Nucleases that selectively digest double-stranded polynucleotides are known and include, for example, lambda nuclease, E. coli. E. coli {nuclease} III and the like. Suitable buffers, times and temperatures for digesting double-stranded polynucleotides are known in the art.
[0126]
Further, as mentioned above, many restriction enzymes cleave only double-stranded polynucleotides. Thus, the present invention provides a novel selective restriction cleavage method that functionally isolates only single-stranded polynucleotides in a mixture of single- and double-stranded polynucleotides. This method involves contacting a mixture of polynucleotides with one or more restriction enzymes under conditions sufficient to digest the double-stranded polynucleotide to form one that cannot serve as a template for nucleotide synthesis. You need to be. In certain embodiments, restriction enzymes, preferably frequent cutters having a four base recognition site, can be used to cut the double stranded polynucleotide into smaller fragments. In consequence, this cleavage functionally isolates the single-stranded polynucleotide, allowing selective synthesis of a polynucleotide product corresponding to the single-stranded polynucleotide. Reaction conditions for digesting the double-stranded polynucleotide, such as buffer, time and temperature, are known for various restriction enzymes suitable for use in the present invention.
[0127]
In a preferred embodiment, the low abundance-enriched test polynucleotide strand and / or the low abundance-enriched reference polynucleotide strand used in the hybridization reaction each have a restriction site near the 5 'end. When one of each of these polynucleotide strands hybridizes, restriction sites are created at one or both ends of the resulting hybrid duplex.
[0128]
Typically, in this embodiment, the hybrid duplex produced when the low abundance-enriched test polynucleotide strand hybridizes with the low abundance-enriched reference polynucleotide strand will have a hybrid duplex at the 5 'restriction site. Not double-stranded. Thus, a standard "filling" reaction is performed and the molecules at these sites translate the double-stranded molecule. The enzymes useful for this filling reaction are not different from those used as filling reactions in other situations. For example, in Example 4, Taq DNA polymerase is conveniently used to fill the 5 'overhang. Buffers and reaction conditions suitable for such enzymes are known.
[0129]
If the low abundance enriched test polynucleotide strand is an antisense test polynucleotide strand, restriction sites can be introduced into the antisense primer and antisense primer complex used to synthesize the antisense test polynucleotide strand. . As described above, a primer or primer complex can be designed such that the restriction site is 3 'of the sequence that serves as a downstream primer site in a subsequent amplification reaction. In this embodiment, cleavage at a restriction site in the double stranded polynucleotide separates the primer site from the residue of the polynucleotide. Cleavage converts the double-stranded polynucleotide to a form that cannot serve as a template for amplification. Thus, restriction digestion functionally isolates the single-stranded polynucleotide and allows for selective nucleotide synthesis, eg, amplification, of the antisense test polynucleotide strand.
[0130]
Similar restriction sites are associated with low abundance-enhancing sense when these strands are manufactured, as described in the section entitled "Preparing Polynucleotide Pools Enriched in Low Abundance Polynucleotide Sequences" below. It can be introduced into a reference polynucleotide chain. These restriction sites can also be 3 'of the sequence which serve as upstream primer sites for amplification. Thus, cleavage at this site also separates the primer site from the rest of the polynucleotide, thereby preventing amplification.
[0131]
In an alternative embodiment, 3 'universal primer sites can be added to both ends of the molecule in the hybridization reaction mixture after the filling reaction and before restriction enzyme digestion. See Example 4. Universal primer sites are typically present in oligonucleotides that ligate or otherwise link to a test polynucleotide strand. Oligonucleotides can be oligodeoxynucleotides, oligoribodeoxynucleotides, or hybrid molecules comprising deoxynucleotides and ribodeoxynucleotides. The universal primer site must have a suitable length and sequence to hybridize to the universal primer. In a preferred embodiment, the universal primer sites serve as "anchors" for the amplification reaction, which is conveniently performed using the polymerase chain reaction ("PCR"). Studies for selecting a suitable universal primer site sequence are known in the art. The usual and preferred lengths for such a sequence are the same as given for the antisense primer above.
[0132]
Universal primer sites can be added to the 3 'end of a polynucleotide chain by any convenient method, such as "oligonucleotide sequencing" or ligation.
[0133]
In oligonucleotide sequencing (also called "homopolymer sequencing"), a particular type of deoxynucleotide, dA, dT, dG or dC, is added to the 3 'end of the DNA strand using a terminal transferase. This reaction produces a DNA strand having an oligonucleotide -dA, -dT, dG or dC sequence that can serve as a universal primer for the oligonucleotide -dT, -dA, dC or dG primer, respectively.
[0134]
Universal primer sites can also be added by ligating to the 3 'end of the antisense polynucleotide strand, for example, as described in Akowitz, Gene '81: 295-306 (1989).
[0135]
In a preferred embodiment, after the filling reaction, oligonucleotide sequencing is performed and an oligonucleotide sequence is added, which can serve as a primer site for a suitable homopolymer primer. Subsequent digestion with a suitable restriction enzyme removes the oligonucleotide sequence from the hybrid duplex, but not from the single stranded polynucleotide, which then serves as a template for amplification.
[0136]
F. Generation of test-specific duplexes from unhybridized low abundance enriched test polynucleotide chains
Removal or digestion of the hybrid duplex formed in the second subtraction hybridization reaction leaves the low abundance enrichment test or reference polynucleotide strand substantially intact. The test or reference polynucleotide strand has the opposite sense. In a preferred embodiment, the reference polynucleotide strand is a sense reference polynucleotide strand and the test polynucleotide strand is an antisense test polynucleotide strand.
[0137]
The method requires that the low abundance enrichment test polynucleotide strand be double stranded, thereby producing a low abundance enrichment test specific duplex. The selective conversion of the low abundance-enriched test polynucleotide strand into a test-specific duplex is such that the low abundance-enriched test polynucleotide strand has a 5 'antisense primer site and a 3' universal primer site. Illustrated herein with reference to a specific example which is an antisense test polynucleotide chain having Preferably, the low abundance-enriched reference polynucleotide strand used in the second hybridization does not have these elements, or if these elements are present, the nucleotide sequence is the antisense test polynucleotide. The nucleotide strand is sufficiently different from that in the antisense test polynucleotide strand that can be selectively amplified. Instead, in a preferred embodiment described in Example 4, the reference polynucleotide strand comprises self-annealable 5 'and 3' primer sites, which prevent amplification. In Example 4, the 5 'poly-G sequence anneals with the 3' poly-C sequence. Other methods for converting low abundance-enriched test polynucleotide chains to test-specific duplexes can be designed by one of skill in the art in light of the guidance herein, and therefore , Within the scope of the present invention.
[0138]
In certain embodiments, the antisense test polynucleotide strand is duplexed using an amplification reaction. Preferably, the amplification is performed by PCR, more preferably by enhanced PCR, both of which are known to those skilled in the art. PCR was described in US Pat. Nos. 4,683,202, 4,683,195, 4,800,159 and 4,965,188, similar to Science # 230: 1350 (1985) by Saiki. be written. PCR requires that two primers hybridize to a substantially complementary sequence that flanks the target sequence in the polynucleotide. A series of repetitions of the reaction steps, including template denaturation, primer annealing, and extension of the primer annealed by DNA polymerase, results in a geometric set of target sequences whose ends are defined by the 5 'end of the primer. When denaturation is typically performed at temperatures that denature most DNA polymerases (eg, about 93-95 ° C.), thermostable polymerases such as those derived from Thermus @ thermofilus, Thermus @ aquaticus (Taq), or Thermus @ flavus are used. It is typically used for extension to prevent the need to add additional polymerase for each extension cycle.
[0139]
In a preferred embodiment, the antisense test polynucleotide strand is amplified using enhanced PCR. Enhanced PCR can be performed, for example, as described in US Pat. No. 5,436,149 (Barnes et al., Issued Jul. 25, 1995), with different Taq or 3'-exonuclease activity lacking 3'-exonuclease activity. Disclosed is the use of a combination of the polymerase, including Thermus flavos DNA polymerase and a lesser amount of a thermostable DNA polymerase having such activity. Similar polymerase combinations that can also be used in the present method are described in US Pat. No. 5,512,462 issued to Cheng et al., Apr. 30, 1996. The considerations that affect the selection of PCR primers and amplification conditions are known, and those skilled in the art can readily determine suitable primers and conditions for a particular application of the method of the invention.
[0140]
For PCR amplification of an antisense test polynucleotide chain having a 5 'antisense primer site and a 3' universal primer site, for example, a universal primer that hybridizes to the universal primer site is used as the 5 'primer and an antisense is used as the 3' primer. Conveniently achieved by using a sense primer or antisense primer complex. Therefore, such primers are preferably selected to serve as an anchor for efficient and sufficient specific PCR amplification.
[0141]
In a preferred embodiment represented in Example 4, the antisense test polynucleotide strand is an antisense cDNA strand comprising a 5 'poly-T sequence and a poly-C sequence added by oligonucleotide sequencing in the selective restriction cleavage method described above. It is. The hybridization reaction mixture also includes a sense reference polynucleotide chain that includes a poly-C sequence, but does not include a poly-T sequence. Antisense test polynucleotide strands can therefore be selectively amplified using poly-G primers as 5 'primers and poly-A primers as 3' primers.
[0142]
In a particularly preferred variation of this embodiment, a restriction site that facilitates cloning of the test-specific duplex is introduced during amplification. Therefore, as described in Example 4, the 5 'primer contains a restriction site such as Not'I, which links to the 5' end of the poly-G primer sequence. Instead of using a poly-A primer as the 3 'primer, a primer containing an Sfi I site is used. The Sfi I primer hybridizes to the Sfi I site at the 5 ′ end of the antisense test polynucleotide strand. Thus, amplification produces a duplex with a 5 'Not I site and a 3' Sfi I site, which can be used to clone the test-specific duplex into a vector of choice. As mentioned above, it is generally desirable to introduce restriction sites for enzymes that rarely appear in DNA and reduce breaks in the test-specific duplex. As the skilled artisan will readily recognize, test duplexes having different restriction sites at each end are preferred for directional cloning, but test duplexes having the same restriction site at each end can also be generated.
[0143]
After amplification, the test-specific duplex produced from the "antisense test polynucleotide strand" is cloned into a vector, typically using standard DNA recombination techniques, In the section entitled "Using Polynucleotide Pools". This cloning step represents a simple method of isolating test-specific sequences from all other sequences in the amplification reaction mixture. The resulting polynucleotide library represents a "reduced polynucleotide pool" that is not present in the reference polynucleotide sample or is present in the reference polynucleotide sample at a substantially lower concentration than the test polynucleotide sample. Low abundance polynucleotide sequences.
[0144]
III. Method for producing a selected polynucleotide pool
The present invention also provides a method for selecting a polynucleotide pool from a polynucleotide sample. These polynucleotide pools can be used to generate drivers for the first and second subtraction hybridization reactions. Other applications for such pools are described in detail below.
[0145]
One selection method exploits the loss of low abundance sequences during dilution to generate a pool of polynucleotides rich in high abundance polynucleotide sequences as compared to a polynucleotide sample. This method can be used, for example, to obtain pools of test or reference polynucleotides that are enriched in high abundance polynucleotide sequences as compared to each test or reference polynucleotide sample. Such a pool can be used as a driver in the first reduction hybridization reaction.
[0146]
The second selection method starts with a high abundance polynucleotide pool generated by the present invention or another dilution method. This method relies on subtractive hybridization using a high abundance polynucleotide pool to generate a low abundance enriched polynucleotide pool. This selection method can be used to obtain a pool of reference polynucleotide chains that are rich in low abundance polynucleotide sequences as compared to a reference polynucleotide sample. The low abundance-enriched reference polynucleotide pool can be used, for example, as a driver in the second trimming hybridization reaction described above.
[0147]
Both methods for producing selected polynucleotide pools are described in application Ser. No. 09/632/898 (filed Aug. 7, 2000), which is expressly incorporated herein by reference. .
[0148]
A. Production of polynucleotide pools rich in high abundance polynucleotide sequences
1. Synthesis of a first antisense polynucleotide strand from a polynucleotide sample
To produce a polynucleotide pool enriched in high abundance polynucleotide sequences, a first antisense polynucleotide strand can be synthesized from a sense polynucleotide strand of a polynucleotide sample or a sense polynucleotide strand produced from a polynucleotide sample.
[0149]
Inevitably, any polynucleotide is used as a starting polynucleotide for the production of a high abundance-enriched polynucleotide pool if each contains a nucleotide sequence that is substantially complementary to the antisense primer. it can. This antisense primer site can be located at the 3 'end of the sense polynucleotide strand (e.g., the poly-A sequence of an mRNA molecule), which produces a full-length antisense polynucleotide strand. Alternatively, the antisense primer sites can be positioned such that the antisense polynucleotide strand is synthesized for only a portion of the sense polynucleotide strand (as if a random primer mixture was used). .
[0150]
Starting polynucleotides useful in the selection methods of the invention can be obtained from any source, as described above for those useful in general subtractive hybridization methods. The starting polynucleotides need not initially be present in pure form: if the other components in the mixture do not substantially interfere with the synthesis of the first antisense polynucleotide strand, they will form a complex mixture. Inside could be a minor fraction.
[0151]
In a preferred embodiment, the synthesis of the first antisense polynucleotide strand is primed using an antisense primer conjugate. As described above, the antisense primer complex comprises: (1) an antisense primer and (2) a specifically adapted RNA polymerase promoter sequence. The antisense primer complex can also include a sequence that is a restriction endonuclease site (restriction site), which facilitates selective restriction cleavage (see above) or cloning of a polynucleotide pool produced according to the methods of the invention. I do.
[0152]
Studies on antisense primers useful in the selection method of the present invention are the same as those described above for antisense primers useful in the general deletion hybridization method of the present invention. Briefly, primers are preferably single-stranded oligonucleotides, most preferably oligodeoxynucleotides. Primers designed to hybridize to a specific sequence motif typically contain from about 10 to about 50 nucleotides, and preferably contain from about 15 to about 25 nucleotides. Can contain fewer nucleotides depending, for example, on the sequence motif. For other applications, oligonucleotide primers are typically, but need not be, shorter, for example, from about 7 to about 15 nucleotides.
[0153]
The second component of the antisense primer complex is an RNA promoter sequence. RNA promoter sequences useful in the methods of the present invention are as described above in connection with general exclusion hybridization methods. Thus, the promoter sequence is typically about the same as that generated from a naturally occurring RNA polymerase promoter, a consensus promoter sequence (Alberts et al., Molecular Biology of the Cell, Second Edition, Garland, NY (1989), or a modified version thereof. It contains 15 to about 250 nucleotides, and preferably contains about 25 to about 60. Particularly suitable for use in the selection methods of the present invention are the T3, T7 and SP6 phage promoters / It is a polymerase system.
[0154]
The RNA promoter sequence links to the antisense primer and facilitates transcription in the presence of ribonucleotides and RNA polymerase under suitable conditions. The primer and promoter components are oriented in a direction that permits transcription of a polynucleotide strand that is complementary to the primer, ie, upstream (5 ′) of the antisense primer so that antisense RNA transcription is in the same direction as primer extension. Link to RNA promoter. Any type of linkage meeting this criterion can be used, but nucleotide linkages are preferred. The linker oligonucleotide between the components, when present, typically contains from about 5 to about 20 bases, but may be at least as large as desired.
[0155]
In a preferred embodiment, the selected polynucleotide pool is generated from RNA. Although total RNA can be used, poly-A @ RNA (ie, mRNA) is preferred in most applications. In each case, multiple antisense primer conjugates can be used, including random sequence antisense primers (ie, random primers). To generate a pool of polynucleotides selected from the mRNA present in the sample, the antisense primer is an oligo-dT sequence (e.g., about 5 to about 50, preferably about 5 to about 20, more preferably about 10 to about 20). 15 T residues). If only RNAs that share a common nucleotide sequence motif are amplified, then the primers are substantially complementary to this sequence motif. When a high abundance-enriched polynucleotide pool is prepared for use as a driver in the first elimination hybridization reaction, the starting polynucleotide is preferably mRNA and the antisense primer complex is preferably Includes random primer.
[0156]
After hybridization of the promoter region operably linked to the antisense primer and the sense polynucleotide in the sample, a first antisense polynucleotide strand is synthesized. If the sense polynucleotide is an mRNA, the first antisense cDNA strand is conveniently generated through the method of reverse transcription, as described above.
[0157]
2. Addition of universal primer site
Following synthesis of the first antisense polynucleotide strand from the polynucleotide sample and from the sense polynucleotide strand produced therefrom, the addition of a universal primer site to the 3 'end of the first antisense polynucleotide strand as described above. It is preferable to continue. The universal primer site must have a suitable length and sequence to hybridize to the universal primer, and is preferably designed to serve as an anchor for an amplification reaction such as PCR. Universal primer sites optionally include restriction sites to facilitate selective restriction cleavage and cloning of polynucleotide pools of the present invention. Universal primer sites can be added to the first antisense polynucleotide strand by any convenient method, including oligonucleotide sequencing and ligation, as in "template exchange" as described above.
[0158]
If the sense polynucleotide strand is mRNA and the first antisense strand is cDNA, template exchange is performed as follows. When a template exchange oligonucleotide is included during reverse transcription, it produces an mRNA-antisense cDNA hybrid. The template exchange oligonucleotide hybridizes to a CAP site at the 5 'end of the mRNA strand and serves as a short extended template for CAP dependent extension of the 3' end of the antisense cDNA strand. Template exchange oligonucleotides typically require small amounts of ribonucleotides at the 3 'end and promote CAP-dependent extension. Therefore, template exchange oligonucleotides usually contain about 1 to about 5 ribonucleotides at their 3 'end.
[0159]
3. Dilution of the first antisense polynucleotide strand
After generation of the first antisense polynucleotide strand, preferably comprising a universal primer site, the reaction mixture is diluted to substantially remove at least some low abundance antisense polynucleotide strands. Serial dilutions are typically made for this purpose, and the degree of dilution depends on the desired abundance limit. Large dilutions remove the polynucleotide chains that are present at multiple copies, whereas minimal dilutions remove the rarest polynucleotides in the mixture. Dilutions useful in standard applications of the method are about 10^-1, About 10^-2, About 10^-3, About 10^-4, About 10^-5, About 10^-6, About 10^-7, About 10^-8, About 10^-9, About 10^-10 , About 10^-11 , About 10^-12 But higher or lower dilutions may be desirable in certain applications. Serial dilution is performed by removing an aliquot of the reaction mixture and transferring the aliquot to an aqueous solution that provides the desired degree of dilution. If desired, multiple transfers are used to achieve a stepwise dilution resulting in the desired degree of dilution. The aqueous solution used for the dilution is preferably compatible with the enzymes used in the next step of the method, producing a double-stranded polynucleotide from the first antisense polynucleotide strand present after dilution.
[0160]
4. Production of first double-stranded polynucleotide from residual first antisense polynucleotide strand
The first double-stranded polynucleotide can be generated from the first antisense polynucleotide strand remaining after dilution by any number of applicable methods. When the first antisense polynucleotide strand is the first antisense cDNA strand, the second strand cDNA is optionally RNaseΔH containing DNA ligase and E. coli. E. coli synthesized using DNA polymerase. The RNase helps break the RNA / first strand cDNA hybrid, and the DNA polymerase synthesizes a complementary DNA strand using the first strand cDNA as a template. The second strand occurs as a deoxynucleotide added to the 3 'end of the growing strand. If the first antisense cDNA contains an RNA promoter sequence at the 5 'end, the single-stranded promoter sequence is replicated in the desired orientation within the double-stranded promoter region.
[0161]
In a preferred embodiment, the first double-stranded polynucleotide is produced by amplification. Where the first antisense polynucleotide strand is a cDNA molecule within an RNA / first strand cDNA hybrid, amplification is performed under conditions where the RNA degrades. Alternatively, the RNA can be removed before being amplified by any suitable technique, such as treatment with sodium hydroxide. Amplification is preferably performed by PCR, more preferably by enhanced PCR. PCR amplification of a first antisense polynucleotide having a 3 'universal primer site uses, for example, a universal primer that hybridizes to the universal primer site as the 5' primer and an antisense primer complex as the 3 'primer. This is conveniently achieved. Therefore, it is preferred that such primers be selected to serve as efficient and fully specific anchors for PCR amplification.
[0162]
If the first antisense polynucleotide strand is generated using an antisense primer complex mixture that includes a random primer, a random sequence linked to the RNA promoter sequence is introduced into the antisense polynucleotide strand. In this embodiment, the RNA promoter sequence can serve as a primer site for amplification. Thus, the amplification reaction can include a 3 'primer at the 5' end of each antisense polynucleotide strand that binds to an RNA promoter sequence.
[0163]
The pool of polynucleotides generated from the first antisense polynucleotide strand is rich in high abundance polynucleotide sequences as compared to the starting polynucleotide sample, and is used, for example, in a subtraction hybridization reaction, and is described in detail below. As such, a selected pool of polynucleotides is generated that is enriched in low abundance polynucleotides as compared to the starting polynucleotide sample.
For clarity, high abundance-enriched polynucleotides are referred to as "first double-stranded polynucleotides" and low abundance-enriched polynucleotides below are referred to as "second double-stranded polynucleotides".
[0164]
If the polynucleotide sample is an mRNA sample, the first double-stranded polynucleotide is called a "cDNA" molecule. In a preferred embodiment, the first double-stranded polynucleotide carries a universal primer site at the 5 'end (compared to the sense strand) and a functional RNA polymerase at the 3' end (compared to the sense strand).
[0165]
5. Synthesis of antisense RNA from first double-stranded polynucleotide
Antisense RNA can be synthesized from a first double-stranded polynucleotide containing an RNA promoter by contacting the polynucleotide with an RNA polymerase capable of binding to an RNA promoter region under conditions suitable for RNA synthesis. The sense strand is transcribed into antisense RNA. Amplification occurs because the polymerase is repeatedly recycled onto the template (ie, reinitiates transcription from the promoter region). This technique allows a wide range of polynucleotides to be copied without having to clone into a vector. Furthermore, recycling the polymerase on the same template prevents erroneous transmission.
[0166]
The RNA polymerase used for transcription must be operably linked to the particular promoter region used in the antisense primer complex described above. Virtually any polymerase / promoter combination can be used; however, bacteriophage RNA polymerases, particularly those from T3, T7 and SP6 phage, are preferred. The most preferred polymerase is T7 RNA polymerase. A particularly high degree of specificity for its promoter site represented by T7 RNA polymerase (Chamberlin et al., In The Enzymes, ed. P. Boyer (Academic Press, New York), pp. 87-108 (1982)) makes this enzyme unique. For use as probes (Melton et al., Nucl. Acids Res., 12: 7035-7056 (1984)), for in vitro translation studies (Krieg et al., Nuc. Acids Res. 12: 7057-7070 (1984)) and For use in the production of synthetic oligoribonucleotides (Milligan et al., Nuc. Acid @ Res. 15: 8783-8798 (1987)). To reagents useful in a variety of recombinant DNA techniques including Toro RNA synthesis. The lack of an effective terminal signal for T7 polymerase also allows this enzyme to transcribe almost any DNA sequence (see Rosenberg et al., Gene 56: 125-135 (1987)). Ultimately, T7 polymerase is available from Promega Biotech, Madison, Wis. Or in concentrated form (1000 units / l) from a number of commercial sources, such as Epicenter Technologies, Madison, Wis. E. FIG. E. coli RNA polymerase is suitable for E. coli. coli RNA polymerase promoter region.
[0167]
The transcription reaction mixture contains the necessary nucleotide triphosphates, which can be modified depending on the ultimate use of the antisense RNA. For example, if antisense is intended for use as an RNA nucleic acid hybridization probe, one or more nucleotides can be labeled as described in detail below.
[0168]
B. Production of polynucleotide pools rich in low abundance polynucleotide sequences
1. Reduced hybridization of polynucleotide samples using antisense polynucleotide strands rich in high abundance polynucleotide sequences
Antisense polynucleotide chains enriched in high abundance polynucleotide sequences can be produced from a polynucleotide sample by any convenient method, including those described above in connection with the general scission hybridization method of the invention. In a preferred embodiment, the high abundance-enriched polynucleotide strand is an antisense RNA transcribed from a first double-stranded polynucleotide as described above. Antisense RNA is hybridized using a polynucleotide sample or a sense polynucleotide produced therefrom. This hybridization reaction produces a double-stranded polynucleotide and an unhybridized sense polynucleotide. Because antisense RNA is rich in high abundance sequences, high abundance sequences are double-stranded and unhybridized sense polynucleotides are rich in low abundance sequences as compared to a polynucleotide sample.
[0169]
Preferably, the antisense polynucleotide strand and the sense polynucleotide strand used in the subtraction hybridization reaction are derived from the same polynucleotide sample. Therefore, in order to generate a low abundance sequence-enriched reference pool of polynucleotides for use in the second hybridization reaction of the general subtraction hybridization described above, the antisense reference polynucleotide strand is It is preferable to hybridize using a polynucleotide chain. However, the selection method of the present invention involves the reduced hybridization of one polynucleotide sample as well as antisense polynucleotide chains derived from sense polynucleotide chains produced from or from different polynucleotide samples. In this case, subtractive hybridization blocks the high abundance sequence separated by the two samples.
[0170]
The antisense polynucleotide strand is usually added in excess to the hybridization reaction to drive hybridization, but this is not a requirement of the method. For most applications, the molar ratio of antisense polynucleotide to other polynucleotide in the reaction mixture will be from about 1: 1 to about 800: 1, preferably from about 1: 1 to about 200: 1, and more preferably about 1: 1. The ratio is from 1: 1 to about 100: 1, but other ratios may be used. Subtractive hybridization is performed as described above for the first and second subscriptive hybridization reactions of the general subscriptive hybridization method of the present invention.
[0171]
In a preferred embodiment, the antisense polynucleotide strand is a pool of abundance-enriched polynucleotides (eg, an abundance-enriched reference polynucleotide) and an antisense RNA molecule produced therefrom, and the sense polynucleotide strand Are mRNA molecules from the same sample. In this case, subtractive hybridization produces an unhybridized mRNA molecule that is rich in low abundance polynucleotide sequences as an unhybridized sense polynucleotide chain, as compared to the sample.
[0172]
2. Synthesis of a second antisense polynucleotide strand from an unhybridized sense polynucleotide strand.
The unhybridized sense polynucleotide strand from the rearrangement hybridization reaction can then be used as a template for the synthesis of another set of antisense polynucleotide strands, if desired. Although any standard strand technology may be used for this purpose, in a preferred embodiment, the second set of antisense polynucleotide strands comprises an antisense primer or an antisense primer complex as described above. Is synthesized using If an antisense primer conjugate is used, the antisense polynucleotide strand contains a primer site at the 5 'end and an RNA promoter sequence. Therefore, if desired, antisense primer conjugates are used to generate a pool of selected polynucleotides, each of which facilitates the synthesis of antisense RNA from the selected polynucleotide containing an RNA promoter. Is preferred. If a primer site and / or an RNA promoter sequence is used to initiate nucleotide synthesis in a reaction mixture that may include an undesired polynucleotide also having a primer site and / or a promoter sequence, the primer site and / or the promoter sequence Preferably, they are sufficiently different to allow specific nucleotide synthesis from the polynucleotide (see eg, Example 4, the primers and RNA promoter sequences used in B.2 are used in A.1. And the primer sites are different from those used in C.2.). Regardless of the method used, the second antisense polynucleotide strand generated from the unhybridized sense polynucleotide strand is rich in low abundance sequences as compared to a polynucleotide sample.
[0173]
Where the unhybridized sense polynucleotide is an mRNA molecule, the mRNA is conveniently reverse transcribed, producing a second antisense cDNA strand as the second antisense polynucleotide strand.
[0174]
3. Addition of universal primer site
In a preferred embodiment of the present invention, a universal primer site is added to the 3 'end of the second antisense polynucleotide strand as described above to allow for the simultaneous synthesis of a low abundance sequence rich second double stranded polynucleotide and Facilitates amplification. Universal primer sites are added by template exchange, oligonucleotide sequencing or ligation. If the primer site is to be used to initiate nucleotide synthesis in a reaction mixture that may include an undesired polynucleotide also having a universal primer site, the primer site introduced into the second antisense polynucleotide strand is specific It is preferably sufficiently different from the desired polynucleotide for efficient nucleotide synthesis (see, eg, Example 4, the universal primer sites used in B.3. Are different from those used in A.2. Is different).
[0175]
4. Generation of a second double-stranded polynucleotide from a second antisense polynucleotide chain
A second double stranded polynucleotide can be generated from the second antisense polynucleotide strand as described above. If a 3 'universal primer site has been added to the antisense polynucleotide strand, the polynucleotide strand is preferably amplified using PCR, more preferably using enhanced PCR. This amplification is conveniently performed using a universal primer that hybridizes to the universal primer site as the 5 'primer and using an antisense primer or antisense primer conjugate as the 3' primer. These primers, if desired, contain restriction sites to facilitate selective restriction cleavage or cloning of a polynucleotide pool of the present invention. The resulting pool of polynucleotides is enriched in low abundance polynucleotide sequences as compared to the starting polynucleotide sample.
[0176]
Where the second antisense polynucleotide strand is the cDNA strand in an RNA / first strand cDNA hybrid, the RNA sequence is preferably prior to amplification by any suitable technique, such as treatment with sodium hydroxide. Is removed. The amplification then produces a double-stranded cDNA molecule.
[0177]
IV. Polynucleotide pool
A. General
The present invention provides a polynucleotide pool that can be produced using the method of the present invention. Such a pool contains a plurality of different polynucleotide sequences, usually at least about 10² , At least about 10³ , At least about 10⁴ , At least about 10⁵ , At least about 10⁶ Or at least about 10⁷ Includes different polynucleotide sequences. In a preferred embodiment, each polynucleotide comprises an RNA promoter sequence and a universal primer site. A polynucleotide can be any form of DNA or RNA and is single-stranded or double-stranded. Preferably, the polynucleotide is a double- or single-stranded cDNA or antisense DNA.
[0178]
In one embodiment, the invention provides a reduced polynucleotide produced by a reduced hybridization between a test and a reference polynucleotide sample, as in the general reduced hybridization method described above. Typically, the reduced polynucleotide pool contains a library of polynucleotide clones. The reduced polynucleotide pool is substantially enriched for polynucleotide sequences that are not present in the reference polynucleotide sample or are present in the reference polynucleotide sample at substantially lower concentrations than the test sample. In a preferred embodiment, the enrichment is about 10³ , About 10⁴ , About 10⁵ , About 10⁶ Or about 10⁷ It is a double order. The reduced polynucleotide pool is also enriched in substantially lower abundance polynucleotide sequences as compared to the test polynucleotide sample. Preferably, the low abundance polynucleotide is about 10³ , About 10⁴ , About 10⁵ , About 10⁶ Or about 10⁷ Twice as rich. The reduced polynucleotide pools of the invention are typically generated as a library of polynucleotides to be cloned into a vector.
[0179]
The selected polynucleotide pool resulting from the selection method of the present invention comprises a plurality of polynucleotides produced from a polynucleotide sample that is substantially rich in high or low abundance polynucleotide sequences as compared to the polynucleotide sample. The selected polynucleotide pool can include a library of polynucleotide clones or a pool of polynucleotides that are not cloned. In a preferred embodiment, the high or low abundance polynucleotide sequence relative to the polynucleotide is about 10%.³ , About 10⁴ , About 10⁵ , About 10⁶ Or about 10⁷ Twice as rich.
[0180]
The polynucleotide of the present invention is useful for various applications. While the following discussion discusses the use of pools, those skilled in the art will appreciate that individual polynucleotides are selected from a pool of polynucleotides and are used essentially as described for pools.
[0181]
B. Use of polynucleotide pool
1. Nucleotide synthesis
Polynucleotide pools produced according to the present invention can be used as templates for cDNA synthesis and / or can be amplified to further extend one or more desired sequences. Amplification is preferably performed by PCR, and more preferably by enhanced PCR. The entire pool can be amplified, preferably using appropriate antisense primers or antisense primer conjugates and universal primers, or a subset of the pool can be amplified. Individual sequences of interest can be amplified using at least one, and preferably two, gene-specific primers. Alternatively, antisense RNA can be synthesized from a pool of polynucleotides as described above.
[0182]
2. Hybridization
Polynucleotide pools or polynucleotides produced therefrom (such as antisense RNA) can be used in hybridizations. If desired, the pools or the polynucleotides produced therefrom are labeled with a detectable label. A wide variety of labeling techniques are known to those skilled in the art and can be used to produce labeled polynucleotides of the present invention according to standard procedures (US Pat. No. 4,755,619). A labeling step can be introduced into one of the above reactions, and the method produces a labeled polynucleotide pool. For example, one or more nucleotide triphosphates can be included in the reaction mixture. Suitable labels are known and include, for example,³²S,³²P, ³Radioactive labels such as H or non-radioactive labels such as fluorescent labels. Labeling may be direct or indirect. In the latter example, one or more biotinylated nucleotides are used to synthesize biotinylated polynucleotides (Sive @ and St. John, Nucl. Acids Res. 16: 10937 (1988) and Duguid et al.). Natl. Acad. Sci. USA 85: 5738-5742 (1988)). Biotinylated polynucleotides can be detected by binding to labeled avidin.
[0183]
3. Drivers in the hybrid hybridization
In other applications, the polynucleotide pools of the present invention are particularly useful for generating polynucleotides intended for use as drivers in subtractive hybridization protocols. Such protocols typically require large amounts of drivers (usually 10 μg). This requirement makes it difficult to test for differential expression of mRNA present in biological material available in small supplies. This difficulty can be addressed by cloning the collection of polynucleotides of interest prior to exclusion, and cloning vectors can amplify the amount of polynucleotide available for hybridization. However, since elimination requires up-front cloning, it is complicated and suffers from under- and over-representation of the sequences depending on the differences in the growth rates of the mixed distributions, And there may be a risk of recombination between sequences in the mixture distribution propagation.
[0184]
The method of the present invention avoids these problems by generating large amounts of antisense RNA from limited amounts of polynucleotides without prior cloning. These methods are superior to PCR, which produces both sense and antisense strands that must be separated before use in subtraction hybridization. The high or low abundance antisense RNA produced as described above can be used in methods of detection and in methods of isolating polynucleotides that vary in abundance between different distributions, e.g. To be compared between different organizations or within the same organization.
[0185]
In a preferred embodiment, the antisense RNA synthesized from a pool of high abundance-enriched polynucleotides derived from a test or reference mRNA sample is a first trimming hybridization method of the general trimming hybridization method of the invention. Used in reactions. Antisense RNA blocks high abundance polynucleotide sequences in the test mRNA, leaving the low abundance-enriched mRNA strand free to serve as a template for nucleotide synthesis.
[0186]
In another preferred embodiment, the antisense RNA synthesized from a pool of low abundance-enriched polynucleotides derived from a reference mRNA sample is used in a second deletion hybridization reaction of a general deletion hybridization method. Is done. More specifically, the low abundance-enriched antisense reference RNA is used as a template to synthesize a low abundance-enriched sense reference cDNA strand. Example 4 illustrates a preferred embodiment of this application where cDNA synthesis is primed using a universal containing restriction site at the 5 'end. The synthesized cDNA strand has a 5 'restriction site, a universal primer site, a poly-A sequence and a 3' promoter sequence. The latter two elements are E. coli. Preferably, it is removed by partial digestion with an exonuclease such as E. coli {exonuclease I. The resulting low abundance-enriched sense reference cDNA strand lacks a poly-A sequence that "non-specifically" hybridizes with a poly-T containing molecule. These sense reference cDNA strands are conveniently used as drivers in subtraction hybridization reactions to block low abundance polynucleotide sequences present in both reference and test polynucleotide samples, and to provide low abundance enrichment assays. The polynucleotide strand is either absent in the reference sample or remains present in smaller amounts. See Example 4, an unblocked, low abundance-enriched test polynucleotide chain serves as a template for nucleotide synthesis, producing a double-stranded polynucleotide.
[0187]
4. Nucleotide array
The polynucleotide pools of the invention, or the polynucleotides generated therefrom, are attached to one or more substrates, if desired, to produce a polynucleotide array, which can be used in hybridization assays. In a preferred embodiment, each type of polynucleotide constitutes a different target element in the array. Preferably, the polynucleotide pools of the present invention are used to generate a DNA microassay.
[0188]
Arrays of the polynucleotides of the invention can be produced according to conventional techniques for making DNA arrays. For example, a sample dispenser attached to a device that can be accurately positioned can be used to spot a sample on a substrate. U.S. Pat. No. 5,807,522 (Brown and Shalon, issued Sep. 15, 1998) is linked to multiple microsized assay areas and each area of an array separated by a distance of 50-200 microns or less. An apparatus is described that facilitates the mass @fabrication of a microarray characterized by a well-defined amount of analyte (typically in picomoles).
[0189]
Another approach to robotic spotting uses an array of pins or capillary-dispensers that are submerged in wells, for example, 96 wells of a microtiter plate for transferring an array of samples onto a substrate. Arrays can also be facilitated by coating elements such as beads or optical fibers with the sample to form target elements. U.S. Patent No. 5,830,645 (Pinkel et al., Issued November 3, 1998) describes the use of beads to generate polynucleotide arrays, and U.S. Patent No. 5,690,894 (Pinkel et al.). , Published November 25, 1997) discloses polynucleotide arrays made from optical fibers.
[0190]
5. Cloning
The polynucleotide pool produced by the above method is cloned into a vector using standard cloning techniques to generate a polynucleotide library. Such libraries necessarily facilitate the investigation of gene expression in any cell or cell distribution. The target cells may be blood (eg, leukocytes such as T or B cells) or brain, spleen, bone, heart, duct, lung, kidney, liver, pituitary gland, endocrine gland, lymph node, dispersed primitive cells, tumor cells, etc. Obtained from other organizations. In the area of neurological research, the identification of mRNAs that change, for example, as a function of alertness, behavior, drug treatment, and improvement, has been hampered by the difficulty of constructing a cDNA library from cerebellar cell nuclei. The use of the polynucleotide pools of the present invention to construct a cDNA library from individual brain cell nuclei results in a larger representation of low abundance mRNA from these tissues compared to their representation in the whole brain cDNA library. Provided to facilitate cloning of important low abundance messages.
[0191]
Suitable vectors for use in cloning are capable of effecting vector replication in a suitable host cell (ie, the origin of replication), as well as sequences encoding selectable markers such as antibiotic resistance genes. Includes replication sequence. Upon introduction of the vector into a suitable host, the vector is replicable and functions independently of or integrates within the host genome. The design of the vector, among other things, depends on the intended use for the vector and the host cell, and the design of the vector of the invention for the particular use and host cell is within the level of ordinary skill in the art.
[0192]
In a preferred embodiment, a polynucleotide of the invention encodes a polypeptide and is cloned into an expression vector. Expression vectors include one or more control sequences capable of effecting and / or enhancing expression of an operably linked protein encoding sequence. Control sequences suitable for expression in prokaryotes, for example, include promoter sequences, operator sequences, and ribosome binding sites. Control sequences for expression in eukaryotes include promoters, enhancers, and transcription termination sequences (ie, include a polyadenylation signal). Expression vectors useful in the methods of the invention also include other sequences, such as, for example, signal sequences or sequences encoding amplifiable genes. The signal sequence directs the secretion of polypeptides fused to them from cells expressing the protein. The inclusion in the vector of a gene that complements the lack of auxotrophy in the selected host cell allows for the selection of host cells to be transformed with the vector. .
[0193]
Vectors of the invention are typically produced by linking the desired elements by ligation at convenient restriction sites. Cloning can be simplified if the antisense primer or antisense primer conjugate used to generate the polynucleotide of the invention and the universal primer site include restriction sites. The inclusion of different restriction sites within the primer or primer complex and universal primer site facilitates directional cloning. Preferably, the restriction sites used occur frequently in the polynucleotides of the original sample, minimizing internal cleavage of the polynucleotides of the present invention. Examples of suitable sites include those recognized by SfiI and NotI.
[0194]
Vectors containing the cloned polynucleotides can be introduced into host cells. Many types of host cells are available for vector propagation and / or expression. Examples of prokaryotes (such as E. coli and Bacillus strains, Pseudomonas, and other bacteria) include advanced eukaryotic cells (such as human immature kidney cells or other mammalian cells) as well. , Yeast or other fungal cells (including S. cerevesiae and P. pastoris), insect cells and plant cells. Host cells of the present invention include cells in media and cells that are present in living organisms (such as transgenic plants or animals).
[0195]
The vector is introduced into the host cell by any convenient method, which will vary depending on the vector-host system used. Usually, the vector is introduced into cells by transformation (known as transfection) or infection with a vector-producing virus (eg, a phage). If the host cell is a prokaryote (or other cell with a cell wall), a convenient transformation method is described by Cohen et al. (1972) Proc. Natl. Acad. Sci. , USA, 69: 2110-14. If a prokaryote is used as the host and the vector is a phagemid vector, the vector can be introduced into the host cell by infection. Yeast cells are described, for example, by Hinnen (1978) in Proc. Natl. Acad. Transformed with polyethylene glycol as taught in Sci, USA, 75: 1929-33. Mammalian cells are conveniently described in Graham et al. (1978) Virology, 52: 546 and Gorman et al. (1990) DNA {and} Prot. Eng. Tech. , 2: 3-10 using the calcium phosphate precipitation method. However, other known methods for introducing DNA into host cells, such as nuclear injection, electroporation and protoplast fusion, are also acceptable for use in the present invention.
[0196]
6. Expression of encoded polypeptide
Host cells transformed with an expression vector can be used to express the polypeptide encoded by the cloned polynucleotide of the present invention. Expression requires culturing the host cells under conditions suitable for cell growth and expression and recovery of the expressed polypeptide from the cell lysate, or, if the polypeptide is secreted, from the medium. In particular, the medium contains nutrients and growth factors appropriate for the cells used. In many cases, nutrients and growth factors are known and can be readily determined, especially by those skilled in the art. Culture conditions suitable for mammalian host cells are described, for example, in Mammarian Cell Culture (Mather ed., Plenum Press 1984) and Barnes and Sato (1980) Cell 22: 649.
[0197]
In addition, culture conditions must allow for transcription, translation and protein transport between cell compartments. Factors affecting these processes are known and include, for example, the number of DNA / RNA copies; factors that stabilize DNA; nutrients, feeds and transcription promoters or repressors present in the medium; culture temperature, pH and Osmolality; and cell density. Regulation of these factors to promote expression in a particular vector-host cell system is within the level of ordinary skill in the art. Principles and practical techniques for maximizing the productivity of mammalian cell cultures in vitro can be found in Mammarian Cell Biotechnology: a Practical Approach (Buttler ed., IRL Press (1991)).
[0198]
Any number of known techniques for large or small scale protein production can be used to express a polypeptide of the invention. These include, but are not limited to, the use of shake flasks, fluidized bed bioreactors, roller bottle culture systems, and stirred tank bioreactor systems. Cell culture can be performed in batch, fed-batch or continuous modes.
[0199]
Methods for recovering recombinant proteins produced as described above are known and will vary depending on the expression system used. The polypeptide containing the signal sequence can be recovered from the medium or the protoplasm. The polypeptide is expressed intracellularly and can be recovered from cell lysates.
[0200]
The expressed polypeptide can be purified from the medium or cell lysate by any method that can separate the polypeptide from the host cell or one or more components of the medium. Typically, the polypeptide is separated from host cells and / or media components that interfere with the intended use of the polypeptide. As a first step, the medium or cell lysate is centrifuged or filtered to remove cell debris. The supernatant is typically concentrated or diluted to the desired volume or diafiltered into a suitable buffer, requiring preparation for further purification.
[0201]
The polypeptide can then be further purified using known techniques. The technique chosen will vary depending on the properties of the expressed polypeptide. For example, where the polypeptide is expressed as a fusion protein that includes an affinity region, purification typically involves the use of an affinity column that includes a cognate binding partner. For example, a polypeptide fused to hexahistidine or a similar metal affinity label can be purified from fractionation on an immobilized metal affinity column.
[0202]
7. Other research and therapeutic uses
The polynucleotide pools of the present invention also have widespread use in both research and therapy. Antisense RNAs function, for example, in various prokaryotic systems, to regulate gene expression. Similarly, antisense RNA can regulate the expression of many prokaryotic genes. This makes it possible to block the expression of undesired or unknown genes in studies intended to identify the function.
[0203]
Investigation and therapeutic use of the polynucleotides of the invention can include in vitro production of the polynucleotides, for example, using subsequent introduction into a cell or subject (in general, Melton, Antisense RNA and). DNA, Cold Cold Spring Harbor (1988)). Polynucleotides can be double-stranded or single-stranded, and single-stranded polynucleotides can be sense or antisense. Polynucleotide fragments can also be used. The use of antisense RNA oligonucleotides to repress transcription and thereby block gene expression is known, and one skilled in the art can readily design antisense oligonucleotides based on the polynucleotides of the present invention. For example, Reeder et al. {Cancer} Res. 58: 3719-26 (1998). In addition, single-stranded sense or double-stranded polynucleotides can be used to prevent gene expression by RNA interference, which can reduce targeted mRNA degradation (also called co-suppression or quelling). I need. Fire et al. , Nature 391: 806-811 (1998); Sharp and Zamore, Science 287: 2431 (2000); and Marx, {Science 288: 1370 (2000).
[0204]
For research and therapeutic applications, the polynucleotide is typically a physiologically acceptable carrier, excipient and / or stabilizer, and / or a component that facilitates introduction of the polynucleotide into cells. Is combined with Physiologically acceptable carriers, excipients and stabilizers are described in Remington's Pharmaceutical Science (1980) 16th edition {Osol} ed. , (1980). Physiologically acceptable carriers, excipients and stabilizers for use in the present invention are non-toxic to cells or objects at the doses used, and include (phosphate buffers, citrate buffers, Buffers (such as buffers made from other organic acids), antioxidants (eg, ascorbic acid), low molecular weight (less than about 10 residues) polypeptides, such as serum albumin, gelatin and immunoglobulins ) Proteins, proteins (such as polyvinylpyrrolidone), hydrophilic polymers, amino acids (such as glycine, glutamine, asparagine, arginine and lysine), monosaccharides, disaccharides, and other carbohydrates (including glucose, mannose and dextrin) ), Chelating reagents (eg, ethylenediaminetetraacetic acid [EDTA]) , Sugar alcohols (such as mannitol and sorbitol), salt-forming counterions (eg sodium), and / or anionic surfactants (such as Tween®, Pluronics® and PEG) Can be. In one embodiment, the physiologically acceptable carrier is an aqueous pH-buffered solution.
[0205]
Components that facilitate intercellular delivery of polynucleotides are known and include, for example, lipids, liposomes, water-oil emulsions, polyethyleneimines and dendrimers, any of which can be used in the compositions of the present invention. Lipids are among the most widely used components of this type, and any available lipid or lipid blend can be used with the polynucleotides of the present invention. Typically, cationic lipids are preferred. Preferred cationic lipids are N- [1- (2,3-dioleyloxy) propyl] -n, n, n, -trimethylammonium chloride (DOTMA), dioleylphosphotidylethanolamine (DOPE), and / or Including dioleyl phosphatidylcholine (DOPC).
[0206]
In another embodiment, a liposomely-entrapped polynucleotide can be used to deliver the polynucleotide to a cell. Liposomes are composed of various types of lipids, phospholipids and / or surfactants. These components are typically arranged in a bilayer formulation that resembles the lipid arrangement of biological membranes. The liposome containing the polynucleotide is produced by a known method, for example, as described in PNAS @ USA 82: 3688-92 (1985) by Epstein et al. And PNAS @ USA 77: 4030-34 (1980) by Hwang et al. Typically, the liposomes in such production are of a small (about 200-800 °) single-layer type, with a lipid content of about 30 mol% cholesterol or higher, and are adjusted to a particular ratio to provide optimal therapy. You. Useful liposomes can be generated by the reverse phase evaporation method using a lipid composition comprising, for example, phosphatidylcholine, cholesterol, and PEG-derived phosphatidylethanolamine (PEG-PE). If desired, the liposomes are extruded through a filter paper of defined pore size to produce liposomes of a particular diameter.
[0207]
The polynucleotide can be conjugated to a dendrimer, which can be used to transfect cells. Dendrimer polycations are highly ordered oligomer or polymer compounds formed by a repetitive reaction sequence that adds oligomers or polymers in three dimensions, typically on a core molecule or well-defined initiator, and it is positively charged. Provide a textured outer surface. Suitable dendrimers include, but are not limited to, "starburst" dendrimers and various dendrimer polycations.
[0208]
The dendrimer polycation is preferably non-covalently associated with the polynucleotide of the present invention. This allows for easy degradation and dissociation once transported into the cell. Typical dendrimer polycations suitably used herein have a molecular weight ranging from about 2,000 to 1,000,000 Da, more preferably from about 5,000 to 500,000 Da. However, other molecular weights can be used. Suitable dendrimer polycations have a hydrodynamic radius of about 11-60 °, and more preferably about 15-55 °. However, other sizes are suitable for use in the present invention. Methods for making and using dendrimers to induce polynucleotides into cells in vivo in vivo are known to those of skill in the art, and include PCT / US83 / 02052, and US Pat. Nos. 4,507,466; 4,558,120. No. 4,568,737; No. 4,587,329; No. 4,631,337; No. 4,694,064; No. 4,713,975; No. 4,737,550; Nos. 4,871,779; 4,857,599; and 5,661,025.
[0209]
For prophylactic or therapeutic uses, the polynucleotides of the invention are formulated in a manner suitable for the particular indication. US Patent No. 6,001,651 to Bennett et al. Describes a number of pharmaceutical compositions and formulations suitable for use with oligonucleotides, as well as methods of administering such oligonucleotides. In a preferred embodiment, a prophylactic or therapeutic composition of the invention comprises a polynucleotide conjugated to a lipid as described above.
[0210]
The compositions of the invention can be stored in any standard form, including, for example, aqueous solutions or lyophilized cakes. Such compositions are typically sterile when administered to cells or recipients. The sterilization of the aqueous solution can be achieved immediately by filtration through a sterilized filtration membrane. If the composition is stored in lyophilized form, the composition can be filtered before or after lyophilization and reconstitution.
[0211]
In some applications, it is advantageous to stabilize the polynucleotides described herein and produce modified polynucleotides to better adapt them for a particular application. For this reason, the polynucleotides of the present invention may contain phosphorothioate, phosphotriester, methylphosphonate, short alkyl or cycloalkyl sugar linkages or short heteroatom or heterocyclic sugar linkages ("backbone"). Including. Most preferred are phosphorothioates and CH₂ -NH-O-CH₂ , CH₂ -N (CH₃ ) -O-CH₂ (Known as methylene (methylimino) or MMI backbone), CH₂ -ON (CH₃ ) -CH₂ , CH₂ -N (CH₃ ) -N (CH₃ ) -CH₂ And ON-N (CH₃ ) -CH₂ -CH backbone (where phosphodiester is OP-O-CH₂ ). Polynucleotides having an ecological backbone structure are also preferred. Summerton, J. et al. E. FIG. And Weller, D .; D. No. 5,034,506. Another preferred embodiment uses a protein-nucleic acid or peptide-nucleic acid (PNA) backbone, wherein the phosphodiester backbone of the polynucleotide is replaced with a polyamide backbone and the base is directly or to the aza nitrogen atom of the polyamide backbone. Join indirectly. P. E. FIG. Nielsen, @M. Egholm, @R. H. Berg, O. Buchardt, {Science} 1991, 254, 1497. The polynucleotides of the present invention may have alkyl or halogen substituted sugar moieties and / or may have sugar analogs such as cyclobutyl instead of pentofuranosyl groups. In other preferred embodiments, the polynucleotide may include at least one modified base form or "universal base" such as inosine. If desired, the polynucleotide may comprise an RNA cleaving group, a cholesteryl group, a reporter group, an intercalator, a group that improves the pharmacokinetic properties of the polynucleotide, and / or a group that improves the pharmacodynamic properties of the polynucleotide. Can be included.
[0212]
V. kit
The materials for use in the method of the invention are ideally suited for the manufacture of kits manufactured according to known procedures. In one embodiment, the kit of the present invention comprises: (1) an antisense primer conjugate comprising an antisense primer linked to an RNA promoter sequence, wherein the RNA promoter sequence is 5 'of the antisense primer. (2) sense primers; and (3) instructions for performing the method of the present invention. The indicating material is a written or printed material, but is not limited thereto. Any medium that can store those instructions and communicate it to the end user is contemplated by the present invention. Such media include, but are not limited to, electrical recording media (eg, magnetic disks, tapes, cartridges, chips), optical media (eg, CD-ROM), and the like. Such a medium may include an address to an Internet site that provides such instructional material. Preferred kits include one or more of the various reagents utilized in the present invention (typically in concentrated form), for example, one or more buffers, a suitable nucleotide triphosphate (eg, dATP, dCTP, dGTP and dTTP; or rATP, rCTP, rGTP and UTP), reverse transcriptase, DNA polymerase and / or RNA polymerase.
[0213]
In another embodiment, a kit of the invention comprises: (1) a polynucleotide pool of the invention, (2) an antisense primer complex as described above, and (3) a sense primer. This kit is useful for producing amplified DNA from a selected pool of polynucleotides. Suitable kits include one or more containers, each containing one or more of reagents for DNA amplification, such as buffers, nucleotide triphosphates and / or DNA polymerases.
[0214]
In yet another embodiment, the kit comprises: (1) a polynucleotide pool of the present invention, and (2) an RNA polymerase capable of transcribing antisense RNA from the selected polynucleotide pool. Suitable kits include one or more containers, each containing one or more of the reagents for antisense RNA production, eg, buffers and / or nucleotide triphosphates.
[0215]
All publications cited herein are expressly incorporated by reference.
[0216]
An example
The following examples are presented by way of illustration, but are not limited to the claimed invention.
[0217]
Example 1
Generation of cDNAs rich in low abundance sequences from human placental mRNA
A. First-strand cDNA and PCR
1. Universal PCR anchor added by template exchange
First strand cDNA was synthesized from human placental poly A + RNA using a random primer linked to the T7 promoter sequence (random-T7 primer): 5'-AAT-TCT-AAT-ACG-ACT-CAC-TAT-AGG. -GNN-NN-NN-3 '(N = A, T, C or G; sequence ID NO: 2). Briefly, 1 μl human placental poly A + RNA (1 μg), 1 μl random-T7 primer (20 μM), 1 μl PCR anchor oligo (also called template exchange oligo:
[Table 1]

(3 '"GGG" shown in bold is a ribonucleotide; sequence ID #NO: 3), and 2 [mu] l of deionized H₂ This was a 5 μl cDNA synthesis mixture containing O. The mixture was incubated at 72 ° C. for 2 minutes and then at 37 ° C. for 2 minutes. The volume was then adjusted to 10 μl with the following reagents: 2 μl of 5 × first strand synthesis buffer (250 mM Tris-HCl, pH 8.3; 30 mM MgCl 2₂ And 375 mM KCl), 0.5 μl dithiothreitol (DTT; 50 mM), 1 μl dNTP mixture (10 mM each dATP, dCTP, dGTP, dTTP), 0.5 μl Rnasin (20 units, promega), 1 μl (200 units) MMLV reverse transcriptase (Superscript II®, Life @ Technologies). Reverse transcription was performed at 42 ° C. for 90 minutes. The reaction was terminated by placing the tube on ice.
[0218]
About 1 pg / μl of the first strand cDNA (about 1 × 10^-6) To remove rare transcripts, and then proceed to PCR with 40 mM Tricine-KOH, pH 9.2; 15 mM KOAc; 3.5 mM Mg (OAc).₂And 0.2 μM 5′-anchor primer (5′-TGC-TGC-GAG-AAG-ACG-ACA-GAA-3 ′); 0.2 μM random-T7 primer; 0.2 mM each dATP, dGTP , DCTP, dTTP; and 2 μl of Advantage® cDNA polymerase mixture (50X; including KlenTaq-1 and Deep @ Vent polymerase; Clontech) in a 100 μl reaction. PCR was performed in DNA Thermal Cycler 480 (PE Biosystems) using the following circulating conditions: 1 minute at 95 ° C; 15 seconds at 95 ° C, 30-35 cycles; 30 seconds at 62 ° C; and 72 ° C. For 6 minutes.
[0219]
After PCR, the double-stranded cDNA was purified by passing through an S-200 spin column (Clontech). Prior to antisense RNA driver synthesis, double-stranded cDNA was tested for glucose 3-phosphate dehydrogenase (G3PDH) and amplification of the c-myc gene. After 25 cycles of PCR amplification, G3PDH was visualized as a dark band on a 1.1% agarose gel stained with ethidium bromide. c-myc was not visualized on ethidium bromide stained gels, even after 35 cycles of amplification.
[0220]
2. Universal PCR anchor added by homopolymer sequencing
First strand cDNA was synthesized on human placental poly A + RNA using the random-T7 primer of Method 1. Briefly, 1 μl human placental poly A + RNA (1 μg), 1 μl random-T7 primer (20 μM), and 3 μl deionized H₂ 5 μl of the cDNA synthesis mixture containing O was incubated at 72 ° C. for 2 minutes and then at 37 ° C. for 2 minutes. The volume was adjusted to 10 μl with the following reagents: 2 μl of 5 × first strand synthesis buffer (250 mM Tris-HCl, pH 8.3; 30 mM MgCl 2₂ And 375 mM KCl), 0.5 μl DTT (50 mM), 1 μl dNTP mixture (10 mM each dATP, dCTP, dGTP, dTTP), 0.5 μl Rnasin (20 units, Promega), 1 μl (200 units) MMLV reverse transcriptase (Superscript II®, Life @ Technologies). Reverse transcription was performed at 42 ° C. for 90 minutes. The reaction was terminated by placing the tube on ice.
[0221]
Homopolymer sequencing was performed as previously reported (DJ Bertoli et al., @BioTechnology, 1994) with the following modifications. 20 μl of deionized H₂ O was added to the first strand cDNA mixture. The diluted first strand mixture was purified using a CHROMA @ SPIN-100 column (Clontech) to remove primers and dNTPs as well as small cDNA fragments. Oligo-dC sequencing was performed by mixing: 30 μl of purified first-strand cDNA; 1 μl of 5 × sequencing buffer (0.5 M potassium cacodylate, pH 7.2, 10 mM @CoCl).₂ 1 mM @DTT), 1 μl of 1 mM @dCTP, 1 μl of terminal transferase (15 units / μl; Life @ Technologies), 8 μl of deionized H₂ O (final volume 50 μl). The reaction mixture was incubated at 37 ° C. for 1 hour, followed by phenol / chloroform extraction and precipitation.
[0222]
About 10 pg / μl of oligo-dC-sequenced cDNA (about 1 × 10^-5) To remove rare transcripts and then proceed to PCR with 40 mM @ Tricine-KOH, pH 9.2; 15 mM @ KOAc; 3.5 mM @ Mg (OAc)₂And 0.2 μM 5′-dG₁₂Primer (5'-d (G)₁₂ -VN-3 ', N = A, G, C or T; V = A, G or C); 0.2 μM random-T7 primer; 0.2 mM of each dATP, dGTP, dCTP, dTTP; 2 μl Advantage The reactions were performed in a 100 μl reaction containing the cDNA polymerase mixture (50X; KlenTaq-1 and Deep @ Vent polymerase; Clontech). PCR was performed in DNA Thermal Cycler 480 (PE Biosystems) using the following circulating conditions: 95 ° C for 1 minute; 95 ° C for 15 seconds @ 30-35 cycles; 52 ° C for 30 seconds; and 72 ° C. 6 minutes.
[0223]
After PCR, the double-stranded cDNA was purified by passing through an S-200 spin column (Clontech). Prior to antisense RNA driver synthesis, double-stranded cDNA was tested for (G3PDH) and amplification of the c-myc gene. After 25 cycles of PCR amplification, G3PDH was visualized as a dark band on a 1.1% agarose gel stained with ethidium bromide. c-myc could not be visualized on ethidium bromide stained gels, even after 35 cycles of amplification.
[0224]
B. Antisense RNA synthesis
Antisense RNA was synthesized by mixing the following: double stranded cDNA (1 μg); 5X RNA translation buffer (200 mM Tris-HCl, pH 8.5; 60 mM MgCl₂ And 350 mM KCl; 25 mM DTT; 2.5 mM ITP; 7.5 mM GTP; 10 mM ATP; 10 mM UTP; 10 mM CTP) and then 40 units of RNasin (Promega) and 80 units of T7 RNA. Polymerase (Boehringer @ Mannheim) was added. H₂ O was added to the final reaction in a volume of 50 μl. The mixture was incubated at 41 ° C. for 90 minutes. Then 10 units of DNase @ I (RNase free; Boehringer @ Mannheim) were added. The reaction was incubated at 37 ° C. for 15 minutes, followed by phenol / chloroform extraction and precipitation.
[0225]
C. Revision hybridization
Regression hybridization was performed using human placental antisense RNA (enriched in high abundance sequences) as a driver and normal human placental mRNA (cognate with antisense RNA) as a “tester”, Table 1 lists the various amounts of antisense RNA and placental mRNA in each mixture.
[0226]

After mixing, the tubes were incubated at 68 ° C for 20 minutes in a PE9600 {Thermal} Cycler. The temperature was then raised to 45 ° C. during the 16 h incubation.
[0227]
D. cDNA synthesis
(Above) Samples A, B, C and D were converted to oligo d (T)₃₀ Primer (5'-d (T)₃₀ -VN-3 ', N = A, G, C or T; V = A, G or C) was used as a template for first strand cDNA synthesis. Briefly, 2 μl of 10 mM oligo d (T)₃₀ Was added to each sample, followed by a 2 minute incubation at 72 ° C. The reaction mixture was then immediately transferred to an ice bath for 2 minutes. The volume was adjusted to 10 μl with the following reagents: 2 μl of 5 × first strand synthesis buffer (250 mM Tris-HCl, pH 8.3; 30 mM MgCl 2₂ And 375 mM KCl), 0.5 μl DTT (50 mM), 1 μl dNTP mixture (10 mM each dATP, dCTP, dGTP, dTTP), 0.5 μl Rnasin (20 units, Promega), 1 μl (200 units) MMLV reverse transcriptase (Superscript II®, Life @ Technologies). Reverse transcription was performed at 42 ° C. for 90 minutes. The reaction was terminated by placing the tube on ice.
[0228]
E. FIG. Oligo-dG sequencing
After first strand synthesis, the RNA was degraded by adding 100 mM @ 1 μl NaOH to each first strand synthesis mixture and incubating at 68 ° C for 30 minutes. Then 19 μl of deionized H₂ O was added to each first-strand mixture and the DNA was purified using a Chroma Spin 100 column (Clontech). Oligo-dG sequencing was performed by mixing: 30 μl of purified first strand cDNA; 1 μl of 5X sequencing buffer (0.5 M potassium cacodylate, pH 7.2, 10 mM @CoCl).₂ , 1 mM @DTT), 1 μl 1 mM @dGTP, 1 μl terminal transferase (15 units / μl; Life Technologies), 8 μl deionized H₂ O (final volume 50 μl). The reaction mixture was incubated at 37 ° C. for 1 hour, followed by phenol / chloroform extraction and precipitation.
[0229]
F. Double cycle cDNA synthesis by low cycle PCR and analysis of PCR products
1. 0.9 kb placental transcript:
The reduction of low abundance sequences in Samples AD was performed by examining the level of cDNA corresponding to 0.9 kb placenta "housekeeping" transcript. The low cycle PCR amplification was performed by pelleting the pellet obtained in the previous step with 40 mM Trinic-KOH, pH 9.2; 15 mM KOAc; 3.5 mM Mg (OAc).₂And 0.2 μM 5′-dC₁₂Primer (5'-d (C)₁₂ -VN-3 ', N = A, G, C or T; V = A, G or C); 0.2 μM oligo d (T)₃₀ Primers; 0.2 mM each of dATP, dGTP, dCTP, dTTP; dissolved in 100 μl reaction containing 2 μl Advantage® cDNA polymerase mixture (50X; KlenTaq-1 and Deep @ Vent polymerase; including Clontech) It was performed by. PCR was performed in DNA Thermal Cycler 480 (PE Biosystems) using the following circulating conditions: 95 ° C for 1 minute; 95 ° C for 15 seconds 8 cycles; 52 ° C for 30 seconds; and 72 ° C for 6 minutes. .
[0230]
The PCR products from Samples A, B, C and D were loaded onto a 1.1% agarose gel (5 μl / well) with 200 ng {1 kb} DNA ladder (Life® Technologies). Staining with ethidium bromide indicated that the banding profile changed with the ratio of antisense mRNA driver to placental mRNA. In particular, the intensity of the band at 0.9 kb, which corresponds to the human placenta housekeeping gene, decreased with increasing antisense RNA / mRNA ratio and was hardly detectable in sample D, because the presence of the antisense RNA driver was high. Shows that they hybridize to the RNA sequences and reduce them.
[0231]
2. Glucose 3-phosphate dehydrogenase (G3PHD):
The deletion of the high abundance sequence in samples AD was also performed by examining the level of the cDNA corresponding to the housekeeping gene G3PHD. Samples A, B, C and D were prepared using 40 mM Tricine-KOH, pH 9.2; 15 mM KOAc; 3.5 mM Mg (OAc).₂And 0.2 μM 5 ′ and 3 ′ G3PHD @ Amplifier set (Clontech); 0.2 mM each of dATP, dGTP, dCTP, dTTP; 2 μl of Advantage® cDNA polymerase mixture (50 ×; KlenTaq-1 and Deep @ Vent Polymerase; including Clontech) in a 50 μl reaction mixture. PCR was performed in DNA Thermal Cycler 480 (PE Biosystems) using the following circulating conditions: 1 minute at 95 ° C; 15 seconds at 95 ° C @ 25 cycles; 2 minutes at 68 ° C.
[0232]
The PCR products from Samples A, B, C and D were loaded onto a 1.1% agarose gel (5 μl / well) with 200 ng {1 kb} DNA ladder (Life® Technologies). In the staining using ethidium bromide, the dark color of the band corresponding to the housekeeping gene G3PHD decreased with an increase in the ratio of antisense RNA / mRNA, and was hardly detectable in sample D. It is confirmed that the mRNA sequence is deleted.
[0233]
3. c-myc:
Using the c-myc gene as a marker, the enrichment of low abundance sequences in samples AD was assayed. Each sample was PCR amplified as described in the G3PHD study, but using the 5 ′ and 3 ′ c-myc @ Amplifier sets (Clontech) as primers, and PCR was performed using the following cycling conditions: 95 C for 1 minute; 95C for 15 seconds @ 30 cycles; 68C for 2 minutes.
[0234]
The PCR products from Samples A, B, C and D were loaded onto a 1.1% agarose gel (5 μl / well) with 200 ng {1 kb} DNA ladder (Life® Technologies). Staining with ethidium bromide showed that the dark color of the c-myc display decreased with increasing ratio of antisense RNA / mRNA. This result indicates that c-myc is present in normal human placental RNA, but not in the antisense RNA driver, and reduced hybridization, followed by amplification at low abundance such as c-myc. Generate a sequence-rich polynucleotide pool.
[0235]
Example 2
Production of cDNAs rich in low abundance sequences from mRNAs produced from various tissues
To test the reproducibility of the method of the invention, antisense RNA drivers were generated from human brain, fetal brain, liver and kidney mRNA as described in Example 1. MRNA samples from each tissue underwent reduced hybridization using an antisense RNA driver made from the same tissue (Group A). A negative control (ie, not treated with antisense RNA) was included for each sample (Group B). After the rounding, low-cycle PCR was performed to generate a double-stranded cDNA corresponding to an unhybridized mRNA sequence.
[0236]
The abundance sequence enrichment was assayed using the P50 gene as a marker. Each sample was prepared using 40 mM Tricine-KOH, pH 9.2; 15 mM KOAc; 3.5 mM Mg (OAc).₂And 0.2 μM of 5 ′ and 3 ′ P50 Amplifier set (Clontech); 0.2 mM of each dATP, dGTP, dCTP, dTTP; 2 μl of Advantage® cDNA polymerase mixture (50 ×; KlenTaq-1 and Deep @ Vent Polymerase; including Clontech) in a 50 μl reaction. The PCR was performed in DNA Thermal Cycler 480 (PE Biosystems) using the following circulating conditions: 1 minute at 95 ° C; 15 seconds at 95 ° C 30 cycles; 2 minutes at 68 ° C.
[0237]
Analysis of PCR products was performed by electrophoresis on a 1.1% agarose gel (5 μl / well), followed by staining with ethidium bromide, group A samples (rich in low abundance sequences) containing the P50 sequence However, the Group B sample (negative control) showed no detectable P50 sequence. The results show that the method of the present invention is effective for producing a selected polynucleotide pool regardless of tissue type.
[0238]
Example 3
Generation of cDNAs rich in variable length low abundance sequences produced from human testis mRNA
To test for enrichment of low-abundance sequences of varying length, antisense RNA drivers were generated from human testis mRNA as described in Example 1. Human testis mRNA samples underwent hybridization using an antisense RNA driver (generating group A samples). Negative controls (ie, not treated with antisense RNA) were included (group B samples were generated). After the rounding, low-cycle PCR was performed to generate a double-stranded cDNA corresponding to an unhybridized mRNA sequence.
[0239]
Using interleukin-6 (IL6, 0.6 kb ORF fragment), P50 (2.8 kb ORF) and insulin growth factor receptor (IGFR; 4.8 kb ORF fragment) as markers, low abundance gene enrichment and size indication Checked. A and B samples were prepared using 40 mM Tricine-KOH, pH 9.2; 15 mM KOAc; 3.5 mM Mg (OAc).₂And 0.2 μM of 5 ′ and 3 ′ IL6-, P50 or IGFR specific primer set (Clontech); 0.2 mM of each dATP, dGTP, dCTP, dTTP; 2 μl of Advantage® cDNA polymerase mixture (50 × PCR amplification in a 50 μl reaction containing KlenTaq-I and Deep @ Vent polymerase; including Clontech). PCR was performed in DNA Thermal Cycler 480 (PE Biosystems) using the following circulating conditions: 1 minute at 95 ° C; 15 seconds at 95 ° C @ 30 cycles; 5 minutes at 68 ° C.
[0240]
Analysis of the PCR products was performed by electrophoresis on a 1.1% agarose gel (5 μl / well), followed by staining with ethidium bromide to give Group A samples (rich in low abundance sequences) to IL6, Although shown to contain bands corresponding to P50 or IGFR sequences, Group B samples (negative controls) showed no detectable bands corresponding to these sequences. The results show that the method of the present invention is enriched for low abundance transcripts of varying sizes.
[0241]
Example 4
Preferred curative hybridization method for identifying low abundance polynucleotides that differ between test and reference samples
This example provides a protocol for a preferred embodiment of the general deletion hybridization method of the present invention. This embodiment uses a high abundance enriched test polynucleotide pool and a low abundance enriched reference polynucleotide pool, which are generated by the exemplary selection method of the invention.
[0242]
A. Production of high-abundance sequencing-rich antisense test RNA pools
High abundance-enriched antisense RNA pools are prepared from test RNA samples as shown in FIG. 1 and described below.
1.First strand (antisense) test cDNA is synthesized from a test RNA sample using a mixture of reverse transcriptase and antisense primer complexes. Each antisense primer complex contains a random sequence, which acts as an antisense primer. Link the T7 RNA promoter sequence to the 5 'end of the random sequence.
2.'Add a universal primer site to the 3' end of the antisense test cDNA strand by oligonucleotide sequencing. In this example, dG sequencing is used to produce a poly-G sequence.
3.連 続 Serially dilute the reaction mixture from step 2 to remove low abundance sequences. Removal of such sequences is monitored by PCR as in the example above.
4.The diluted reaction mixture is then subjected to PCR using a 5 'universal primer that hybridizes to the 3' universal primer site of the newly synthesized antisense test cDNA strand. As shown in FIG. 1B (4), the 3 'primer hybridizes to the T7 RNA promoter sequence at the 5' end of the antisense test cDNA strand. PCR produces a pool of double-stranded test cDNA molecules that are rich in high abundance sequences.
5.Antisense test RNA is synthesized from high abundance sequence-rich cDNA molecules using T7 RNA polymerase to generate a driver for use in the first trimming hybridization of Procedure C (Step C.1, below). Let it. This driver is rich in high abundance test RNA sequences.
[0243]
B. Production of a pool of sense reference cDNA strands rich in low abundance sequences
The antisense reference RNA was prepared using A. Produced as described for antisense test RNA in 5 (above). Antisense reference RNA enriched in high abundance sequences is used to generate a low abundance enriched pool of sense reference cDNA strands, as shown in FIG. 2 and described below.
1.Antisense reference RNA is used as a driver in a subtraction hybridization reaction using a reference mRNA to block high abundance mRNA, leave low abundance reference mRNA molecules free, and provide a template for first strand cDNA synthesis. Work as First strand (antisense) reference cDNA is synthesized using a reverse transcriptase and an antisense primer conjugate containing an oligo-dT sequence linked to the SP6 RNA promoter sequence at the 5 'end. (These primer and promoter sequences must be different from those used in A.1 above.)
2.'A universal primer site is added to the 3' end of the antisense reference cDNA strand by oligonucleotide sequencing. The universal primer site is described in the above A.I. 2. Must be different from that added to the antisense test cDNA strand, and therefore dC sequencing is used to generate poly dC sequences.
3.Then B. 2. Is treated with 100 mM NaOH at 68 ° C. for 60 minutes to degrade the reference mRNA. Leave the low abundance-enriched antisense reference cDNA strand.
4.The antisense reference cDNA strand is then hybridized to the 3 'universal primer site at the 5' universal primer (here poly-G), and the antisense reference cDNA (here poly-A) linked to the SP6 promoter sequence. PCR is performed using a 3 'antisense primer that hybridizes to the 5' antisense primer site. PCR produces a pool of double-stranded reference cDNA molecules that are rich in low abundance sequences.
5.Antisense reference RNA is synthesized from a low abundance-enriched reference cDNA molecule using SP6 polymerase.
6.The 'sense reference cDNA strand is synthesized from the antisense reference RNA using reverse transcriptase and a 5' universal primer containing a restriction site at the 5 'end (e.g., Alu I linking to poly-G). This restriction site is cleaved by an enzyme specific to double-stranded DNA. This embodiment facilitates the selective cleavage of the hybridized sequence after the second hybridization reaction of the general hybridization method of C (below).
7.Then B. The reaction mixture of 6 is treated with 100 mM NaOH at 68 ° C. for 60 minutes to degrade the antisense reference RNA, leaving the sense reference cDNA.
8.参照 The resulting reference cDNA strand is rich in low abundance sequences and can be used in the second trimming hybridization of C's general trimming hybridization method. In order to reduce non-specific hybridization between the poly-A and poly-T sequences, the poly-A sequence of the sense reference cDNA strand is used before the second trimming hybridization reaction of the present invention to use . Elimination by partial digestion with E. coli exonuclease I. Suitable reaction conditions are determined (eg, by setting up multiple reactions of about 5 to about 3 minutes and monitoring poly-A removal by amplification using oligonucleotide-dT primers). Determine empirically.
[0244]
C. General curative hybridization between test and reference polynucleotides
1.に お い て In the first reduction hybridization reaction using the test mRNA, 5. The low abundance test mRNA molecule is left free to block the high abundance test mRNA molecule and serve as a template for first-strand cDNA synthesis using the antisense test RNA as a driver. The low abundance-enriched first-strand (antisense) test cDNA was isolated from an unhybridized test mRNA molecule by the use of reverse transcriptase and antisense Synthesize using primers (primer sequence must be different from that used in B.2, but may be the same or different from that used in A.1) There can be). One restriction site (eg, AluΔI) is cleaved by an enzyme specific for double-stranded DNA. This embodiment is used to facilitate the selective cleavage of the hybridized sequence after the second hybridization reaction of the general hybridization of method C (below). Other restriction sites are used to clone the test-specific polynucleotide into a cloning vector after the second hybridization reaction.
2.Then C. 2. Is treated with 100 mM NaOH at 68 ° C. for 60 minutes to degrade the test mRNA. Leave the low abundance-enriched antisense test cDNA strand.
3.B. 7. Used as a driver in a subtraction hybridization reaction using the antisense test cDNA strand of step 3 to block the antisense test cDNA strand corresponding to the polynucleotide sequence present in both the test and reference samples. I do. This second hybridization reaction produces a double-stranded cDNA molecule, an unhybridized low abundance enriched antisense test cDNA strand and an unhybridized low abundance enriched sense reference cDNA strand (ie, driver). I do. The unhybridized antisense test cDNA strand corresponds to the test-specific sequence, while the hybrid duplex corresponds to the sequence present in both the test and reference samples.
4.The “sticky ends” of the hybrid duplex are filled in using Taq polymerase to generate a double-stranded Alu I restriction site.
5.Oligonucleotide sequencing is performed using dC to add a poly-C sequence to all molecules in the reaction mixture.
6.The hybrid duplex is digested with Alu I, which cleaves the 3 'poly-C sequence at both ends of the duplex. This reaction leaves intact unhybridized low abundance enriched antisense test cDNA strand, and unhybridized low abundance enriched sense reference cDNA strand (ie, driver). However, the driver has 5 'poly-G and 3' poly-C sequences that self-anneal, preventing amplification.
7.変 換 Convert the low abundance-enriched antisense test cDNA strand into a double-stranded test cDNA molecule by selective PCR using suitable primers. In this example, the 5 'primer contains poly-G and the 3' primer contains an Sfi I sequence that binds to the Sfi I sequence at the 5 'end of the antisense test cDNA strand. The 5 'primer also contains a restriction site at the 5' end (here, Not I). PCR produces a pool of reduced cDNA molecules rich in low abundance test sequences and different sequences between the starting test and reference RNA samples. Each double-stranded molecule has a Not I restriction site at the 5 'end and an Sfi I restriction site at the 3' end.
8.The mixture of 7 is digested using Sfi I and Not I and ligated into a suitable vector to generate a library of test-specific clones with low abundance enrichment. Sfi @ I and Not @ I are "infrequent @cutte", leaving almost all test-specific sequences intact, thus enhancing recovery of full-length cDNA. The use of two different restriction sites in this procedure facilitates directional cloning into an expression vector and facilitates rapid analysis of subsequently expressed genes.
[Brief description of the drawings]
FIG. 1A is a schematic diagram of a preferred embodiment of a selection method of the invention that is useful for producing a pool of polynucleotides that are enriched in high abundance sequences relative to a polynucleotide sample.
FIG. 1B is a schematic diagram of a preferred embodiment of the selection method of the present invention that is useful for producing a pool of polynucleotides that are enriched in high abundance sequences as compared to a polynucleotide sample.
FIG. 2A is a schematic diagram of a preferred embodiment of the second selection method of the present invention.
FIG. 2B is a schematic diagram of a preferred embodiment of the second selection method of the present invention.
FIG. 2C is a schematic diagram of a preferred embodiment of the second selection method of the present invention.
FIG. 3A is a schematic diagram of a preferred embodiment of the general hybridization method of the present invention.
FIG. 3B is a schematic diagram of a preferred embodiment of the general hybridization method of the present invention.
FIG. 3C is a schematic diagram of a preferred embodiment of the general hybridization method of the present invention.
FIG. 3D is a schematic view of a preferred embodiment of the general hybridization method of the present invention.

Claims (94)

A method of reducing hybridization for identifying one or more polynucleotides in a test sample that is absent or has a low abundance in a reference sample, the method comprising:
a) providing a high abundance-enriched polynucleotide chain of or produced from a pool of test or reference polynucleotides that is enriched in the high abundance polynucleotide sequence as compared to a test or reference polynucleotide sample, respectively;
b) contacting the high abundance-enriched polynucleotide strand with a test polynucleotide strand of a test polynucleotide sample or produced therefrom under hybridization conditions to form a first hybridization mixture; Generating an unhybridized test polynucleotide strand that is enriched in the low abundance polynucleotide sequence as compared to the nucleotide sample;
c) synthesizing a polynucleotide chain from the unhybridized test polynucleotide chain, thereby producing a low abundance-enriched test polynucleotide chain;
d) providing a low abundance-enriched reference polynucleotide strand of or a reference pool of polynucleotides enriched in the low abundance polynucleotide sequence compared to a reference polynucleotide sample;
e) contacting the low abundance-enriched reference polynucleotide strand with the low abundance-enriched test polynucleotide strand under hybridization conditions to form a second hybridization mixture, whereby the hybrid duplex, hybrid Generating an unsoyered low abundance-enriched reference polynucleotide strand and an unhybridized low abundance-enriched test polynucleotide strand;
f) removing or digesting the hybrid duplex; and g) producing a test-specific duplex from the unhybridized low abundance-enriched test polynucleotide strand;
The method comprising: 2. The method of claim 1, wherein the high abundance-enriched polynucleotide strand is of or produced from a pool of test polynucleotides that are enriched in high abundance polynucleotide sequences as compared to a test polynucleotide sample. The high abundance-enriched polynucleotide strand of (a) comprises a high abundance-enriched antisense polynucleotide chain;
(B) the test polynucleotide strand comprises a sense test polynucleotide strand;
The low abundance-enriched test polynucleotide strand of (c) comprises an antisense test polynucleotide strand; and the low abundance-enriched reference polynucleotide strand of (d) comprises a sense reference polynucleotide strand;
The method of claim 1. 4. The method of claim 3, wherein the antisense test polynucleotide strand is synthesized from the unhybridized sense test polynucleotide strand using a first antisense primer or a first antisense primer complex. The method wherein the one antisense primer complex comprises a first antisense primer operably linked to a first RNA promoter sequence, wherein the first RNA promoter sequence is 5 'of the first antisense primer. The first antisense primer or first antisense primer complex comprises:
a) a sequence that binds to a primer site in the unhybridized sense test polynucleotide strand;
b) The first restriction site 5 'of the sequence of (a), wherein the first restriction site cleaves the double-stranded polynucleotide but leaves the single-stranded polynucleotide substantially intact by a restriction endonuclease. C) being cleaved; and c) the first universal primer site 5 'of the restriction site of (b),
5. The method of claim 4, comprising: 6. The method of claim 5, wherein the antisense test polynucleotide strand is synthesized from the unhybridized sense test polynucleotide strand using a first antisense primer, wherein the first universal primer site of (c) is The method comprising a second restriction site, wherein the second restriction site is different from the first restriction site. The sense reference polynucleotide strand comprises a third restriction site previously added to the 5 'end of the sense reference polynucleotide strand, which cleaves the double stranded polynucleotide but replaces the single stranded polynucleotide. 6. The method of claim 5, which is cleaved by a restriction endonuclease that remains substantially intact. The method of claim 1, wherein the hybrid duplex is digested with at least one enzyme. 9. The method of claim 8, wherein said enzyme is a restriction endonuclease. Removal or digestion of the hybrid duplex of (f):
i) The second hybridization mixture is an enzyme that causes the single-stranded portion of the hybrid duplex to double-stranded, thereby creating double-stranded first, second and third restriction sites within the hybrid duplex. Processing;
ii) adding a second universal primer site to the 3 'end of the polynucleotide strand in the second hybridization mixture; and iii) treating the second hybridization mixture with one or more restriction endonucleases, wherein The restriction endonuclease cleaves the hybrid duplex at the duplex first and third restriction sites, but leaves the antisense test polynucleotide strand and the sense reference polynucleotide strand substantially intact. The method of claim 7 comprising: 11. The method of claim 10, wherein the test-specific duplex is generated from an unhybridized antisense test polynucleotide strand by amplification. The test-specific duplex is hybridized using a first universal primer that hybridizes as a 3 'primer to the first universal primer site and a second universal primer that hybridizes to the second universal primer as the 5' primer. 12. The method of claim 11, wherein the antisense test polynucleotide is synthesized from an unsensed polynucleotide strand. 12. The method of claim 11, wherein said amplification is performed by an enhanced polymerase chain reaction. The method of claim 1, wherein the molar ratio of the abundant enriched polynucleotide strand to the test polynucleotide strand in the first hybridization mixture is from about 1 to about 100: 1. The method of claim 1, wherein the molar ratio of the low abundance-enriched reference polynucleotide chain to the low abundance-enriched test polynucleotide chain in the second hybridization mixture is from about 1 to about 100: 1. 2. The method of claim 1, wherein the test and reference polynucleotide samples are mRNA samples. 4. The method of claim 3, wherein the high abundance-enriched antisense polynucleotide strand is an antisense RNA molecule. 4. The method of claim 3, wherein the sense test polynucleotide strand is an mRNA molecule. 4. The method of claim 3, wherein the antisense test polynucleotide strand is an antisense cDNA molecule. 4. The method of claim 3, wherein the sense reference polynucleotide strand is a sense cDNA molecule. 4. The method of claim 3, wherein the synthesis of the antisense test polynucleotide strand is primed using oligonucleotide-dT priming. The test and reference polynucleotide samples are test and reference mRNA samples, and the high abundance-enriched antisense polynucleotide strand is:
a) synthesizing a first antisense cDNA strand from a test or reference mRNA sample using a second antisense primer complex that includes a second antisense primer operably linked to a second RNA promoter sequence. And the second RNA promoter sequence is 5 'of the second antisense primer;
b) adding a third universal primer site to the 3 'end of the first antisense cDNA strand;
c) diluting the first antisense cDNA strand to substantially remove at least some low abundance antisense antisense cDNA strands;
d) generating a first double-stranded cDNA molecule from the remaining first antisense cDNA strand, wherein the first double-stranded cDNA molecule is rich in high abundance polynucleotide sequences as compared to the starting mRNA sample;
6. The method of claim 5, which is a test or reference antisense RNA molecule produced by a method comprising: e) synthesizing a high abundance-enriched antisense RNA molecule from a double-stranded cDNA molecule. The sense reference polynucleotide is:
a) providing a high abundance-enriched antisense RNA molecule of or produced from a pool of reference polynucleotides that is enriched in high abundance polynucleotide sequences as compared to a reference polynucleotide sample;
b) contacting the high abundance-enriched antisense RNA molecule with a reference mRNA sample under hybridization conditions to form a hybridization mixture, thereby hybridizing with a low abundance polynucleotide sequence compared to the reference mRNA sample. To generate unmRNA molecules;
c) synthesizing a second antisense cDNA strand from the unhybridized mRNA molecule using a third antisense primer or a third antisense primer conjugate, wherein the third antisense primer conjugate is a third RNA promoter sequence A third antisense primer operably linked to a third RNA promoter sequence 5 'of the third antisense primer;
d) adding a fourth universal primer site to the 3 'end of the second antisense cDNA strand;
e) generating a second double-stranded cDNA molecule from the second antisense cDNA strand;
f) synthesizing a second antisense RNA molecule from the second double-stranded cDNA molecule; and g) synthesizing a first sense cDNA strand from the second antisense RNA molecule.
23. The method of claim 22, which is a sense cDNA strand produced by a method comprising: 2. The method of claim 1, further comprising cloning at least one test-specific duplex into a vector. 25. The method of claim 24, further comprising generating a polynucleotide library from said test-specific duplex. 25. The method of claim 24, wherein the cloned test-specific duplex encodes a polypeptide and the vector is an expression vector. 27. The method of claim 26, further comprising directing the expression vector into a host cell and expressing the protein encoded by the cloning test specific duplex. 5. The method of claim 4, wherein the test-specific duplex is synthesized from an unhybridized antisense test polynucleotide strand using a first antisense primer complex, wherein the method further comprises one additional step. The method comprising synthesizing comprising synthesizing one or more antisense RNA molecules from a test-specific duplex. 29. The method of claim 28, further comprising directing one or more antisense RNA molecules synthesized from the test-specific duplex into the cell. 30. The method of claim 29, wherein the cells are in vitro. 2. The method of claim 1, comprising using one or more test-specific duplexes or one or more polynucleotides directly or indirectly generated therefrom in a hybridization reaction. 32. The method of claim 31, wherein at least one of the test-specific duplexes or a polynucleotide produced therefrom is labeled with a detectable label. 2. The method of claim 1, further comprising binding a plurality of test-specific duplexes or polynucleotides generated therefrom to the substrate to generate a polynucleotide array. 2. The method of claim 1, further comprising amplifying one or more test-specific duplexes. 35. The method of claim 34, wherein the one or more test-specific duplexes are amplified using one or more gene-specific primers. The method of claim 1, wherein the test and reference polynucleotide samples are different samples. Test and reference polynucleotide samples include:
MRNA from a first cell or tissue and mRNA from a second different cell or tissue;
MRNA from a cell or tissue in a first stage of differentiation or growth and mRNA from a cell or tissue in a second different stage of differentiation or growth;
MRNA from a cell or tissue treated with an activating reagent and mRNA from a cell or tissue not treated or treated with a second different activating reagent;
MRNA from normal cells or tissues and mRNA from diseased cells or tissues,
37. The method of claim 36 selected from one of the following: 38. The method of claim 37, wherein the test and reference polynucleotide samples are mRNA from normal cells or tissues and mRNA from diseased cells or tissues, wherein the disease is an infection or cancer. A method for functionally isolating a single-stranded polynucleotide in a mixture of single- and double-stranded polynucleotides, wherein the mixture of single- and double-stranded polynucleotides comprises one or more The method comprising contacting a restriction endonuclease with a form that cannot be used as a template for a nucleotide synthesis reaction using the single-stranded polynucleotide as a template under conditions sufficient to digest the double-stranded polynucleotide. 40. The method of claim 39, wherein said restriction endonuclease cleaves primer sites from double-stranded polynucleotides in said mixture. 41. The method of claim 40, wherein the uncleaved single-stranded polynucleotide is converted to a double-stranded polynucleotide by amplification. A plurality of polynucleotides produced by subtractive hybridization between a test and a reference polynucleotide sample according to the method of claim 1, wherein the plurality of polynucleotides are at least 10 ^3. Comprising different polynucleotides, and:
Being present in the reference polynucleotide sample at a substantially lower concentration than not present in the reference polynucleotide sample or present in the test polynucleotide sample; and
Low abundance sequences compared to test polynucleotide samples,
The plurality of polynucleotides substantially enriched in a sequence that is: 43. The plurality of polynucleotides of claim 42, wherein each polynucleotide comprises an RNA promoter sequence and a universal primer site. 43. The plurality of polynucleotides of claim 42, wherein the polynucleotide is a double-stranded cDNA molecule. 43. The plurality of polynucleotides of claim 42, wherein said polynucleotide is an antisense RNA molecule. a)
i) a sequence that binds to a primer site in an unhybridized sense test polynucleotide strand;
ii) a first restriction site 5 'of the sequence of (i), wherein the first restriction site cleaves the double-stranded polynucleotide but leaves the single-stranded polynucleotide substantially intact; Said first restriction site 5 ′ cleaved by;
iii) the first universal primer site 5 'of the restriction site of (iii),
A kit comprising: an antisense primer or an antisense primer complex comprising: and b) instructions for performing the method of claim 3. a) the plurality of polynucleotides of claim 42;
b) an antisense primer complex comprising an antisense primer operably linked to an RNA promoter sequence, wherein the RNA promoter sequence is 5 'of the antisense primer; and c) a sense primer;
Kit containing. a) a plurality of polynucleotides of claim 42; and b) an RNA polymerase capable of transcribing antisense RNA from the plurality of polynucleotides;
Kit containing. A method of producing a polynucleotide pool selected from a polynucleotide sample,
a) comprising an antisense primer operably linking the first antisense polynucleotide strand from a polynucleotide sample or sense polynucleotide produced therefrom to an RNA promoter sequence, wherein the RNA promoter sequence is 5 ′ of the antisense primer. Synthesized using an antisense primer conjugate that is:
b) adding a universal primer site to the 3 'end of the first antisense polynucleotide strand;
c) diluting the first antisense polynucleotide strand to substantially remove at least some low abundance first antisense polynucleotide strand;
d) generating from the remaining first antisense polynucleotide strand a first double-stranded polynucleotide that is enriched in high abundance polynucleotide sequence as compared to the polynucleotide sample;
The method comprising: The polynucleotide sample is an mRNA sample, the first antisense polynucleotide strand is a first antisense cDNA strand, and the first double-stranded polynucleotide is a first double-stranded cDNA molecule. 49 methods. 51. The method of claim 50, wherein the synthesis of the first antisense cDNA strand is primed using a random primer or an oligonucleotide dT-primer. 51. The method of claim 50, wherein a universal primer site is added to the 3 'end of the first antisense cDNA strand by template exchange, oligonucleotide sequencing or ligation. 50. The method of claim 49, wherein the RNA promoter sequence is an RNA promoter sequence recognized by a bacteriophage RNA polymerase selected from the group consisting of T7, T3, and SP6. A first double-stranded polynucleotide is generated by amplifying the remaining first antisense polynucleotide strand, and the amplification hybridizes to the universal primer site as a 5 'primer and as a 3' primer. 50. The method of claim 49, wherein the method is performed using an antisense primer complex of 55. The method of claim 54, wherein said amplification is performed by an enhanced polymerase chain reaction. 50. The method of claim 49, wherein the first double-stranded polynucleotide is produced using an enzyme mixture comprising DNA polymerase, DNA ligase and RNase. 50. The method of claim 49, further comprising synthesizing a first antisense RNA molecule from the first double-stranded polynucleotide. a) contacting a first antisense RNA molecule with a polynucleotide sample or a sense polynucleotide strand produced therefrom under hybridization conditions to form a hybridization mixture, thereby comparing the polynucleotide sample to Generating an unhybridized sense polynucleotide strand enriched in the low abundance polynucleotide sequence;
b) synthesizing a second antisense polynucleotide from the unhybridized sense polynucleotide strand using an antisense primer or an antisense primer complex, wherein the antisense primer complex is operable with an RNA promoter sequence The RNA promoter sequence is 5 'of the antisense primer;
c) adding a universal primer site to the 3 'end of the second antisense polynucleotide strand; and d) generating a second double-stranded polynucleotide from the second antisense polynucleotide strand;
58. The method of claim 57, further comprising: 59. The method of claim 58, wherein the molar ratio of the first antisense RNA molecule to the other polynucleotide in the hybridization mixture is from about 1 to about 100: 1. The polynucleotide sample is an mRNA sample, the sense polynucleotide strand is an mRNA molecule in the mRNA sample, the second antisense polynucleotide strand is a second antisense cDNA strand, and the second duplex 59. The method of claim 58, wherein said polynucleotide is a second double-stranded cDNA molecule. 61. The method of claim 60, wherein the synthesis of the second antisense cDNA strand is primed using oligonucleotide dT-priming. 61. The method of claim 60, wherein a universal primer site is added to the 3 'end of the second antisense cDNA strand by template exchange, oligonucleotide sequencing or ligation. A second antisense polynucleotide strand is synthesized from the unhybridized sense polynucleotide strand using an antisense primer complex, and the RNA promoter sequence of (b) is selected from the group consisting of T7, T3 and SP6. 59. The method of claim 58, comprising an RNA promoter sequence for the RNA polymerase. A second double stranded polynucleotide is generated by amplifying the second antisense polynucleotide strand, and the amplification hybridizes to the universal primer site as a 5 'primer and an antihybrid as a 3' primer. 59. The method of claim 58, wherein said method is performed using a sense primer or antisense primer complex. 65. The method of claim 64, wherein said amplification is performed by an enhanced polymerase chain reaction. 59. The method of claim 58, wherein the second double-stranded polynucleotide is produced using an enzyme mixture comprising DNA polymerase, DNA ligase and RNase. 59. The method of claim 58, wherein each of the universal primer and / or the antisense primer or antisense primer complex comprises a restriction site. 59. The method of claim 58, wherein the second antisense polynucleotide strand is synthesized from the unhybridized sense polynucleotide strand using an antisense primer complex, and further comprising the antisense from the second double stranded polynucleotide. The method comprising synthesizing a sense RNA molecule. 59. The method of claim 58, comprising using a second double-stranded polynucleotide, or a polynucleotide produced directly or indirectly therefrom, in a hybridization reaction. A method of producing a polynucleotide pool selected from a polynucleotide sample,
a) hybridizing a first antisense polynucleotide strand produced from the first polynucleotide sample to a second polynucleotide sample or to a sense polynucleotide strand produced therefrom, wherein the first antisense polynucleotide strand Produces an unhybridized sense polynucleotide strand that is rich in high abundance polynucleotide sequences as compared to the first polynucleotide sample, thereby rich in low abundance polynucleotide sequences compared to the second polynucleotide sample ;
b) synthesizing a second antisense polynucleotide strand from the unhybridized sense polynucleotide strand using an antisense primer or an antisense primer complex, wherein the antisense primer complex is an RNA promoter Including an antisense primer operably linked to the sequence, wherein the RNA promoter sequence is 5 'of the antisense primer;
c) adding a universal primer site to the 3 'end of the second antisense polynucleotide strand;
d) generating a double-stranded polynucleotide from the second antisense polynucleotide chain;
The method comprising: 71. The method of claim 70, wherein the molar ratio of the first antisense polynucleotide strand to other polynucleotides in the hybridization mixture is from about 1 to about 100: 1. The first and second polynucleotide strands are mRNA samples, the sense polynucleotide strand is an mRNA molecule, the first antisense polynucleotide strand is an antisense RNA, and the second antisense polynucleotide strand is an antisense cDNA strand. 70. The method of claim 70, wherein the double-stranded polynucleotide is a double-stranded cDNA molecule. A double-stranded polynucleotide is generated by amplifying a second antisense polynucleotide strand, and the amplification hybridizes to a universal primer site as a 5 'primer and an antisense primer as a 3' primer or 71. The method of claim 70, which is performed using an antisense primer conjugate. 71. The method of claim 70, wherein the universal primer and / or the antisense primer or antisense primer complex each comprise a restriction site. 71. The method of claim 70, further comprising cloning at least one double stranded polynucleotide into a vector. 76. The method of claim 75, wherein the cloned polynucleotide encodes a polypeptide and the vector is an expression vector. 77. The method of claim 76, further comprising directing the expression vector into a host cell and expressing the protein encoded by the cloned double-stranded polynucleotide. 71. The second antisense polynucleotide strand is synthesized from the unhybridized sense polynucleotide strand using an antisense primer complex, further comprising synthesizing the antisense RNA molecule from the double-stranded polynucleotide. the method of. 80. The method of claim 79, comprising using one or more double-stranded polynucleotides, or polynucleotides produced directly or indirectly therefrom in a hybridization reaction. 80. The method of claim 79, wherein at least one double-stranded polynucleotide or a polynucleotide produced therefrom is labeled with a detectable label. 71. The method of claim 70, further comprising adding a plurality of double-stranded polynucleotides or polynucleotides generated therefrom to the substrate to generate a polynucleotide array. 71. The method of claim 70, further comprising amplifying at least one double-stranded polynucleotide. 83. The method of claim 82, wherein the one or more double-stranded polynucleotides are amplified using one or more gene-specific primers. 71. The method of claim 70, wherein the first and second polynucleotide samples are different samples. A plurality of polynucleotides produced from the polynucleotide sample, wherein at least 10 ³ The plurality of polynucleotides comprising different species of polynucleotides and substantially enriched in high abundance polynucleotide sequences as compared to the polynucleotide sample, wherein each of the polynucleotides comprises an RNA promoter sequence and a universal primer site. A plurality of polynucleotides produced from the polynucleotide sample, wherein at least 10 ³ The plurality of polynucleotides comprising different species of polynucleotides and being substantially rich in low abundance polynucleotide sequences as compared to the polynucleotide sample. 87. The plurality of polynucleotides of claim 86, wherein each of the polynucleotides comprises an RNA promoter sequence and a universal primer site. 87. The plurality of polynucleotides of claim 86, wherein the polynucleotide is a double-stranded cDNA. 87. The plurality of polynucleotides of claim 86, wherein the polynucleotide is an antisense RNA. a) an antisense primer complex comprising an antisense primer operably linked to an RNA promoter sequence, wherein the RNA promoter sequence is 5 ′ of the antisense primer;
b) a sense primer; and c) instructions for performing the method of claim 49;
Kit containing. a) an antisense primer complex comprising an antisense primer operably linked to an RNA promoter sequence, wherein the RNA promoter sequence is 5 ′ of the antisense primer;
b) a sense primer; and c) instructions for performing the method of claim 70;
Kit containing. a) the plurality of polynucleotides of claim 86;
b) an antisense primer complex comprising an antisense primer operably linked to an RNA promoter sequence, wherein the RNA promoter sequence is 5 ′ of the antisense primer; and c) a sense primer;
Kit containing. a) a plurality of polynucleotides of claim 86; and b) an RNA polymerase capable of transcribing antisense RNA from the plurality of polynucleotides;
Kit containing. A method of producing a polynucleotide pool selected from a polynucleotide sample,
a) synthesizing a first antisense polynucleotide strand from a polynucleotide sample or from a sense polynucleotide produced therefrom;
b) diluting the first antisense polynucleotide strand to substantially remove at least some low abundance first antisense polynucleotide strand; and c) high abundance polynucleotide sequence as compared to the polynucleotide sample. Synthesizing a first double-stranded polynucleotide enriched from the remaining first antisense polynucleotide strand;
d) generating a second antisense polynucleotide chain from the first double-stranded polynucleotide;
e) contacting the second antisense polynucleotide strand with a polynucleotide sample or a sense polynucleotide strand produced therefrom under hybridization conditions to form a hybridization mixture, thereby comparing the polynucleotide sample to the polynucleotide sample. Producing an unhybridized sense polynucleotide strand enriched in the low abundance polynucleotide sequence;
f) synthesizing a third antisense polynucleotide strand from the unhybridized sense polynucleotide strand;
g) generating a second double-stranded polynucleotide from the third antisense polynucleotide strand;
The method comprising:

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