JP2004000154A - Sequence specific detection of nucleic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an alternative method for sequence specific detection of a nucleic acid; to provide a material suitable for the method; and to provide a method for sequence specifically detecting the nucleic acid by using a solid carrier. <P>SOLUTION: The solid carrier holds two or more nucleic acid analogues bonded to different sites on the surface of the solid carrier and having different base sequences. At least one of the nucleic acid analogues has a base sequence complementary to one base sequence of a nucleic acid of the detection object, and at least one of the other nucleic acid analogues has a base sequence noncomplementary to the base sequence. The method determines the bond caused at the previously determined site when the detection is carried out under a condition under which the nucleic acid of the detection object binds to the nucleic acid analogue by using the carrier. The method for selectively detecting a nucleic acid variant in the presence of large excess of nonmutated nucleic acid involves using the carrier and determining the bond of the mutated nucleic acid and/or nonmutated nucleic acid at different sites. The method for determining the nucleic acid involves using the carrier and determining the relative amounts of the mutated nucleic acid and the normal nucleic acid by comparing bound mutated nucleic acid and nonmutated nucleic acid. <P>COPYRIGHT: (C)2004,JPO

Description

【００６６】
実施例５：
定量
実施例１に記載のように、１００μＭの濃度で、異なる塩基配列を有する３つの（Ａｄｏ）_６−ＰＮＡ分子（図１ａ、配列番号１、２、３）を用いて、膜片を誘導化する。次いで、２０ｍｌのハイブリダイゼーション容器中、１０ｍｌのハイブリダイゼーション緩衝液（実施例２を参照）を用いて、４５℃でそれらをプレハイブリダイズする。３０分後、緩衝液を交換する。実施例１〜７を通して、添加する緩衝液は、ＤＩＧ標識成分−オリゴヌクレオチド１、２および３、配列番号４、５、６の被検体の濃度により異なる。該片を、４５℃で６０分間ハイブリダイズし、次いで、１０ｍｌの洗浄緩衝液で１０分間２回洗浄する。実施例２に記載の手法を用いて、検出を行なう。ルミネセンスシグナルを、ルミネセンスイメージャーを用いて記録し、次いで、評価する（図４）。
【００６７】
見出されたシグナル強度を用いて、被検体複合体組成物に関して定性的または半定量的発見を達成することができる。シグナル強度を更正した後、絶対的に定量的な発見を達成することができる。
【００６８】
実施例６：
ＰＣＲアンプリコンの検出
Ａ．好適な被検体（増幅物）を得ること
その配列がＰＮＡプローブ、ＰＮＡ１に相補的な二本鎖ＤＮＡ断片をｐＵＣ１９プラスミドに連結する。該プラスミドを大腸菌に形質転換し、クローニングし、次いでシーケンスする。次のハイブリダイゼーション実験には、プラスミド配列の一部分を増幅し、増幅反応中にＤＩＧ標識する。５０μｌの全体積中で増幅を行なう。増幅複合体は、１μｌ　プラスミド（１ｎｇ／μｌ）、１μｌ　プライマーＦ１（１０μＭ）、１μｌ　ＤＩＧプライマーＲ１（１０μＭ）、５μｌ１０×ＰＣＲ緩衝液（１００ｍＭ　Ｔｒｉｓ／ＨＣｌ、１５ｍＭ　ＭｇＣｌ_２、５００ｍＭ　ＫＣｌ、ｐＨ８．３）、２μｌ　ｄＮＴＰ溶液（蒸留水中、１０ｍＭ　ｄＡＴＰ、１０ｍＭ　ｄＣＴＰ、１０ｍＭ　ｄＧＴＰ、１０ｍＭ　ｄＴＴＰ、ｐＨ７．０）、０．５μｌ　Ｔａｑポリメラーゼ（５ユニット／μｌ）および３８．５μｌ水からなる。
【００６９】
プライマーＦ１：５’−ＧＴＡ　ＡＡＡ　ＣＧＡ　ＣＧＧ　ＣＣＡ　ＧＴ−３’（配列番号１２）
プライマーＲ１：５’−ＤＩＧ−ＡＡＣ　ＡＧＣ　ＴＡＴ　ＧＡＣ　ＣＡＴ　ＧＡ−３’（配列番号１３）
【００７０】
各反応混合物を、９６℃で３分間温める。次の工程で、３段階のＰＣＲサイクル（９６℃、４５秒、４８℃、３０秒、７２℃、１分）を３０回行なう。最終サイクルで、７２℃、５分間、伸長工程を増やす。
【００７１】
Ｂ．ハイブリダイゼーション反応
実施例１に記載のように、１００μＭ〜０．１μＭの濃度範囲のＰＮＡ溶液を用いて、塩基配列が１つまたは２つの位置でそれぞれ異なる３つの（Ａｄｏ）_６−ＰＮＡ配列（図１ａ、配列番号：１、２、３を参照）で、膜を誘導化する。２０ｍｌのハイブリダイゼーション容器中、５ｍｌのハイブリダイゼーション緩衝液を用いて４５℃で該膜を前処理する。３０分後緩衝液を交換し、被検体溶液を添加する。被検体溶液を作るために、１ｍｌのハイブリダイゼーション緩衝液で、増幅複合体を直接（ｄｓアンプリコン）、および５分間の熱変性後（ｓｓアンプリコン）希釈する。４５℃で１時間、２時間３０分および４時間ハイブリダイゼーション後、各回５ｍｌの洗浄緩衝液を用いて、１０分間２回、膜を洗浄する。実施例２に記載のように、ハイブリダイゼーション事象を検出する（図３）。
【００７２】
図３に、９個の区画を示す。各列間の相違は、インキュベーションの時間（４時間、２．５時間および１時間）である。各カラム間の相違は、検出対象の核酸の型である。３つの重複する列のスポットをカラムＩの３つの区画それぞれに適用する。列間の相違は、ＰＮＡの配列であるが、各区画のカラム間の相違は、濃度である。オリゴヌクレオチドを検出する核酸として用いた場合に対して、特異性および定量能力をカラムＩに示す。
【００７３】
図は、インキュベーション時間の影響を示す。たった１時間のハイブリダイゼーション後、ＯＤＮ１ａと増幅物に対する優れた配列識別性が得られることが明らかである。図３におけるカラムＩＩとＩＩＩ　との相違は、前もって一本鎖にした増幅物を、検出対象の核酸として１　つの場合で用いることである。カラムＩＩＩ　において、前もって一本鎖にしなかった増幅物を、検出対象の核酸として用いる。二本鎖核酸を固体担体に適用する前に変性させることは必ずしも必要でないことが、シグナルにより明らかに示される。このことにより、作業工程数が減少（加熱工程、一本鎖の分離、洗浄工程）し、したがって、コンタミネーションの危険が減少する。したがって、低塩条件と組み合わせたＰＮＡプローブは、ＤＮＡプローブよりも明らかな有利性を提供する。
【００７４】
実施例７：
ＰＮＡ／ＤＮＡハイブリダイゼーションの比較
実施例１に記載のように、５０μＭの溶液を用いて、塩基配列が１つまたは２つの位置で異なる（図１ａおよび１ｂ参照）、それぞれ３つの（Ａｄｏ）_６−ＰＮＡ配列（配列番号：１、２、３）およびそれぞれ３つのＤＮＡ分子（配列番号：８、１０、１１）で、膜片を誘導化する。実施例１とは異なり、スポット体積は、２００ｎｌの代わりに４００ｎｌである。２０ｍｌのハイブリダイゼーション容器中、５ｍｌの低塩緩衝液（実施例２を参照）または高塩緩衝液（６×ＳＳＣ；０．９Ｍ　ＮａＣｌ、９０ｍＭ　クエン酸ナトリウム、０．１％ＳＤＳ、ｐＨ７．０）のいずれかを用いて、３７℃または４５℃いずれかで３０分間、該膜片をプレハイブリダイズする。次の工程で、３つのＤＩＧ標識オリゴヌクレオチド（図１ｃ、配列番号：４、５、６）の１つを添加する。６０分間のハイブリダイゼーション工程後、３７℃または４５℃で、各回５ｍｌの洗浄緩衝液を用いて１０分間２回、該片を洗浄する。低塩実験には、実施例２由来の洗浄緩衝液を用いる。高塩実験には、０．０２％ＳＤＳを有する１×ＳＳＣ緩衝液、ｐＨ７．０を用いる。実施例２に記載のように、ハイブリダイゼーションの結果を検出する。定量的に評価を行ない、表２に示す。
【００７５】
ＤＮＡおよびＰＮＡ両プローブは、一重および二重ミスマッチ配列の相補的一本鎖標的配列を完全に識別することが可能である。ＰＮＡプローブは、ある種の型のミスマッチに対して、特にミスマッチが配列の中間に存在しないが、むしろ末端にシフトしている場合に、ＤＮＡプローブよりも明らかな有利性を示す。このことは、同一の配列を有するＰＮＡプローブよりもＤＮＡプローブによりずっと強力に寛容される分散したＧ／Ｔミスマッチ（プローブ１／ＯＤＮ３）の例において、特に明解になる。
【００７６】
【表２】

【００７７】
実施例８：
膜およびＰＮＡプローブ間のリンカーの長さの影響
実施例１に記載のように、１００μＭ〜１μＭの濃度範囲でＰＮＡ溶液を用いて、リンカーの長さ（Ａｄｏ_３、Ａｄｏ_６またはＡｄｏ_９）により異なるＰＮＡ分子（図１ａ、配列番号７、１、９を参照）で、膜片を誘導化する。実施例１とは異なり、スポット体積は、２００ｎｌの代わりに１μｌである。１０ｍｌのハイブリダイゼーション緩衝液（５、１０または２５ｍＭリン酸ナトリウム、０．１％ＳＤＳ、ｐＨ７．０）を含むハイブリダイゼーション容器中、３５℃で３０分間、該膜片をプレハイブリダイズする。次の工程で、１０ｐＭｏｌの^３２Ｐ標識オリゴヌクレオチド（図１ｃ；ＯＤＮ１ｂ、配列番号４を参照）を添加し、５０℃で６０分間、調製物をハイブリダイズする。５０ｍｌの洗浄緩衝液（５ｍＭリン酸ナトリウム、０．１％ＳＤＳ、ｐＨ７．０）を用いて、５０℃で１０分間２回、該片を洗浄する。オートラジオグラフィーを用いて、ハイブリダイゼーション事象を検出する（図５）。図は、より長いリンカーがハイブリダイゼーションを大きく改良することを示す。
【００７８】
実施例９：
ＰＮＡ膜の再利用
実施例１に記載のように、１００μＭ〜１μＭの濃度範囲でＰＮＡ溶液を用いて、（Ａｄｏ）_６−ＰＮＡ分子（図１ａ；ＰＮＡ１ｂ、配列番号１を参照）で、膜を誘導化する。５つの同一の濃度の連続で、ＰＮＡを適用する。実施例８に記載のように、スポット体積は１μｌである。１０ｍｌのハイブリダイゼーション緩衝液（１０ｍＭリン酸ナトリウム、０．１％ＳＤＳ、ｐＨ７．０）を含むハイブリダイゼーション容器中、３５℃で３０分間、該膜をプレハイブリダイズする。次の工程で、１０ｐＭｏｌの^３２Ｐ標識オリゴヌクレオチド（図１ｃ；ＯＤＮ１ｂ、配列番号４）を添加し、５０℃で６０分間、調製物をハイブリダイズする。５０ｍｌの洗浄緩衝液（５ｍＭリン酸ナトリウム、０．１％ＳＤＳ、ｐＨ７．０）を用いて、５０℃で１０分間２回、該膜を洗浄する。オートラジオグラフィーを用いて、ハイブリダイゼーション事象を検出する（図６ａ）。オートラジオグラフィーを行なった後、該膜を５つの同一片に切断する。これらの膜片を、実験休止中、それぞれ別々に取り扱う。膜１は、インキュベートせずに対照膜として供する。膜２は、５０ｍｌの０．１Ｍ水酸化ナトリウム溶液を用いて室温で６０分間インキュベートする。膜３は、５０ｍｌの１Ｍ水酸化ナトリウム溶液を用いて同様に６０分間インキュベートする。膜４は、５０ｍｌの蒸留水を用いて７０℃で６０分間インキュベートする。膜５は、５０ｍｌの０．１Ｎ水酸化ナトリウム溶液を用いて７０℃で６０分間インキュベートする。次いで、蒸留水を用いて１０分間２回、すべての膜を洗浄する。この操作が終了後、もう１度オートラジオグラフィーを行なう（図６ｂ）。次いで、これらの膜片を、記載したように、２回目のハイブリダイゼーション反応に用いて、オートラジオグラフィーを用いて、ハイブリダイゼーション事象を検出する（図６ｃ）。
【００７９】
図６ｂに示すように、異なる処理方法により非常に異なる結果を得る。７０℃で再蒸留水での処理（膜４）は、膜をほとんど完全に再生させる。予期せぬ発見は、核酸の変性に通常用いられる条件を使用した場合、再生の成功がより不成功になるという事実であった。室温で１Ｍ水酸化ナトリウム溶液との膜３のインキュベーションそれ自体が、事実上再生効果を生じない。１Ｍから０．１Ｍへ水酸化ナトリウム溶液の濃度を下げることは、室温（膜２）および７０℃（膜５）での再生度を高める。しかし、これらの条件は、再蒸留水を用いた場合（膜４）のように、どれも再生度を少しでも良好にするという結果を生じない。これらの条件が膜結合ＰＮＡ／ＤＮＡ二本鎖の効率的な変性に対する重要なパラメーターであることが、実施例により示される。
【００８０】
再生方法に関わらず、すべての膜は、シグナル対ノイズ比をかなり悪化させることなく、ハイブリダイゼーションに再利用できる（図６ｃ）。再生または再ハイブリダイズするＰＮＡ膜の能力の顕著な効果を失うことなく、６回までの再ハイブリダイゼーションを行なうことができる。
【００８１】
【発明の効果】
本発明によって、核酸の配列特異的検出のための択一的方法を提供することおよびこの方法のための好適な材料を提供することができる。また固体担体の表面上の前もって決定された部位に結合した、異なる塩基配列を有する２以上の核酸類似体を有する固体担体を提供することができる。さらに、本発明によって、この固体担体を用いる核酸の配列特異的検出方法が提供できる。
【００８２】
【配列表】
（１）　配列番号：１の情報：
（ｉ）配列の特徴：
（Ａ）　配列の長さ：１６アミノ酸
（Ｂ）　配列の型：アミノ酸
（Ｃ）　鎖の数：一本鎖
（Ｄ）　トポロジー：直鎖状
（ｉｉ）配列の種類：ぺプチド
（ｉｉｉ）ハイポセティカル：　ＮＯ
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：１
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−チミニル）　アセチル）−Ｎ−（２−　アミノ（Ｎ’−ヘキサ（８−　アミノ−３，６−　ジオキサ−　オクタノ−１−　イル）−エチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：２
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−グアニニル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：３
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−チミニル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：４
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−アデニニル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：５
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−シトシル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：６
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−グアニニル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：７
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−チミニル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：８
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−シトシル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：９
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−アデニニル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：１０
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−シトシル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：１１．．１２
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−アデニニル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：１３
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−シトシル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：１４
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−チミニル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：１５
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−アデニニル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」

（１）　配列番号：２の情報
（ｉ）配列の特徴：
（Ａ）　配列の長さ：１６アミノ酸
（Ｂ）　配列の型：アミノ酸
（Ｃ）　鎖の数：一本鎖
（Ｄ）　トポロジー：直鎖状
（ｉｉ）配列の種類：ぺプチド
（ｉｉｉ）ハイポセティカル：　ＮＯ
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：１
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−チミニル）　アセチル）−Ｎ−（２−　アミノ（Ｎ’−ヘキサ（８−　アミノ−３，６−　ジオキサ−　オクタノ−１−　イル）−エチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：２
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−グアニニル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：３
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−チミニル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：４
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−アデニニル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：５
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−シトシル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：６
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−グアニニル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：７
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−チミニル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：８
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−グアニニル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：９
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、　　　　　　　　　　　　　　　　　　　　　　　Ｎ−（（１−アデニニル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：１０
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−シトシル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：１１．．１２
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−アデニニル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：１３
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−シトシル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：１４
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−チミニル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：１５
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−アデニニル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」

（１）　配列番号：３の情報：
（ｉ）配列の特徴：
（Ａ）　配列の長さ：１６アミノ酸
（Ｂ）　配列の型：アミノ酸
（Ｃ）　鎖の数：一本鎖
（Ｄ）　トポロジー：直鎖状
（ｉｉ）配列の種類：ぺプチド
（ｉｉｉ）ハイポセティカル：ＮＯ
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：１
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−チミニル）　アセチル）−Ｎ−（２−　アミノ（Ｎ’−ヘキサ（８−　アミノ−３，６−　ジオキサ−　オクタノ−１−　イル）−エチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：２
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−グアニニル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：３
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−チミニル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：４
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−アデニニル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：５
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−シトシル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：６
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−アデニニル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：７
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−チミニル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：８
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−シトシル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：９
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−アデニニル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：１０
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−シトシル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：１１．．１２
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−アデニニル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：１３
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−シトシル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：１４
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−チミニル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：１５
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−アデニニル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」

（１）　配列番号：４の情報：
（ｉ）配列の特徴：
（Ａ）　配列の長さ：１５塩基対
（Ｂ）　配列の型：核酸
（Ｃ）　鎖の数：一本鎖
（Ｄ）　トポロジー：直鎖状
（ｉｉ）配列の種類：他の核酸
（Ａ）　種類：　／ｄｅｓｃ　＝　「オリゴデオキシリボヌクレオチド」
（ｉｉｉ）ハイポセティカル：ＮＯ
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：ｍｉｓｃ＿ｆｅａｔｕｒｅ
（Ｂ）　存在位置：１５
（Ｄ）　他の情報：／ｎｏｔｅ＝「アミノリンカー（Ｂｏｅｈｒｉｎｇｅｒ　Ｍａｎｎｈｅｉｍ　ＧｍｂＨ　　　　　　　，　ＢＲＤ）を介するジゴキシゲニンまたは３２−Ｐで５’−　リン酸基を標識」

（１）　配列番号：５の情報：
（ｉ）配列の特徴：
（Ａ）　配列の長さ：１５塩基対
（Ｂ）　配列の型：核酸
（Ｃ）　鎖の数：一本鎖
（Ｄ）　トポロジー：直鎖状
（ｉｉ）配列の種類：他の核酸
（Ａ）　種類：　／ｄｅｓｃ　＝　「オリゴデオキシリボヌクレオチド」
（ｉｉｉ）ハイポセティカル：ＮＯ
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：ｍｉｓｃ＿ｆｅａｔｕｒｅ
（Ｂ）　存在位置：１５
（Ｄ）　他の情報：／ｎｏｔｅ＝「アミノリンカー（Ｂｏｅｈｒｉｎｇｅｒ　Ｍａｎｎｈｅｉｍ　ＧｍｂＨ　　　　　　　，　ＢＲＤ）を介するジゴキシゲニンで５’−　リン酸基を標識」

（１）　配列番号：６の情報：
（ｉ）配列の特徴：
（Ａ）　配列の長さ：１５塩基対
（Ｂ）　配列の型：核酸
（Ｃ）　鎖の数：一本鎖
（Ｄ）　トポロジー：直鎖状
（ｉｉ）配列の種類：他の核酸
（Ａ）　種類：　／ｄｅｓｃ　＝　「オリゴデオキシリボヌクレオチド」
（ｉｉｉ）ハイポセティカル：ＮＯ
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：ｍｉｓｃ＿ｆｅａｔｕｒｅ
（Ｂ）　存在位置：１５
（Ｄ）　他の情報：／ｎｏｔｅ＝「アミノリンカー（Ｂｏｅｈｒｉｎｇｅｒ　Ｍａｎｎｈｅｉｍ　ＧｍｂＨ　　　　　　　，　ＢＲＤ）を介するジゴキシゲニンで５’−　リン酸基を標識」

（１）　配列番号：７の情報：
（ｉ）配列の特徴：
（Ａ）　配列の長さ：１５塩基対
（Ｂ）　配列の型：核酸
（Ｃ）　鎖の数：一本鎖
（Ｄ）　トポロジー：直鎖状
（ｉｉ）配列の酒類：ぺプチド
（ｉｉｉ）ハイポセティカル：　ＮＯ
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：１
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−チミニル）　アセチル）−Ｎ−（２−　アミノ（Ｎ’−トリ（８−アミノ　　−３，６−　ジオキサ−　オクタノ−１−　イル）−エチル）−ベータ−　アラニン　　である」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：２
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−グアニニル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：３
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−チミニル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：４
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−アデニニル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：５
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−シトシル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：６
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−グアニニル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：７
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−チミニル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：８
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−シトシル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：９
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−アデニニル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：１０
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−シトシル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：１１．．１２
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−アデニニル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：１３
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−シトシル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：１４
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−チミニル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：１５
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−アデニニル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」

（１）　配列番号：８の情報：
（ｉ）配列の特徴：
（Ａ）　配列の長さ：３０塩基対
（Ｂ）　配列の型：核酸
（Ｃ）　鎖の数：一本鎖
（Ｄ）　トポロジー：直鎖状
（ｉｉ）配列の種類：他の核酸
（Ａ）　種類：　／ｄｅｓｃ　＝　「オリゴデオキシリボヌクレオチド」
（ｉｉｉ）ハイポセティカル：ＮＯ

（１）　配列番号：９の情報：
（ｉ）配列の特徴：
（Ａ）　配列の長さ：１６アミノ酸
（Ｂ）　配列の型：アミノ酸
（Ｃ）　鎖の数：一本鎖
（Ｄ）　トポロジー：直鎖状
（ｉｉ）配列の酒類：ぺプチド
（ｉｉｉ）ハイポセティカル：　ＮＯ
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：１
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−チミニル）　アセチル）−Ｎ−（２−　アミノ（Ｎ’−ノナ（８−アミノ　　−３，６−　ジオキサ−　オクタノ−１−　イル）−エチル）−ベータ−　アラニン　　である」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：２
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−グアニニル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：３
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−チミニル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：４
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−アデニニル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：５
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−シトシル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：６
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−グアニニル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：７
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−チミニル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：８
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−シトシル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：９
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−アデニニル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：１０
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−シトシル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：１１．．１２
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−アデニニル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：１３
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−シトシル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：１４
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−チミニル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：Ｍｏｄｉｆｉｅｄ−ｓｉｔｅ
（Ｂ）　存在位置：１５
（Ｄ）　他の情報：／ｐｒｏｄｕｃｔ＝　「その他」
／ｎｏｔｅ＝「Ｘａａ　は、
Ｎ−（（１−アデニニル）　アセチル）−Ｎ−（２−　アミノエチル）−ベータ−　アラニンである」

（１）　配列番号：１０の情報：
（ｉ）配列の特徴：
（Ａ）　配列の長さ：３０塩基対
（Ｂ）　配列の型：核酸
（Ｃ）　鎖の数：一本鎖
（Ｄ）　トポロジー：直鎖状
（ｉｉ）配列の種類：他の核酸
（Ａ）　種類：　／ｄｅｓｃ　＝　「オリゴデオキシリボヌクレオチド」
（ｉｉｉ）ハイポセティカル：ＮＯ

（１）　配列番号：１１の情報：
（ｉ）配列の特徴：
（Ａ）　配列の長さ：３０塩基対
（Ｂ）　配列の型：核酸
（Ｃ）　鎖の数：一本鎖
（Ｄ）　トポロジー：直鎖状
（ｉｉ）配列の種類：他の核酸
（Ａ）　種類：　／ｄｅｓｃ　＝　「オリゴデオキシリボヌクレオチド」
（ｉｉｉ）ハイポセティカル：ＮＯ

（１）　配列番号：１２の情報：
（ｉ）配列の特徴：
（Ａ）　配列の長さ：１７塩基対
（Ｂ）　配列の型：核酸
（Ｃ）　鎖の数：一本鎖
（Ｄ）　トポロジー：直鎖状
（ｉｉ）配列の種類：他の核酸
（Ａ）　種類：　／ｄｅｓｃ　＝　「オリゴデオキシリボヌクレオチド」
（ｉｉｉ）ハイポセティカル：ＮＯ

（１）　配列番号：１３の情報：
（ｉ）配列の特徴：
（Ａ）　配列の長さ：１７塩基対
（Ｂ）　配列の型：核酸
（Ｃ）　鎖の数：一本鎖
（Ｄ）　トポロジー：直鎖状
（ｉｉ）配列の種類：他の核酸
（Ａ）　種類：　／ｄｅｓｃ　＝　「オリゴデオキシリボヌクレオチド」
（ｉｉｉ）ハイポセティカル：ＮＯ
（ｉｘ）配列の特徴：
（Ａ）　特徴を表わす記号：ｍｉｓｃ＿ｆｅａｔｕｒｅ
（Ｂ）　存在位置：１
（Ｄ）　他の情報：／ｎｏｔｅ＝「５’−　末端のＡ　は、アミノ修飾剤（ＢｏｅｈｒｉｎｇｅｒＭａｎｎｈｅｉｍ　ＧｍｂＨ）　を介してジゴキシゲニンに結合する」

【図面の簡単な説明】
【図１】図１ａは、ＰＮＡ分子の配列を示す。それらを、本発明の方法を説明する例として用いる。ＰＮＡを、国際公開第９２／２０７０２号パンフレットに記載のように調製した。
図１ｂは、図１ａ由来のＰＮＡ配列と相同な、ＤＮＡ／ＯＤＮハイブリダイゼーション実験に用いたＤＮＡ分子の配列を示す。
図１ｃは、５’−ＤＩＧ　Ｅｎｄ　Ｌａｂｅｌｌｉｎｇ　Ｋｉｔ（ＢｏｅｈｒｉｎｇｅｒＭａｎｎｈｅｉｍ）を用いて５’−リン酸末端をジゴキシゲニンで標識し、ポリヌクレオチドキナーゼおよび^３２Ｐ−γ−ＡＴＰ５’を用いてリン酸化した、用いた相補的オリゴヌクレオチド（ＯＤＮ）の配列を示す。
図１ｄは、ハイブイッドを形成するオリゴヌクレオチド（ＯＤＮ）とＰＮＡとの可能な組み合わせを示す。同一の組み合わせを、ＤＮＡプローブ（ＤＮＡ、図１ｂを参照）とオリゴヌクレオチド（ＯＤＮ、図１ｃを参照）との間のハイブリダイゼーションに適用する。
【図２】図２は、本発明の方法の選択性を示す実施例４由来のハイブリダイゼーションの結果を示す。条件は：２００ｎｌのスポット体積（１００；１０；１；０．１μＭ　ＰＮＡ、開裂当たり１つの濃度）、４５℃でのインキュベーションであった。
【図３】図３は、実施例６由来のハイブリダイゼーションの結果を示す。それらは、固定したＰＮＡプローブによりＰＣＲアンプリコンを検出することを証明する。条件は：２００ｎｌのスポット体積（１００；１０；１；０．１μＭ　ＰＮＡ、開裂当たり１つの濃度）、４５℃でのインキュベーションであった。図３の符号は、以下を意味する：
Ｉ　対照実験、ＯＤＮ１ａ（１ｐＭｏｌ、１ｎＭ）、列１（ＰＮＡ１）、列２（ＰＮＡ２）、列３（ＰＮＡ３）
ＩＩ　　ｓｓ増幅物（１１８ｂｐ、１倍のＤＩＧ標識）
９４℃で５分間の熱変性
（１ｍｌのハイブリダイゼーション緩衝液で希釈した５０μｌ　ＰＣＲ調製物）
列１（ＰＮＡ１）、列２（ＰＮＡ２）、列３（ＰＮＡ３）
ＩＩＩ　ｄｓ増幅物（１１８ｂｐ、１倍のＤＩＧ標識）
（１ｍｌのハイブリダイゼーション緩衝液で直接希釈した５０μｌ　ＰＣＲ調製物）
列１（ＰＮＡ１）、列２（ＰＮＡ２）、列３（ＰＮＡ３）。
【図４】図４は、ＰＮＡアレイにより分析混合物の定性的定量的分析の結果を示す。条件は：２００ｎｌのスポット体積（１００μＭ　ＰＮＡ、列当たり１つのＰＮＡ、開裂当たり１つの分析混合物）であった。
【図５】図５は、ハイブリダイゼーションにおけるリンカーの長さの効果を示す。条件は：１μｌのスポット体積（１００；４０；２０；１０；５；１μＭ　ＰＮＡ、開裂当たり１つの濃度、列１では（Ａｄｏ）_３−ＰＮＡ、列２では（Ａｄｏ）_６−ＰＮＡ、列３では（Ａｄｏ）_９−ＰＮＡ）であった。図５の符号は、以下を意味する：
Ａ．５ｍＭ　リン酸ナトリウム、０．１％ＳＤＳ，ｐＨ７．０でのプレハイブリダイゼーション／ハイブリダイゼーション
Ｂ．１０ｍＭ　リン酸ナトリウム、０．１％ＳＤＳ，ｐＨ７．０でのプレハイブリダイゼーション／ハイブリダイゼーション
Ｃ．２５ｍＭ　リン酸ナトリウム、０．１％ＳＤＳ，ｐＨ７．０でのプレハイブリダイゼーション／ハイブリダイゼーション。
【図６】図６Ａは、１回目のハイブリダーゼーション後のシグナル強度を示す。図６Ａは、ＰＮＡで誘導化した膜が、再生後何回も使用できることを示唆する。条件は：１μｌのスポット体積（１００；４０；２０；１０；５；１μＭ　ＰＮＡ、開裂当たり１つの濃度）であった。
図６Ｂは、再生手法後のシグナル強度を示す。図６Ｂは、ＰＮＡで誘導化した膜が、再生後何回も使用できることを示唆する。条件は：１μｌのスポット体積（１００；４０；２０；１０；５；１μＭ　ＰＮＡ、開裂当たり１つの濃度）であった。
膜１：再生なし（対照）
膜２：０．１Ｍ水酸化ナトリウム溶液、ＲＴ、１時間、２×１０分　再蒸留水　ＲＴを用いる再生
膜３：１Ｍ水酸化ナトリウム溶液、ＲＴ、１時間、２×１０分　再蒸留水　ＲＴを用いる再生
膜４：蒸留水、７０℃　１時間、２×１０分　再蒸留水　ＲＴを用いる再生
膜５：０．１Ｍ水酸化ナトリウム溶液、７０℃　１時間、２×１０分　再蒸留水ＲＴを用いる再生
図６Ｃは、再ハイブリダイゼーション後のシグナル強度を示す。図６Ｃは、ＰＮＡで誘導化した膜が、再生後何回も使用できることを示唆する。条件は：１μｌのスポット体積（１００；４０；２０；１０；５；１μＭ　ＰＮＡ、開裂当たり１つの濃度）であった。[0001]
TECHNICAL FIELD OF THE INVENTION
The subject of the present invention is a solid support carrying two or more nucleic acid analogues having different base sequences, bound to a predetermined site on the surface of the solid support. The present invention also provides a method for detecting a nucleic acid using a carrier having this property.
[0002]
[Prior art]
In the last decade, sample analysis has undergone rapid development. The analytes were first detected by reactions with ordinary chemical reagents and later detected with enzymes, but tests that use the immunological properties of the analytes have recently become the standard, especially in medical diagnostics. It has become. This is especially true in the field of infectious diseases. However, immunological techniques can basically only detect only those analytes for which immunologically active compounds such as antigens or antibodies play a role. These approaches have resulted in promising adaptations to many infections caused by viruses or bacteria. However, genetic disorders or predispositions that do not express as a change or insufficient content in the pattern of the protein are difficult or impossible to detect using immunological techniques. Therefore, nucleic acids have recently been detected in many cases. The presence of certain nucleic acids can infer the presence of an infectious agent or the genetic status of an organism. In recent years, in which methods for amplifying nucleic acids present in a small number have become available, detection methods based on the presence of specific nucleic acid sequences have become particularly easy. Due to the large amount of sequence information and the fact that two nucleotide sequences with completely different functions often differ only by one base unit, the specific detection of nucleotide sequences is based on the detection of nucleic acid sequences by reagents and assays. Still taking a considerable challenge on the method. Although the nucleotide sequence is often unknown, it is rather detected only by the nucleic acid detection method itself.
[0003]
A method for detecting a nucleotide sequence of an HLA gene capable of detecting a clinically relevant point mutation is described (for example, see Patent Document 1). In this method, an oligonucleotide having a nucleotide sequence that is bound to a membrane and that is exactly complementary to one of the two nucleic acids to be identified is contacted with the sample. While certain conditions are maintained, only exactly complementary nucleic acids will bind to the solid phase-bound oligonucleotide and will be detectable.
[0004]
A method is described for simultaneously detecting all known allelic variants of an amplified region of a nucleic acid using multiple oligonucleotides attached to separate predetermined sites on a nylon membrane by poly dT. (For example, see Non-Patent Document 1).
[0005]
A method is described in which a nucleic acid sequence can be theoretically determined by bringing an oligonucleotide having a known sequence determined in advance into contact with an unknown nucleic acid sample under hybridization conditions (for example, see Patent Document 2). This requires that all possible substitutions of the nucleic acid sequence be fixed at known sites on the solid phase. By determining the site where the nucleic acid containing the sequence to be determined hybridizes, it is possible to theoretically determine which sequence is present in the nucleic acid.
[0006]
Prior art in the field of genetic polymorphism analysis using "oligonucleotide arrays" has been described (see, for example, Non-Patent Documents 2 and 3).
[0007]
[Patent Document 1]
European Patent EP-B-0Ｂ237 362
[Patent Document 2]
U.S. Pat. No. 5,202,231
[Non-patent document 1]
"Proc. Natl. Acad. Sci. USA", 1989, Vol. 86, p. 6230-6234
[Non-patent document 2]
"Nucleic Acids Research", 1994, Vol. 22, p. 5456-5465
[Non-Patent Document 3]
"Clin. {Chem.}", 1995, Vol. 41, No. 5, p. 700-6
[0008]
[Problems to be solved by the invention]
A major problem with the prior art is the fact that the melting temperatures of the sequence or selected sequence-specific oligonucleotides containing the nucleic acids to be detected are different. To remedy this situation, a complex method must be performed to select the length of the oligonucleotide and its base composition, and to optimize the location of mismatches in the oligonucleotide and the salt concentration of the hybridization complex. . However, in many cases, it is practically impossible to simultaneously distinguish sequences closely related to each other. Hybridization temperature is another critical parameter. With a small change of about 1 to 2 ° C., it is possible to change the strength and obtain a result without error. Wrong analytical results based on the presence of point mutations have important implications for diagnosis.
[0009]
It is therefore an object of the present invention to provide an alternative method for sequence-specific detection of nucleic acids and to provide suitable materials for this method.
[0010]
This object has been achieved by providing a solid support having two or more nucleic acid analogs having different base sequences attached to predetermined sites on the surface of the solid support. Another object of the present invention is a method for sequence-specific detection of a nucleic acid using the solid carrier.
[0011]
[Means for Solving the Problems]
That is, the gist of the present invention is:
(1) A method for regenerating a solid support having at least two different peptide nucleic acid probes bound to a surface to which a nucleic acid hybridizes,
a) treating the carrier under conditions for denaturation of the peptide nucleic acid / nucleic acid complex; and
b) washing the solid support to remove the modified nucleic acid polymer;
Including, the method,
(2) The method according to the above (1), wherein denaturation of the peptide nucleic acid / nucleic acid complex is caused by using distilled water at an elevated temperature.
(3) The method according to the above (1), wherein denaturation of the peptide nucleic acid / nucleic acid complex is caused by using a solution containing sodium hydroxide at an increased temperature.
(4) The method according to the above (1), wherein the solid carrier is basically flat.
(5) The method according to (1), wherein the peptide nucleic acid probe has a backbone of 2-aminoethylglycine subunit.
(6) a solid support comprising at least two single-stranded peptide nucleic acid probes of different base sequences covalently linked to the solid support in separate and distinct regions;
(7) The solid support according to (6), wherein the separate and distinct regions are each separated by a region to which a probe is not bound.
(8) The solid carrier according to (6), wherein all the probes have the same base length.
(9) the solid support according to (6), wherein all the probes have a length of 10 to 50 bases;
(10) The solid support according to (6), wherein the solid support is basically flat, and
(11) The solid carrier according to (6), wherein the peptide nucleic acid probe has a 2-aminoethylglycine subunit backbone.
About.
[0012]
BEST MODE FOR CARRYING OUT THE INVENTION
In the present invention, the “solid carrier” refers to a carrier having a surface large enough to allow a specific region to be distinguished on the surface. This surface is preferably flat, 5 mm²Larger than 10mm²~ About 100cm²It is preferable that Preferably, the carrier material is not liquid or gaseous and does not dissolve or incompletely dissolve in the sample liquid or the reaction preparation used to immobilize the nucleic acids on the surface. Examples of such materials are glass, plastic (eg, polystyrene, polyamide, polyethylene, polypropylene, etc.), gold, and the like. The material itself need not necessarily be completely solid, but rather can be made solid by the addition of a support material.
[0013]
The appearance of the solid support basically depends on the method used to detect the presence of the nucleic acid on the solid support. For example, it has been found that it is appropriate to select a basically flat mold such as a chip.
[0014]
Thus, particularly suitable solid supports are, for example, 1-5 mm thick, 1-5 cm²Is a polystyrene chip having a surface area of. 4 × 2.5cm in size²Has been found to be particularly well suited for use with the present invention. Two or more nucleic acid analogs having different base sequences are bound to different sites on the surface of the carrier. Preferably, these sites or regions do not overlap with each other. They are preferably separated from each other by surface regions to which the nucleic acid analog is not bound. The site to which the nucleic acid analog binds is hereinafter referred to as “binding region”. The coupling regions can have different shapes. These forms are basically determined by the method of preparation of the solid support or the method used to determine the nucleic acid analog at the binding region. The minimum size of the binding region is basically determined by the instrument that detects the event—binding of the nucleic acid to the nucleic acid analog of the region. Instruments are already available that can detect binding to areas of about 1 μm in size. The upper bound on the size of the coupling region is determined by cost efficiency and operational considerations.
[0015]
The size of the binding region is also basically determined by the method used for applying the nucleic acid analog to the surface. Such a method will be described later.
[0016]
The number of binding regions on a solid support depends on the intended use of the solid support. In the simplest case, exactly two binding regions are needed to detect certain point mutations. In this case, the binding region includes a nucleic acid analog having a base at the position where the point mutation is detected. This base is complementary to the base at the position in the normal sequence. On the other hand, other binding regions include nucleic acid analogs having, at corresponding positions, bases complementary to bases of the mutated sequence. Alternatively, a solid support having two completely different nucleic acid analogs attached to a surface can be used to simultaneously detect two nucleic acids or nucleic acid sequences that are only marginally related to each other.
[0017]
"Nucleic acid analog" refers to a non-natural molecule capable of detecting a nucleic acid by base pairing. Therefore, they include a specific base sequence completely complementary to the base sequence of the nucleic acid to be detected. Therefore, it is preferable that the base sequence is composed of two natural nucleobases. However, modifications to nucleobases are possible as long as the specificity of base pairing is not lost.
[0018]
These nucleic acid analogs are considered to be complementary to a nucleic acid having a base sequence that forms a hydrogen bond with the base sequence of the nucleic acid when bound to the nucleic acid by the principle of base pairing. This sequence is preferably at least 8 bases long, more preferably 8 to 25 bases long.
[0019]
Furthermore, nucleic acid analogs are defined, at least in the term backbone, by the fact that they are structurally different from nucleic acids. The “backbone” in a nucleic acid or a nucleic acid analog refers to a structure composed of basically the same units each containing a base. In natural nucleic acids, the backbone is the sugar phosphate backbone. In nucleic acid analogs, the backbone is structurally modified, for example, where the sugar or phosphate moiety is completely or partially replaced with another chemical unit, such as an acyclic moiety. Basically, identical units can be replaced by one another in the backbone.
[0020]
Some properties of the nucleic acid analogs are described below to facilitate selection of suitable nucleic acid analogs for use in the present invention. It is advantageous for a nucleic acid analog that the nucleic acid analog has a higher affinity for a sequence-complementary nucleic acid than an oligonucleotide having the same base sequence. Also preferred are nucleic acid analogs that can retain less charge than a corresponding oligonucleotide of the same length or compensate for the charge with an opposite charge. Basically, uncharged nucleic acid analogs are particularly preferred. Particularly preferred nucleic acid analogs are those whose affinity for the complementary nucleic acid is essentially independent of the salt content of the hybridization complex.
[0021]
Particularly suitable nucleic acid analogs are WO 92/20702 and WO 92/20703 (peptide nucleic acids, PNAs such as Nature {365, {566-568 (1993)} and Nucl. Acids Res. 5332-5 (1993)). These patent applications are referred to for the description of the structure of nucleic acid analogs. Preferred nucleic acid analogs are compounds having a polyamide backbone that includes many bases attached along the backbone at each base attached to the backbone nitrogen atom. However, the nucleic acid analogues may also contain compounds as described in EP-A-0 672 677. Additional nucleic acid analogs are described in Recueil 91, {1069-1080 (1971)}, Methods in Molecular Biology 29, {355-389 (1993), Tetrahedron 31, 31, 73-75 (1975), J. Am. {Org. {Chem. {52, {4202-4206 (1987)}, Nucl. {Acids} Res. 17, 6129-6141 (1989), Unusual Properties of New Polymers (Springer Verlag 1983), 1-16, Specialty Polymers (Springer Verlag 1981), 1-208, International Publication No. 92, International Publication No. No. 06815 pamphlet, WO 86/05518 pamphlet and WO 86/05519 pamphlet. Additional nucleic acid analogs can be found in Proc. {Natl. {Acad. {Sci. USA 91, 7864-7868 (1994), Proc. {Natl. {Acad. {Sci. USA 92, 6097-6101 (1995) and J.C. {Am. {Chem. {Soc. No. 117, 6140-6141 (1995). The nucleic acid analogs described are 8 to 30 bases in length, with 10 to 25 bases being particularly advantageous. What is termed a nucleic acid analog binds, directly or indirectly, to the surface of a solid support. The type of linkage depends on which reactive groups are available for binding on the solid support, and without limiting the ability of the nucleic acid analog to bind to complementary nucleic acids, It basically depends on what is available. The type of linkage also depends on whether the nucleic acid analog is to be bound to different sites at the same time, or whether it is built on them. It is also appropriate to coat the surface of the solid support with a layer of a material having a stronger binding force, or to activate the surface by a chemical reaction. The reactive groups on the surface of the solid support are usually -OH, NH₂And SH groups. The reactive group of the nucleic acid analog is -OH, -NH₂, -SH, -COOH, -SO₃H and -PO₃H₂It is preferred to be selected from the group.
[0022]
In a particularly preferred embodiment, the surface and the reactive group of the nucleic acid analog are covalently linked to each other by a linker having a length of more than 15 atoms and less than 200 atoms. "Linker" basically refers to a portion of a molecule that is sterically available on the surface of a solid support and has the function of removing nucleic acid analogs. A linker with a hydrogen atom that facilitates dissolution (eg, in an alkylene unit) and a number of heteroatoms (eg, —O— or —NH— or —NR—) is usually chosen. Preferably, the linker comprises one or more ethyleneoxy units and / or peptide groups. In a particularly preferred embodiment, the linker comprises one or more units as described in DE-A-3924705. The unit described in the examples hereinafter referred to as Ado (8-amino-3,6-dioxaoctanoic acid) is particularly preferred. By using a longer linker between the PNA and the solid support, the dependence of nucleic acid binding on the PNA surface can be somewhat reduced.
[0023]
The nucleic acid analog attached to the site may be a mixture of two or more analogs having different but known sequences. As a result, the number of sites required for majority determination can be reduced.
[0024]
The surface of the carrier is preferably uncharged and preferably hydrophilic. The present invention has shown that the use of an essentially uncharged surface in detecting nucleic acids is advantageous.
[0025]
The solid supports provided with nucleic acid analogs at different sites provided by the present invention can be produced by various methods. In one embodiment, an appropriate amount of solution, each containing a different nucleic acid analog, is applied to different sites on the surface of the solid support, for example, via a pipette. Liquid samples must not mix with each other on the surface of the solid support. This can be done, for example, by positioning the application sites apart from each other or by using a hydrophobic barrier to stop the spread of liquid between the various sites. Preferably, either the nucleic acid analog or the surface of the solid support is activated for the reaction. This activation is achieved, for example, when one of the groups has been activated by creation of a reactive species. In the case of a carboxyl group, this is an activated ester (eg, N-hydroxysuccinimide ester, etc.) that quickly becomes an ester bond at the hydroxyl group without further activation. Suitably activated polyamide membranes retain, for example, triazine groups capable of reacting with the amino groups of the nucleic acid analog in the formation of covalent bonds. Activation can also be carried out with a squaric acid derivative from WO 95/15983 or a bifunctional reagent of glutaraldehyde (GB 2197720).
[0026]
Analog binding can also be recognized by coating a carrier surface having a nucleotide sequence complementary to a portion of the nucleotide analog sequence. The binding of different nucleic acid analogs to the binding region can be performed simultaneously or sequentially.
[0027]
After a sufficient time has elapsed for binding to occur, it is advantageous to wash away any unbound or poorly bound nucleic acid analogs, along with any binding reagents used. This is preferably performed under conditions where the unbound nucleic acid analog cannot bind to the nucleic acid analog bound to other regions.
[0028]
Basically, it is also possible to construct nucleic acid analogs at different sites on the surface by monomer units. The techniques described in WO 92/10092 or WO 90/15070 can be used for this purpose. Suitable monomers are described, for example, in WO 92/20702.
[0029]
Another subject of the present invention is a method for the sequence-specific detection of nucleic acids using a solid support provided by the present invention.
[0030]
Detectable nucleic acids are natural or artificial nucleic acids. Thus, "nucleic acid" also refers to nucleic acid analogs. However, nucleic acids to be detected are specific to organisms containing nucleic acids such as, for example, viruses, bacteria, multicellular organisms, plasmids or genetic conditions such as predisposition or propensity for certain diseases or spontaneous gene mutations. Especially RNA or DNA. In this case, RNA and DNA are basically of genomic or derived origin. In the context of the present invention, an important class of nucleic acids is the result of nucleic acid amplification. These results are hereinafter also referred to as “amplified products” or “amplicons”. Nucleic acids can exist in unpurified, purified or processed forms. Purification can also be performed by separating nucleic acids from cell components in a preparation step such as an affinity separation step. Nucleic acids can also be extended enzymatically, specifically, amplified or transcribed.
[0031]
The carrier provided by the present invention for sequence-specific detection of a nucleic acid has a nucleotide sequence complementary to the nucleotide sequence of the nucleic acid to be detected, and retains a nucleic acid analog bound to a certain site. This base sequence is intentionally selected so as to specifically indicate the presence of the nucleic acid. In the usual case, this means that the mixture contains as few additional nucleic acids as possible, with identical total sequences, and preferably no such nucleic acids. However, it has to be mentioned that the carriers provided by the present invention can also be used for specifically detecting a group of nucleic acids. For example, detecting any member of a certain taxonomic group, such as a family of bacteria, by its nucleic acid can be an object of the methods of the present invention. In this case, the nucleotide sequence of the nucleic acid analog can be deliberately selected such that it is in a conserved region but only present in members of this taxonomic group.
[0032]
Preferably, additional nucleic acid analogs having a base sequence that is not complementary to the same base sequence are bound to different sites on the surface. It may be shorter or longer in its base sequence, or may be a nucleic acid analog that differs from the first nucleic acid analog by one or more bases. The difference in the base sequence depends on the problem to be solved. The differences may include, for example, point mutations or smaller deletions and insertions. In many genetic diseases, such as cystic fibrosis, the sequence of nucleic acid analogs differs in terms of individual positions (point mutations) and many positions (eg, deletions at Δ508, etc.).
[0033]
The mutation to be detected is preferably located close to the middle of the nucleotide sequence of the analog.
[0034]
The sequences of the analogs can also be deliberately selected such that they are identical (duplicate) in length, but differ by one base from each other at the position of hybridization. The sequences can also be intentionally selected such that the regions of hybridization are adjacent to one another on the nucleic acid to be detected.
[0035]
By selecting nucleic acid analogs having sequences complementary to those of these nucleic acids, many mutations can be detected in the same or different nucleic acids.
[0036]
In a particularly easy case, where the aim is to determine which of the two alleles is present in the complex, the two nucleic acid analogues bind to different sites on the surface of the solid support and their base sequences are Alleles also differ exactly in different positions. Thus, one selects a nucleic acid analog that is complementary to one sequence of one allele, while the other nucleic acid analog is complementary to the sequence of the other allele. The length of the nucleic acid analog and the site of hybridization are the same.
[0037]
For example, all alleles for cystic fibrosis and the like are usually detected. Wild type contains two healthy alleles. Heterozygotes contain one mutation and one wild-type sequence, and homozygous mutations contain two mutated nucleic acids. In this case, the intention is not only to determine if the mutation is present, but also whether it is heterozygous or homozygous. According to the present invention, it is possible to quantitatively detect both alleles simultaneously, and it is therefore possible to distinguish the three cases.
[0038]
In many cases, particularly in the determination of oncology and infection parameters, mutated cells / particles are usually present in the background of non-mutated / normal cells. In these cases, selective detection cannot be reliably performed, or the methods provided by the state of the art cannot be used at all. For example, analysis of ras mutations from DNA from stool samples requires the reliable detection of mutant sequences in the presence of about 100 normal sequences (Science {256, {102-105} (1992)). . In the field of infectious diseases, it is desirable to determine different HIV populations in one infected patient. However, the amount of many variants in these HIV populations is less than 2% for all HIV sequences. Thus, the method provided by the present invention makes it particularly perfectly possible to examine nucleic acid mixtures very similar to each other, even if one nucleic acid is present in a much larger amount than the nucleic acid to be determined.
[0039]
Preferably, the lengths of the bound nucleic acid analogs are the same. Suitable lengths have been found to be 10-100 bases, preferably 10-50 bases. Particularly good results are obtained with nucleic acid analogs that are 10 to 25 bases in length.
[0040]
To carry out the method provided by the present invention, a sample containing nucleic acids is contacted with a site on the surface of a carrier that has bound a nucleic acid analog. This can be done, for example, by bringing the solid carrier into the sample solution or by pouring the sample solution into one or more portions on the solid carrier. Prior to contact with the carrier, the nucleic acid in the sample solution can be denatured (single-stranded). However, a major advantage of the present invention is the fact that a preliminary denaturation step can be eliminated, for example, by using PNA. PNA pushes a strand out of the double-stranded nucleic acid to be determined. The only important requirement is that a nucleic acid analog complementary to one sequence of the nucleic acid to be determined is contacted with the solid support under conditions in which the nucleic acid to be detected specifically binds to a suitable site on the surface. That is. Of course, these conditions may be different for different types of nucleic acid analogs, but are easily determined for a given nucleic acid analog by performing tests. In general, these conditions are based on conditions known for carriers loaded with oligonucleotides. However, if the nucleic acid analog is used as described in WO 92/20702, conditions can be chosen which are significantly different from the hybridization conditions for the corresponding oligonucleotide. However, it has been found appropriate to use much smaller amounts of salt than with the corresponding oligonucleotide. For example, the presence of less than 100 mM salt, more preferably less than 50 mM salt and most preferably less than 10 mM salt is recommended. Under these conditions, oligonucleotides having the same sequence will not be sufficient to distinguish nucleic acids having similar sequences.
[0041]
The sample is contacted with the surface as long as necessary to achieve sufficient binding of the nucleic acid to the appropriate site on the surface. This period is typically a few minutes.
[0042]
The next step is to determine whether the nucleic acid has bound to the surface and, if so, to which site. This is regarded as an indicator of the presence of a nucleic acid containing a nucleotide sequence complementary to the nucleic acid analog bound to this site. A variety of methods can be used to determine binding.
[0043]
Devices are already available that can directly determine changes at specific sites on the surface. For methods using these types of devices, it is not necessary to remove a sample containing nucleic acids from the surface after application. However, it is usually preferable to remove the liquid from the surface and use a cleaning solution to remove any residual reagent still adhering to the surface. This step offers the advantage of also washing out sample components that can interfere with the binding determination.
[0044]
In a preferred embodiment, the binding between the nucleic acid analog and the nucleic acid to be detected is determined by the label inserted into the nucleic acid to be determined in a step performed before the sample is brought into contact with the surface. The label may be, for example, a detectable group such as a fluorescent group. This determination can optionally be made using a microscope or in a measuring cell provided for this purpose. Although the site at which binding occurs is an indicator of the presence of a nucleic acid having a certain sequence, the amount of label at a site determined in advance can be used as an indicator of the amount of nucleic acid to be determined.
[0045]
In a particularly preferred embodiment, the nucleic acid to be determined is a nucleic acid amplification method such as the polymerase chain reaction described in European Patent EP-B-0 {202} 362 or the NASBA or the like described in European Patent Application No. 0 {329} 822. Is the product of It is important that nucleic acid sequences that bind to the nucleic acid analog are amplified by the amplification method. The better the amplification method retains the original sequence, ie, the fewer errors that are incorporated into the sequence during amplification, the better the amplification method. The polymerase chain reaction has been found to be particularly suitable. Amplification primers are particularly chosen such that the nucleic acid sequence to be detected is located in the region between the hybridization sites.
[0046]
It has also proved advantageous during the amplification to insert the labels necessary for the detection reaction into the amplification. For example, this can be done by using a labeled primer or a labeled mononucleoside triphosphate.
[0047]
The binding that has occurred can be detected directly without the insertion of a label, for example, by using an intercalating agent. These agents have the property of selectively precipitating on base-containing double-stranded compounds containing complexes of nucleic acid and nucleic acid bound in a sequence-specific manner. The presence of the complex can be detected using a special property of the intercalating agent such as a fluorescent reagent. Ethidium bromide is a particularly preferred agent.
[0048]
Another method of detecting hybrids without inserting a label is based on surface plasmon resonance, for example as described in EP-A-0 618 441.
[0049]
According to another possible method of determining binding, the surface is contacted with a labeled antibody solution for detection. This antibody is directed against a complex consisting of the nucleic acid analog and the nucleic acid to be determined. Antibodies of this nature are described, for example, in WO 95/17430.
[0050]
Hybrid detection depends on the type of label used. For example, it can be detected using a scanner, a CCD camera or a microscope.
[0051]
The present invention offers many advantages. In particular, differences in the sequence of the nucleic acid region located in the secondary structure can be detected. The present invention can also be used to amplify the sensitivity of the detection, since larger absolute amounts of the binding nucleic acid to be determined can be used compared to conventional methods. In particular, it is possible to increase the signal-to-noise ratio as compared to the method using oligonucleotides. In a first attempt, a signal to noise ratio of less than 1: 1000 was obtained.
[0052]
The invention can be used in at least two fields. In the first case, known mutations and polymorphism are detected using a solid support. In this case, the number of mutations and polymorphisms to be detected is an indicator of the number or site of various nucleic acid analogs required on the surface. The sequence of the nucleic acid analog is specifically comparable to the sequence of the nucleic acid near the mutation and polymorphism. The sequence is preferably selected in such a way that different bases due to nucleic acid analogs having a similar sequence are located at or near the sequence.
[0053]
The carriers provided by the present invention can be used in the following fields:
In the study of infectious diseases, simultaneous determination of different analytes / parameters and the status of genes in bacteria or viruses for eg multidrug resistance studies.
Oncology (mutation detection in tumor suppressor and oncogenes and determination of correlation between mutant and normal cells).
Study of hereditary diseases (cystic fibrosis, sickle cell anemia, etc.).
Tissue and bone marrow typing (MHC complex) (see Clin. {Chem. {41/4, {553-5 (1995)}).
[0054]
In a second possible application, the sequence of short nucleic acid fragments can be determined using the so-called "sequence by hybridization" method. In this method, the same number of different nucleic acid analogs in which there is a permutation of the selected sequence length are immobilized. In order to achieve a sufficient level of sensitivity, N is equal to the number of bases in each nucleic acid analog.^NAre required. It is preferable that N is 5 to 12. To sequence very short DNA fragments requires significantly fewer sites. A method of sequencing an unknown nucleic acid using the “sequence by hybridization” method is described in WO 92/10588.
An advantage of the present invention is the fact that the specificity of hybridization hardly depends on the conditions of the sample. This facilitates simultaneous binding of nucleic acids to different regions of the surface.
[0055]
Surprisingly, nucleic acid analogs such as PNA have been shown to have excellent ability to discriminate surface sequences. This discrimination was better than expected from the melting temperature of similar dissolved compounds.
[0056]
Surprisingly, the carriers provided by the present invention are also suitable for use in determining a number of consecutive nucleic acids. After making the determination, the carrier in contact with the liquid is subjected to a heat treatment. The temperature at which the bond between the nucleic acid analog and the nucleic acid dissolves is selected. The carrier can then be utilized to make another determination. Using the methods provided by the present invention, it is possible to first determine the relative amounts of very similar nucleic acids located in close proximity to one another in a sample, at concentrations in the range of at least 100-fold. For example, it was possible to quantitatively determine the mutant using a sequencing method. However, the maximum discrimination available using this method was 1:10.
[0057]
Using the methods provided by the present invention, it is also possible to bind double-stranded DNA to a nucleic acid analog immobilized on a solid support without a denaturation step. Comparative studies have shown that this is not possible with immobilized DNA. It was also shown that mismatches that were not at the center of the hybrid could be identified with high selectivity.
[0058]
【Example】
The following examples illustrate the invention in more detail.
[0059]
General:
The nucleic acid analogs used were prepared as described in WO 92/20702. Unless otherwise indicated, compounds and reagents were products of Boehringer @ Mannheim @ GmbH.
[0060]
Example 1:
Covalent bond derivatization of nylon membrane
A 200 nl solution containing PNA at the desired concentration in 0.5 M sodium carbonate pH 9.0 is applied to the Immunodyne ABC membrane (Pall) using a pipette. After the spots have dried, the membrane is washed with 0.1 M sodium hydroxide solution to inactivate functional reactive groups on the surface that may still be present. The membrane is washed twice with water and then dried.
[0061]
Example 2:
Detection of hybridization events using luminescence
The membrane is derivatized as described in Example 1 using 100 μM, 10 μM, 1 μM, and 0.1 μM @PNA solution. Then, the membrane was placed in a hybridization oven at 45 ° C. using 50 ml of a hybridization buffer (10 mM sodium phosphate, pH 7.2, 0.1% SDS (sodium dodecyl sulfate)) in a 50 ml hybridization container. Prehybridize. After 30 minutes, 10 μl of a solution containing the DIG-labeled oligonucleotide at a concentration of 1 μM is added and the complex is allowed to hybridize for another 60 minutes. The membrane is then washed twice at 45 ° C. for 10 minutes with 25 ml of wash buffer (5 mM sodium phosphate, pH 7.2, 0.05% SDS) each time. The detection reaction is performed according to a protocol for detecting digoxigenin (DIG {Detection Kit, {Boehringer Mannheim}, GmbH, BRD)}. The anti-DIG-AP conjugate is used at a dilution of 1: 10000. CDP-Star as a substrate for alkaline phosphatase^TMIs used at a dilution of 1: 10000.
[0062]
Example 3:
Detection of hybridization events using fluorescence
Membranes are derivatized as described in Example 1 with 100 μM, 10 μM and 1 μM @PNA solutions. Pre-hybridize the membrane in a 45 ° C. oven with 10 ml of hybridization buffer (see Example 2) in a 50 ml screw-cap vessel. After 30 minutes, 10 μl of a solution containing the fluorescently labeled oligonucleotide at a concentration of 1 μM is added and the preparation is allowed to hybridize for another 60 minutes. The membrane is then washed twice with 25 ml of wash buffer (see Example 2) twice at 45 ° C. for 10 minutes. The membrane is dried and then the fluorescence intensity is measured.
[0063]
Example 4:
Method selectivity
As described in Example 1, using a PNA solution in a concentration range of 100 μM to 0.1 μM, three (Ado) base sequences differing at one or two positions, respectively.₆-Derivatize three membrane pieces with the PNA molecule (see Fig. 1a, SEQ ID NOs: 1, 2, 3). The membrane pieces are prehybridized with 10 ml of hybridization buffer for 30 minutes in a 50 ml screw-top container. In the next step, one of the three DIG-labeled oligonucleotides (FIG. 1b, SEQ ID NOs: 4, 5, 6) is added. After hybridizing for 60 minutes, the membrane is washed twice with 25 ml of wash buffer each time for 10 minutes. A hybridization event is detected as described in Example 2.
[0064]
All possible double-stranded hybrids of PNA molecules and oligonucleotides involved are shown in FIG. 1d. FIG. 2 shows that in almost all cases, only the oligonucleotide detected is exactly the complement of the immobilized nucleic acid analog (PNA1, PNA2, PNA3). The signal to noise ratio (S / N) can also be estimated from the figure. They were quantitatively evaluated and the results are shown in Table 1.
[0065]
[Table 1]

[0066]
Example 5:
Quantitative
As described in Example 1, at a concentration of 100 μM, three (Ado) s having different base sequences₆Derivatize the membrane pieces using the PNA molecule (FIG. 1a, SEQ ID Nos. 1, 2, 3). They are then prehybridized at 45 ° C. in a 20 ml hybridization vessel with 10 ml of hybridization buffer (see Example 2). After 30 minutes, change the buffer. Throughout Examples 1-7, the buffer added depends on the concentration of the DIG-labeled component—the analytes of oligonucleotides 1, 2, and 3, SEQ ID NOs: 4, 5, and 6. The pieces are hybridized at 45 ° C. for 60 minutes and then washed twice with 10 ml of wash buffer for 10 minutes. Detection is performed using the method described in the second embodiment. The luminescence signal is recorded using a luminescence imager and then evaluated (FIG. 4).
[0067]
The signal strength found can be used to achieve a qualitative or semi-quantitative finding on the analyte complex composition. After correcting the signal intensity, absolutely quantitative findings can be achieved.
[0068]
Example 6:
PCR amplicon detection
A. Obtaining a suitable analyte (amplified product)
A double-stranded DNA fragment whose sequence is complementary to the PNA probe, PNA1, is ligated to the pUC19 plasmid. The plasmid is transformed into E. coli, cloned and then sequenced. For the next hybridization experiment, a portion of the plasmid sequence is amplified and DIG-labeled during the amplification reaction. Amplification is performed in a total volume of 50 μl. The amplification complex was composed of 1 μl plasmid (1 ng / μl), 1 μl primer F1 (10 μM), 1 μl DIG primer R1 (10 μM), 5 μl 10 × PCR buffer (100 mM Tris / HCl, 15 mM MgCl2).₂, 500 mM KCl, pH 8.3), 2 μl dNTP solution (10 mM dATP, 10 mM dCTP, 10 mM dGTP, 10 mM dTTP, pH 7.0 in distilled water), 0.5 μl Taq polymerase (5 units / μl) and 38.5 μl water Become.
[0069]
Primer F1: 5'-GTA AAA CGA CGG CCA GT-3 '(SEQ ID NO: 12)
Primer R1: 5'-DIG-AAC \ AGC \ TAT \ GAC \ CAT \ GA-3 '(SEQ ID NO: 13)
[0070]
Warm each reaction mixture at 96 ° C. for 3 minutes. In the next step, three PCR cycles (96 ° C., 45 seconds, 48 ° C., 30 seconds, 72 ° C., 1 minute) are performed 30 times. In the final cycle, increase the extension step for 5 minutes at 72 ° C.
[0071]
B. Hybridization reaction
As described in Example 1, using a PNA solution in a concentration range of 100 μM to 0.1 μM, three (Ado) nucleotide sequences having different nucleotide sequences at one or two positions, respectively.₆-Derivatize the membrane with the PNA sequence (see Fig. La, SEQ ID NOs: 1, 2, 3). The membrane is pretreated at 45 ° C. with 5 ml of hybridization buffer in a 20 ml hybridization vessel. After 30 minutes, the buffer is replaced and the analyte solution is added. To make the analyte solution, dilute the amplification complex directly (ds amplicon) and after 5 minutes heat denaturation (ss amplicon) with 1 ml of hybridization buffer. After hybridization at 45 ° C. for 1 hour, 2 hours 30 minutes and 4 hours, the membrane is washed twice with 5 ml of wash buffer each time for 10 minutes. A hybridization event is detected as described in Example 2 (FIG. 3).
[0072]
FIG. 3 shows nine sections. The difference between each row is the time of incubation (4 hours, 2.5 hours and 1 hour). The difference between each column is the type of nucleic acid to be detected. The spots in three overlapping rows are applied to each of the three compartments of column I. The difference between the columns is the sequence of the PNA, while the difference between the columns in each compartment is the concentration. Column I shows the specificity and quantification ability for the case where the oligonucleotide was used as the nucleic acid to be detected.
[0073]
The figure shows the effect of incubation time. It is clear that after only one hour of hybridization, excellent sequence discrimination between ODN1a and the amplicon is obtained. The difference between the columns II and III in FIG. 3 is that a single-stranded amplified product is used as a nucleic acid to be detected in one case. In column III, an amplified product that has not been made into a single strand in advance is used as a nucleic acid to be detected. The signal clearly indicates that it is not necessary to denature the double-stranded nucleic acid before applying it to the solid support. This reduces the number of working steps (heating step, single strand separation, washing step) and thus reduces the risk of contamination. Thus, PNA probes in combination with low salt conditions offer distinct advantages over DNA probes.
[0074]
Example 7:
Comparison of PNA / DNA hybridization
As described in Example 1, using a 50 μM solution, the base sequence differs at one or two positions (see FIGS. 1 a and 1 b), each with three (Ado)₆-Derivatize a membrane piece with the PNA sequence (SEQ ID NO: 1, 2, 3) and three DNA molecules each (SEQ ID NO: 8, 10, 11). Unlike Example 1, the spot volume is 400 nl instead of 200 nl. 5 ml of low salt buffer (see Example 2) or high salt buffer (6 × SSC; 0.9 M NaCl, 90 mM sodium citrate, 0.1% SDS, pH 7.0) in a 20 ml hybridization vessel The membrane pieces are prehybridized at either 37 ° C. or 45 ° C. for 30 minutes using either In the next step, one of the three DIG-labeled oligonucleotides (FIG. 1c, SEQ ID NOs: 4, 5, 6) is added. After a 60 minute hybridization step, the strips are washed twice at 37 ° C. or 45 ° C. with 5 ml of wash buffer each time for 10 minutes. For low salt experiments, the wash buffer from Example 2 is used. For high salt experiments, 1 × SSC buffer with 0.02% SDS, pH 7.0 is used. As described in Example 2, the results of the hybridization are detected. The evaluation was performed quantitatively and shown in Table 2.
[0075]
Both DNA and PNA probes are capable of completely discriminating the complementary single-stranded target sequences of single and double mismatched sequences. PNA probes offer a distinct advantage over DNA probes for certain types of mismatches, especially when the mismatch is not in the middle of the sequence but rather shifted to the ends. This is particularly evident in the example of a dispersed G / T mismatch (probe 1 / ODN3), which is much more tolerated by DNA probes than by PNA probes having identical sequences.
[0076]
[Table 2]

[0077]
Example 8:
Effect of linker length between membrane and PNA probe
As described in Example 1, the linker length (Ado) was determined using a PNA solution in a concentration range of 100 μM to 1 μM.₃, Ado₆Or Ado₉) Induce membrane fragments with different PNA molecules (see FIG. 1a, SEQ ID NOs: 7, 1, 9). Unlike Example 1, the spot volume is 1 μl instead of 200 nl. The membrane pieces are prehybridized at 35 ° C. for 30 minutes in a hybridization vessel containing 10 ml of hybridization buffer (5, 10 or 25 mM sodium phosphate, 0.1% SDS, pH 7.0). In the next step, 10 pMol³²A P-labeled oligonucleotide (FIG. 1c; ODN1b, see SEQ ID NO: 4) is added and the preparation is hybridized at 50 ° C. for 60 minutes. The pieces are washed twice with 50 ml of wash buffer (5 mM sodium phosphate, 0.1% SDS, pH 7.0) at 50 ° C. for 10 minutes. Hybridization events are detected using autoradiography (FIG. 5). The figure shows that longer linkers greatly improve hybridization.
[0078]
Example 9:
Reuse of PNA membrane
As described in Example 1, using a PNA solution in a concentration range of 100 μM to 1 μM, (Ado)₆-Derivatize the membrane with a PNA molecule (Fig. 1a; PNA1b, see SEQ ID NO: 1). The PNA is applied in five identical concentrations in succession. As described in Example 8, the spot volume is 1 μl. The membrane is prehybridized at 35 ° C. for 30 minutes in a hybridization vessel containing 10 ml of hybridization buffer (10 mM sodium phosphate, 0.1% SDS, pH 7.0). In the next step, 10 pMol³²A P-labeled oligonucleotide (FIG. 1c; ODN1b, SEQ ID NO: 4) is added and the preparation is hybridized at 50 ° C. for 60 minutes. The membrane is washed twice with 50 ml of wash buffer (5 mM sodium phosphate, 0.1% SDS, pH 7.0) at 50 ° C. for 10 minutes. Hybridization events are detected using autoradiography (FIG. 6a). After performing autoradiography, the membrane is cut into five identical pieces. These pieces of membrane are handled separately during the rest of the experiment. Membrane 1 serves as a control membrane without incubation. Membrane 2 is incubated with 50 ml of 0.1 M sodium hydroxide solution for 60 minutes at room temperature. Membrane 3 is likewise incubated with 50 ml of a 1 M sodium hydroxide solution for 60 minutes. The membrane 4 is incubated at 70 ° C. for 60 minutes with 50 ml of distilled water. The membrane 5 is incubated with 70 ml of a 0.1N sodium hydroxide solution at 70 ° C. for 60 minutes. Then wash all membranes twice with distilled water for 10 minutes. After this operation is completed, another autoradiography is performed (FIG. 6B). These pieces of membrane are then used in a second hybridization reaction as described to detect hybridization events using autoradiography (FIG. 6c).
[0079]
As shown in FIG. 6b, different processing methods yield very different results. Treatment with double distilled water at 70 ° C. (membrane 4) almost completely regenerates the membrane. An unexpected finding was the fact that successful regeneration was less successful using conditions commonly used for nucleic acid denaturation. Incubation of membrane 3 with a 1 M sodium hydroxide solution at room temperature by itself produces virtually no regeneration effect. Reducing the concentration of the sodium hydroxide solution from 1 M to 0.1 M increases the regeneration at room temperature (membrane 2) and at 70 ° C. (membrane 5). However, none of these conditions results in any good regeneration, as in the case of using double distilled water (membrane 4). The examples show that these conditions are important parameters for efficient denaturation of the membrane-bound PNA / DNA duplex.
[0080]
Regardless of the regeneration method, all membranes can be reused for hybridization without significantly degrading the signal-to-noise ratio (FIG. 6c). Up to six rehybridizations can be performed without losing the significant effect of the ability of the PNA membrane to regenerate or rehybridize.
[0081]
【The invention's effect】
According to the present invention, it is possible to provide an alternative method for sequence-specific detection of a nucleic acid and to provide suitable materials for this method. It is also possible to provide a solid support having two or more nucleic acid analogs having different base sequences bound to predetermined sites on the surface of the solid support. Further, the present invention can provide a method for detecting a sequence of a nucleic acid using the solid carrier.
[0082]
[Sequence list]
(1) Information of SEQ ID NO: 1
(I) Sequence features:
(A) Sequence length: 16 amino acids
(B) Sequence type: amino acid
(C) Number of chains: single-stranded
(D) Topology: linear
(Ii) Sequence type: peptide
(Iii) Hypothetical: NO
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) Location: 1
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-thyminyl) acetyl) -N- (2- amino (N′-hexa (8- amino-3,6- dioxa- octano-1- yl) -ethyl) -beta- alanine)
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) @ Location: 2
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-guaninyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) @ Location: 3
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-thyminyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) @ Location: 4
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-adenylyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) @ Location: 5
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-cytosyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) @ Location: 6
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-guaninyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) Location: 7
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-thyminyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) Location: 8
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-cytosyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) @ Location: 9
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-adenylyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) Location: 10
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-cytosyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) @ Location: 11. . 12
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-adenylyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) Location: 13
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-cytosyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) Location: 14
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-thyminyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) Location: 15
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-adenylyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”

(1) Information of SEQ ID NO: 2
(I) Sequence features:
(A) Sequence length: 16 amino acids
(B) Sequence type: amino acid
(C) Number of chains: single-stranded
(D) Topology: linear
(Ii) Sequence type: peptide
(Iii) Hypothetical: NO
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) Location: 1
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-thyminyl) acetyl) -N- (2- amino (N′-hexa (8- amino-3,6- dioxa- octano-1- yl) -ethyl) -beta- alanine)
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) @ Location: 2
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-guaninyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) @ Location: 3
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-thyminyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) @ Location: 4
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-adenylyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) @ Location: 5
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-cytosyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) @ Location: 6
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-guaninyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) Location: 7
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-thyminyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) Location: 8
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-guaninyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) @ Location: 9
(D) Other information: / product = “Others”
/ Note = “Xaa} is {N-((1-adenyl)} acetyl) -N- (2- {aminoethyl) -beta- {alanine”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) Location: 10
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-cytosyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) @ Location: 11. . 12
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-adenylyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) Location: 13
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-cytosyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) Location: 14
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-thyminyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) Location: 15
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-adenylyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”

(1) Information of SEQ ID NO: 3
(I) Sequence features:
(A) Sequence length: 16 amino acids
(B) Sequence type: amino acid
(C) Number of chains: single-stranded
(D) Topology: linear
(Ii) Sequence type: peptide
(Iii) Hypothetical: NO
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) Location: 1
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-thyminyl) acetyl) -N- (2- amino (N′-hexa (8- amino-3,6- dioxa- octano-1- yl) -ethyl) -beta- alanine)
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) @ Location: 2
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-guaninyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) @ Location: 3
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-thyminyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) @ Location: 4
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-adenylyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) @ Location: 5
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-cytosyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) @ Location: 6
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-adenylyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) Location: 7
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-thyminyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) Location: 8
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-cytosyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) @ Location: 9
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-adenylyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) Location: 10
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-cytosyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) @ Location: 11. . 12
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-adenylyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) Location: 13
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-cytosyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) Location: 14
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-thyminyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) Location: 15
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-adenylyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”

(1) Information of SEQ ID NO: 4:
(I) Sequence features:
(A) Sequence length: 15 base pairs
(B) Sequence type: nucleic acid
(C) Number of chains: single-stranded
(D) Topology: linear
(Ii) Sequence type: other nucleic acid
(A) {type: {/ desc} = {"oligodeoxyribonucleotide"]
(Iii) Hypothetical: NO
(Ix) Sequence features:
(A) Symbol representing feature: misc_feature
(B) Location: 15
(D) {other information: / note = "label the 5 '-{phosphate} group with digoxigenin or 32-P via an aminolinker (Boehringer \ Mannheim \ GmbH \, \ BRD)"

(1) Information of SEQ ID NO: 5:
(I) Sequence features:
(A) Sequence length: 15 base pairs
(B) Sequence type: nucleic acid
(C) Number of chains: single-stranded
(D) Topology: linear
(Ii) Sequence type: other nucleic acid
(A) {type: {/ desc} = {"oligodeoxyribonucleotide"]
(Iii) Hypothetical: NO
(Ix) Sequence features:
(A) Symbol representing feature: misc_feature
(B) Location: 15
(D) {Other information: / note = "Labeling the 5 '-{phosphate" group with digoxigenin via an aminolinker (Boehringer @ Mannheim \ GmbH, \ BRD) "

(1) Information of SEQ ID NO: 6:
(I) Sequence features:
(A) Sequence length: 15 base pairs
(B) Sequence type: nucleic acid
(C) Number of chains: single-stranded
(D) Topology: linear
(Ii) Sequence type: other nucleic acid
(A) {type: {/ desc} = {"oligodeoxyribonucleotide"]
(Iii) Hypothetical: NO
(Ix) Sequence features:
(A) Symbol representing feature: misc_feature
(B) Location: 15
(D) {other information: / note = "Labeling the 5 '-{phosphate" group with digoxigenin via an aminolinker (Boehringer @ Mannheim \ GmbH, \ BRD) "

(1) Information on SEQ ID NO: 7:
(I) Sequence features:
(A) Sequence length: 15 base pairs
(B) Sequence type: nucleic acid
(C) Number of chains: single-stranded
(D) Topology: linear
(Ii) Alcohols of the sequence: peptide
(Iii) Hypothetical: NO
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) Location: 1
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-thyminyl) {acetyl) -N- (2- {amino (N'-tri (8-amino} -3,6- {dioxa- {octano-1- {yl) -ethyl) -beta- {alanine} ")
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) @ Location: 2
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-guaninyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) @ Location: 3
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-thyminyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) @ Location: 4
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-adenylyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) @ Location: 5
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-cytosyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) @ Location: 6
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-guaninyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) Location: 7
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-thyminyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) Location: 8
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-cytosyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) @ Location: 9
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-adenylyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) Location: 10
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-cytosyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) @ Location: 11. . 12
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-adenylyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) Location: 13
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-cytosyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) Location: 14
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-thyminyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) Location: 15
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-adenylyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”

(1) Information of SEQ ID NO: 8:
(I) Sequence features:
(A) Sequence length: 30 base pairs
(B) Sequence type: nucleic acid
(C) Number of chains: single-stranded
(D) Topology: linear
(Ii) Sequence type: other nucleic acid
(A) {type: {/ desc} = {"oligodeoxyribonucleotide"]
(Iii) Hypothetical: NO

(1) Information of SEQ ID NO: 9:
(I) Sequence features:
(A) Sequence length: 16 amino acids
(B) Sequence type: amino acid
(C) Number of chains: single-stranded
(D) Topology: linear
(Ii) Alcohols of the sequence: peptide
(Iii) Hypothetical: NO
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) Location: 1
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-thyminyl) {acetyl) -N- (2- {amino (N'-nona (8-amino} -3,6- {dioxa- {octano-1- {yl) -ethyl) -beta- {alanine} ")
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) @ Location: 2
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-guaninyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) @ Location: 3
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-thyminyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) @ Location: 4
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-adenylyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) @ Location: 5
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-cytosyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) @ Location: 6
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-guaninyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) Location: 7
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-thyminyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) Location: 8
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-cytosyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) @ Location: 9
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-adenylyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) Location: 10
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-cytosyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) @ Location: 11. . 12
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-adenylyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) Location: 13
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-cytosyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) Location: 14
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-thyminyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”
(Ix) Sequence features:
(A) Symbol representing feature: Modified-site
(B) Location: 15
(D) Other information: / product = “Others”
/ Note = “Xaa is
N-((1-adenylyl) acetyl) -N- (2- aminoethyl) -beta- alanine ”

(1) Information of SEQ ID NO: 10:
(I) Sequence features:
(A) Sequence length: 30 base pairs
(B) Sequence type: nucleic acid
(C) Number of chains: single-stranded
(D) Topology: linear
(Ii) Sequence type: other nucleic acid
(A) {type: {/ desc} = {"oligodeoxyribonucleotide"]
(Iii) Hypothetical: NO

(1) Information of SEQ ID NO: 11:
(I) Sequence features:
(A) Sequence length: 30 base pairs
(B) Sequence type: nucleic acid
(C) Number of chains: single-stranded
(D) Topology: linear
(Ii) Sequence type: other nucleic acid
(A) {type: {/ desc} = {"oligodeoxyribonucleotide"]
(Iii) Hypothetical: NO

(1) Information of SEQ ID NO: 12:
(I) Sequence features:
(A) Sequence length: 17 base pairs
(B) Sequence type: nucleic acid
(C) Number of chains: single-stranded
(D) Topology: linear
(Ii) Sequence type: other nucleic acid
(A) {type: {/ desc} = {"oligodeoxyribonucleotide"]
(Iii) Hypothetical: NO

(1) Information of SEQ ID NO: 13:
(I) Sequence features:
(A) Sequence length: 17 base pairs
(B) Sequence type: nucleic acid
(C) Number of chains: single-stranded
(D) Topology: linear
(Ii) Sequence type: other nucleic acid
(A) {type: {/ desc} = {"oligodeoxyribonucleotide"]
(Iii) Hypothetical: NO
(Ix) Sequence features:
(A) Symbol representing feature: misc_feature
(B) Location: 1
(D) {other information: / note = "5 '-{terminal A} binds to digoxigenin via amino modifier (Boehringer Mannheim {GmbH)}"

[Brief description of the drawings]
FIG. 1a shows the sequence of the PNA molecule. They are used as examples to illustrate the method of the invention. PNA was prepared as described in WO 92/20702.
FIG. 1b shows the sequence of the DNA molecule used in the DNA / ODN hybridization experiments that is homologous to the PNA sequence from FIG. 1a.
FIG. 1c shows that the 5′-phosphate end is labeled with digoxigenin using the 5′-DIG End Labelling Kit (Boehringer Mannheim), and the polynucleotide kinase and³²The sequence of the complementary oligonucleotide (ODN) used, phosphorylated with P-γ-ATP5 ', is shown.
FIG. 1d shows the possible combinations of oligonucleotides (ODN) and PNA to form a hybrid. The same combination applies to the hybridization between a DNA probe (DNA, see FIG. 1b) and an oligonucleotide (ODN, see FIG. 1c).
FIG. 2 shows the results of hybridization from Example 4 showing the selectivity of the method of the present invention. Conditions were: 200 nl spot volume (100; 10; 1; 0.1 μM @PNA, one concentration per cleavage), incubation at 45 ° C.
FIG. 3 shows the results of hybridization from Example 6. They demonstrate that immobilized PNA probes detect PCR amplicons. Conditions were: 200 nl spot volume (100; 10; 1; 0.1 μM @PNA, one concentration per cleavage), incubation at 45 ° C. The symbols in FIG. 3 mean the following:
I control experiment, ODN1a (1 pMol, 1 nM), row 1 (PNA1), row 2 (PNA2), row 3 (PNA3)
II s amplified product (118 bp, 1x DIG label)
Thermal denaturation at 94 ° C for 5 minutes
(50 μl @ PCR preparation diluted with 1 ml hybridization buffer)
Row 1 (PNA1), Row 2 (PNA2), Row 3 (PNA3)
III ds amplification product (118 bp, 1 × DIG label)
(50 μl @ PCR preparation directly diluted in 1 ml hybridization buffer)
Row 1 (PNA1), Row 2 (PNA2), Row 3 (PNA3).
FIG. 4 shows the results of qualitative and quantitative analysis of the analysis mixture by PNA array. Conditions were: 200 nl spot volume (100 μM @PNA, one PNA per row, one analysis mixture per cleavage).
FIG. 5 shows the effect of linker length on hybridization. Conditions were: 1 μl spot volume (100; 40; 20; 10; 5; 1 μM @ PNA, one concentration per cleavage, (Ado) in row 1₃-PNA, column 2 (Ado)₆-PNA, column 3 (Ado)₉-PNA). The symbols in FIG. 5 mean the following:
A. Prehybridization / hybridization with 5 mM sodium phosphate, 0.1% SDS, pH 7.0
B. Prehybridization / hybridization with 10 mM sodium phosphate, 0.1% SDS, pH 7.0
C. Prehybridization / hybridization with 25 mM sodium phosphate, 0.1% SDS, pH 7.0.
FIG. 6A shows the signal intensity after the first hybridization. FIG. 6A suggests that membranes derivatized with PNA can be used many times after regeneration. Conditions were: 1 μl spot volume (100; 40; 20; 10; 5; 1 μM @ PNA, one concentration per cleavage).
FIG. 6B shows the signal intensity after the regeneration technique. FIG. 6B suggests that the PNA-derivatized membrane can be used many times after regeneration. Conditions were: 1 μl spot volume (100; 40; 20; 10; 5; 1 μM @ PNA, one concentration per cleavage).
Membrane 1: no regeneration (control)
Membrane 2: 0.1 M sodium hydroxide solution, RT, 1 hour, 2 × 10 minutes {regeneration using double distilled water} RT
Membrane 3: Regeneration using 1M sodium hydroxide solution, RT, 1 hour, 2 × 10 minutes {double-distilled water} RT
Membrane 4: Regeneration using distilled water, 70 ° C., 1 hour, 2 × 10 minutes, double distilled water, RT
Membrane 5: 0.1 M sodium hydroxide solution, 70 ° C. for 1 hour, 2 × 10 minutes, regeneration using double distilled water RT
FIG. 6C shows the signal intensity after rehybridization. FIG. 6C suggests that the PNA-derivatized membrane can be used many times after regeneration. Conditions were: 1 μl spot volume (100; 40; 20; 10; 5; 1 μM @ PNA, one concentration per cleavage).

Claims (11)

A method for regenerating a solid support having at least two different peptide nucleic acid probes attached to a surface to which nucleic acids hybridize, comprising:
a) treating the carrier under conditions for denaturing the peptide nucleic acid / nucleic acid complex; and b) washing the solid carrier to remove the modified nucleic acid polymer;
Including, methods. 2. The method of claim 1, wherein the elevated temperature of distilled water is used to cause denaturation of the peptide nucleic acid / nucleic acid complex. 2. The method of claim 1, wherein the elevated temperature sodium hydroxide containing solution is used to cause denaturation of the peptide nucleic acid / nucleic acid complex. The method of claim 1, wherein the solid support is essentially flat. The method of claim 1, wherein the peptide nucleic acid probe has a 2-aminoethylglycine subunit backbone. A solid support comprising at least two single-stranded peptide nucleic acid probes of different base sequences covalently linked to the solid support in separate and distinct regions. 7. The solid support according to claim 6, wherein the separate and distinct regions are each separated by a region to which no probe is bound. 7. The solid support according to claim 6, wherein the probes are all identical in base length. The solid support according to claim 6, wherein all the probes have a length of 10 to 50 bases. 7. The solid support according to claim 6, wherein the solid support is essentially flat. The solid support according to claim 6, wherein the peptide nucleic acid probe has a backbone of 2-aminoethylglycine subunit.

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