JP3626782B2 - Oligonucleotide and nucleic acid sequence analysis method - Google Patents

Description

【００３２】
上記オリゴヌクレオチド及び試料ＤＮＡの合成は標準のプロトコール通りに行い、５’末端の保護基であるトリチル基は除かないサイクルにて合成を終了した。上記（９）の試料ＤＮＡをＯＰＣカートリッジ（ＡＢＩ社製）を用いて精製を行った。上記（１）〜（９）のオリゴヌクレオチド及び試料ＤＮＡを濃縮乾固した後、（１）〜（８）については０．５Ｍ重炭酸ナトリウム緩衝液（ｐＨ８．４）に、（９）についてはＴＥ緩衝液に懸濁し、２６０ｎｍの吸光度測定により定量し、１ｎｍｏｌ／μｌに調整した。
【００３３】
（Ｂ）オリゴヌクレオチドの固定化
オリゴヌクレオチドの固定化は、表面に高密度の陰イオン性カルボキシル基を有するナイロン膜にアミノ修飾オリゴヌクレオチドのアミノ基をアミド結合させることにより、以下の通り行った。
【００３４】
バイオダインＣ（商標；ポール社製）膜を０．１Ｎ ＨＣｌによりすすぎ、酸性化した後、２０％ＥＤＣ（１−エチル−３−ジメチルアミノプロピルカルボジイミド塩酸塩）に室温１５〜３０分間浸した。脱イオン水及び０．５Ｍ重炭酸ナトリウム緩衝液（ｐＨ８．４）で軽くすすいだ後、ドットブロット装置（Ｂｉｏ−Ｒａｄ社製）にセットした。０．５Ｍ重炭酸ナトリウム緩衝液（ｐＨ８．４）に懸濁されたアミノ修飾オリゴヌクレオチドを１５分間室温で膜と反応させた。ＴＢＳ（Ｔｒｉｓ−緩衝食塩水）／０．１％ Ｔｗｅｅｎ−２０によりすすいだ膜を、０．１Ｎ ＮａＯＨにて１０分間処理し、脱イオン水で軽くすすいだ後風乾した。
【００３５】
（Ｃ）試料ＤＮＡの標識化
試料ＤＮＡの標識化は［γ−３２Ｐ］ＡＴＰによって、５’末端を放射性ラベルした。反応は、ＤＮＡ５’末端標識キット（ＭＥＧＡＬＡＢＥＬＴＭ；宝酒造社製）により行った。
【００３６】
（Ｄ）ハイブリダイゼーション反応
前記のオリゴヌクレオチド固定化フィルターと、放射性標識した試料ＤＮＡとを、５× ＳＳＣ（７５０ｍＭ塩化ナトリウム・７５ｍＭクエン酸三ナトリウム）／０．１％ （ドデシル硫酸ナトリウム）緩衝液中にて、４〜１０℃ １時間〜一晩ハイブリダイゼーションさせた。ハイブリダイゼーション反応後、２× ＳＳＣ／０．１％ ＳＤＳ緩衝液にて、４〜１０℃、５分間の洗浄を３回行った。風乾した後、オートラジオグラフィーにて、各ドットの放射線量を測定し、ハイブリダイゼーション強度を算出した。
【００３７】
その結果を表２に示す。結果は対照とした比較例のオリゴヌクレオチドのハイブリダイゼーション強度を１とする相対値で示した。特異的領域の配列が異なる場合にハイブリダイゼーション量も変化するが、イノシン（非特異的領域）の導入によりいずれの場合もハイブリダイゼーション量が増大することが判明した。結合の種類及び数が全く同じ場合では、イノシンを３’、５’両末端に導入した場合の方が５’末端のみにイノシンを導入した場合よりもハイブリダイゼーション量が高いことが判明した。３’、５’両末端にイノシンを導入することで、高感度のハイブリダイゼーションが実現される。
【００３８】
【表２】

【００３９】
【実施例Ｂ】
ミスマッチの判別
（Ａ）オリゴヌクレオチドの合成
アプライド・バイオシステムズ社（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ ；ＡＢＩ社）製のＤＮＡ合成装置（装置名３９４ ＤＮＡ／ＲＮＡ Ｓｙｎｔｈｅｓｉｓｅｒ）によって表３に示す配列を有する本発明のオリゴヌクレオチド、比較対象用オリゴヌクレオチド及び試料ＤＮＡを、上記実施例Ａの（Ａ）と同様の方法で合成した。
【００４０】

【００４１】
（Ｂ）オリゴヌクレオチドの固定化
実施例Ａの（Ｂ）と同様にして行った。
（Ｃ）試料ＤＮＡの標識化
実施例Ａの（Ｃ）と同様にして行った。
（Ｄ）ハイブリダイゼーション反応
実施例Ａの（Ｄ）と同様にして行った。
（Ｅ）ミスマッチ判別能の計算
ハイブリダイゼーション反応によって得られた結果より、ミスマッチ判別能を算出した。ミスマッチ判別能とは、下記のＤ値（Ｄｉｓｃｒｉｍｉｎａｔｉｏｎ ｖａｌｕｅ）を指標とする概念であり、ミスマッチの区別し易さをさす。
【００４２】
【数１】

【００４３】
試料ＤＮＡと完全に相補的な配列のハイブリダイゼーション量とは、対照例の配列のハイブリダイゼーション量を指し、ミスマッチを有する配列のハイブリダイゼーション量とは、その対照例に対応する比較例あるいは実施例の配列のハイブリダイゼーション量を指す。Ｄ値は、対照例の配列に対する比較例あるいは実施例の配列のハイブリダイゼーション量の比である。即ち、対照例の配列のＤ値は１であり、比較例及び実施例に関してはＤ値が高ければその配列が対照例の配列と区別し易い（ハイブリダイゼーション量が対照例に対して小さい）ことを表す。
【００４４】
表４に各配列のＤ値を示す。イノシンを導入したオリゴヌクレオチドの方がＤ値が高く、特に３’，５’両末端にイノシンを導入したオリゴヌクレオチドの方がＤ値が高い。ミスマッチの判別能は、イノシンを導入したオリゴヌクレオチド、特に３’，５’両末端にイノシンを導入したオリゴヌクレオチドが優れていることが判明した。
【００４５】
【表４】

【００４６】
【実施例Ｃ】
界面活性剤のハイブリダイゼーション反応への影響
（Ａ）ＤＮＡの合成
アプライド・バイオシステムズ社（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ ；ＡＢＩ社）製のＤＮＡ合成装置（装置名３９４ ＤＮＡ／ＲＮＡ Ｓｙｎｔｈｅｓｉｓｅｒ）によって表５に示す配列を有する本発明のオリゴヌクレオチド、比較対称用オリゴヌクレオチド及び試料ＤＮＡを、上記実施例Ａの（Ａ）と同様の方法で合成した。
【００４７】
【表５】

【００４８】
（Ｂ）ＤＮＡの固定化
実施例Ａの（Ｂ）と同様にして行った。
（Ｃ）ＤＮＡの標識化
実施例Ａの（Ｃ）と同様にして行った。
（Ｄ）ハイブリダイゼーション反応
前記のオリゴヌクレオチド固定化フィルターに対して、放射性標識した試料ＤＮＡを、５× ＳＳＣ（７５０ｍＭ塩化ナトリウム・７５ｍＭクエン酸三ナトリウム）／７％ Ｎ−ラウロイルサルコシン酸ナトリウム緩衝液中にて、４〜１０℃１時間〜一晩ハイブリダイゼーション反応させた。ハイブリダイゼーション反応後、２× ＳＳＣ／０．１％ ＳＤＳ緩衝液にて、４〜１０℃、５分間の洗浄を３回行った。また、対照実験として、同様に作製したオリゴヌクレオチド固定化フィルターに対して、５× ＳＳＣ（７５０ｍＭ塩化ナトリウム・７５ｍＭクエン酸三ナトリウム）緩衝液中にて、４〜１０℃ １時間〜一晩ハイブリダイゼーション反応させた。ハイブリダイゼーション反応後、２× ＳＳＣ緩衝液にて、４〜１０℃・５分間の洗浄を３回行った。風乾した後、オートラジオグラフィーにて、各ドットの放射線量を測定し、ハイブリダイゼーション強度を算出した。
【００４９】
その結果を表６に示す。結果は、界面活性剤（Ｎ−ラウロイルサルコシン酸ナトリウム）を用いた場合の背景の放射線量を１とする相対値で示した。ハイブリダイゼーション反応にＮ−ラウロイルサルコシン酸ナトリウムを添加させるとフィルターへの非特異的な吸着によるバックグラウンドのシグナルを低下させる効果とともに、ハイブリダイゼーション強度が増大し、結果としてハイブリダイゼーション感度の向上が認められた。
【００５０】
【表６】

【００５１】
【発明の効果】
本発明によれば、ハイブリダイゼーション感度を上昇させることが可能となるとともに、完全に相補的なハイブリッドとミスマッチを有するハイブリッド、特にハイブリッドの末端部でのミスマッチが区別し易くなり、それによってハイブリダイゼーション法によるＤＮＡ塩基配列解析が容易になる。
【００５２】
【配列表】
配列番号：１
配列の長さ：８
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成オリゴヌクレオチド
配列

【００５３】
配列番号：２
配列の長さ：８
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成オリゴヌクレオチド
配列

【００５４】
配列番号：３
配列の長さ：８
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成オリゴヌクレオチド
配列

【００５５】
配列番号：４
配列の長さ：８
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成オリゴヌクレオチド
配列

【００５６】
配列番号：５
配列の長さ：１２
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成オリゴヌクレオチド
配列

【００５７】
配列番号：６
配列の長さ：１２
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成オリゴヌクレオチド
配列

【００５８】
配列番号：７
配列の長さ：１２
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成オリゴヌクレオチド
配列

【００５９】
配列番号：８
配列の長さ：１２
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成オリゴヌクレオチド
配列

【００６０】
配列番号：９
配列の長さ：２１
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成オリゴヌクレオチド
配列

【００６１】
配列番号：１０
配列の長さ：８
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成オリゴヌクレオチド
配列

【００６２】
配列番号：１１
配列の長さ：１２
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成オリゴヌクレオチド
配列

【００６３】
配列番号：１２
配列の長さ：１６
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成オリゴヌクレオチド
配列

【００６４】
配列番号：１３
配列の長さ：１６
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成オリゴヌクレオチド
配列

【００６５】
配列番号：１４
配列の長さ：２４
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成オリゴヌクレオチド
配列

【００６６】
配列番号：１５
配列の長さ：２６
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成オリゴヌクレオチド
配列

【００６７】
配列番号：１６
配列の長さ：８
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成オリゴヌクレオチド
配列

【００６８】
配列番号：１７
配列の長さ：８
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成オリゴヌクレオチド
配列

【００６９】
配列番号：１８
配列の長さ：８
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成オリゴヌクレオチド
配列

【００７０】
配列番号：１９
配列の長さ：８
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成オリゴヌクレオチド
配列

【００７１】
配列番号：２０
配列の長さ：１２
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成オリゴヌクレオチド
配列

【００７２】
配列番号：２１
配列の長さ：１２
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成オリゴヌクレオチド
配列

【００７３】
配列番号：２２
配列の長さ：１２
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成オリゴヌクレオチド
配列

【００７４】
配列番号：２３
配列の長さ：１２
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成オリゴヌクレオチド
配列

【００７５】
配列番号：２４
配列の長さ：１６
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成オリゴヌクレオチド
配列

【００７６】
配列番号：２５
配列の長さ：１６
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成オリゴヌクレオチド
配列

【００７７】
配列番号：２６
配列の長さ：１６
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成オリゴヌクレオチド
配列

【００７８】
配列番号：２７
配列の長さ：１６
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成オリゴヌクレオチド
配列

【００７９】
配列番号：２８
配列の長さ：１６
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成オリゴヌクレオチド
配列

【００８０】
配列番号：２９
配列の長さ：１６
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成オリゴヌクレオチド
配列

【００８１】
配列番号：３０
配列の長さ：１６
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成オリゴヌクレオチド
配列

【００８２】
配列番号：３１
配列の長さ：１６
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成オリゴヌクレオチド
配列

【００８３】
配列番号：３２
配列の長さ：２４
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成オリゴヌクレオチド
配列

【００８４】
配列番号：３３
配列の長さ：２４
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成オリゴヌクレオチド
配列

【００８５】
配列番号：３４
配列の長さ：２４
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成オリゴヌクレオチド
配列

【００８６】
配列番号：３５
配列の長さ：２４
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成オリゴヌクレオチド
配列

【図面の簡単な説明】
【図１】通常のオリゴヌクレオチドと核酸とのハイブリッドを示す模式図。
【図２】本発明のオリゴヌクレオチドと核酸とのハイブリッドを示す模式図。[0001]
[Industrial application fields]
The present invention relates to a method for analyzing nucleotide sequences of oligonucleotides and nucleic acids, and more particularly, for hybridization that can be used for nucleotide sequence analysis of nucleic acids, diagnosis of infectious diseases and genetic diseases, genome mapping, etc. The present invention relates to an oligonucleotide having a novel structure that can be suitably used as a probe, and a method for analyzing a base sequence using the oligonucleotide.
[0002]
[Prior art]
Nucleic acid base sequence analysis by hybridization is widely performed, for example, in base sequence determination, diagnosis of infectious diseases and genetic diseases, genome mapping, and the like. In addition, SBH (sequencing by hybridization) [R. Drmanac, et al. Science, 260, 1649 (1993)], that is, a method for determining a base sequence by hybridization is expected to be put to practical use as a high-speed and low-cost method.
[0003]
In these base sequence analyses, there is a technology that distinguishes between the hybrids between the probe and target sequences generated by hybridization (hybridization) that have mismatches and those that are completely complementary without mismatches. is necessary.
[0004]
Hybridization reaction is a complex reaction governed by many factors such as ionic strength of reaction solution, base composition of probe and sample DNA, reaction temperature and time, etc. Since hybrids with mismatches are unstable compared to completely complementary hybrids, a system that can detect only completely complementary hybrids can be constructed by appropriately setting these conditions. It is considered theoretically possible to do.
[0005]
However, conventionally, a technique of chemically modifying DNA bases to obtain highly sensitive hybridization [Proc. Natl. Acad. Sci. USA, 90, 11460 (1993)] and a method [DNA and Cell Biology, 9, 527 (1990)] in which washing after hybridization is performed at a low temperature for a long time as hybridization conditions to enhance mismatch discrimination ability. However, sufficient results have not been obtained.
[0006]
In particular, it is difficult to accurately and highly sensitively discriminate between hybridization when there is a mismatch near the end of the probe and hybridization that does not contain a mismatch, and this becomes a barrier to the practical application of nucleotide sequence analysis. Yes.
[0007]
[Problems to be solved by the invention]
The purpose of the present invention is to obtain a highly sensitive hybridization result in order to increase the accuracy of nucleic acid base sequence analysis by the hybridization method, and to clearly distinguish hybrids having mismatches at the ends from completely complementary hybrids. It is to provide an oligonucleotide and a base sequence analysis method that can be used.
[0008]
[Means for Solving the Problems]
As a result of intensive studies to achieve the above object, the present inventors have bound a region composed of a certain nucleotide at the end of a region having a specific base sequence to hybridize with a target sequence of a nucleic acid. It was found that the use of the oligonucleotides yielded a highly sensitive hybridization result and a base pair mismatch could be detected with high sensitivity, thereby completing the present invention.
[0009]
That is, the present invention
Consists of nucleotides containing a base selected from adenine (A), guanine (G), cytosine (C), thymine (T) or uracil (U)Link to a specific region and at least one of the 5 'end or the 3' end of this specific regionConsists of nucleotides containing bases other than adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U)An oligonucleotide having a non-specific region,
The specific region is the target sequence in the sample nucleic acid to be hybridized with the oligonucleotide.Complementary toHaving a specific base sequence,
The non-specific region isIncluded in nucleotides constituting specific regionsCan form base pairs with each of the bases,Other than adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U)Consists of at least one nucleotide having another base or oligomer thereofIt is characterized byIt is an oligonucleotide.
[0010]
The method of the present invention also includes
A specific region composed of nucleotides containing a base selected from adenine (A), guanine (G), cytosine (C), thymine (T) or uracil (U), and the 5 ′ end and 3 of this specific region 'Nonspecific consisting of nucleotides containing other bases other than adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U) linked to at least one of the ends A specific region has a specific base sequence complementary to a target sequence in a sample nucleic acid to be hybridized with the oligonucleotide, and a non-specific region is Which can form base pairs with each of the bases contained in the nucleotides constituting the specific region, adenine (A), guanine (G), cytosine (C), thymine At least one nucleotide or oligonucleotide consisting oligomer thereof having a T) and uracil (U) other than the other basesHybridizing to a sample nucleic acid;
Determining the presence or absence of a target sequence having a sequence complementary to the base sequence of a specific region in the oligonucleotide in the sample nucleic acid according to the hybridization intensity or the presence or absence of hybridization.It is characterized byThis is a nucleic acid base sequence analysis method.
Yet another method of the present invention provides:
A specific region composed of nucleotides containing a base selected from adenine (A), guanine (G), cytosine (C), thymine (T) or uracil (U), and the 5 ′ end and 3 of this specific region 'Nonspecific consisting of nucleotides containing other bases other than adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U) linked to at least one of the ends A specific region has a specific base sequence complementary to a target sequence in a sample nucleic acid to be hybridized with the oligonucleotide, and a non-specific region is Which can form base pairs with each of the bases contained in the nucleotides constituting the specific region, adenine (A), guanine (G), cytosine (C), thymine A step of hybridizing the nucleic acid sample at least one nucleotide or oligonucleotide consisting oligomer thereof having a T) and uracil (U) other than the other bases,
Measuring the hybridization intensity;
A method for discriminating a nucleobase mismatch, comprising: calculating a mismatch discriminating ability between a target sequence and a sequence in a sample nucleic acid based on the measured hybridization intensity.
[0011]
Hereinafter, the present invention will be described in more detail.
Several terms are used herein, but when used herein, these terms have the following meanings.
[0012]
The “sample nucleic acid” means a nucleic acid that is hybridized with the oligonucleotide of the present invention for the purpose of analyzing the base sequence, and may be DNA or RNA. The “specific region” means a region having a complementarity between a target sequence and a base sequence in a sample nucleic acid that can hybridize, and each base can form a pair with the target sequence. “A base constituting a normal nucleic acid” means a base contained in a nucleotide constituting DNA or RNA, that is, in the case of DNA, adenine (A), guanine (G), cytosine (C), thymine (T). In RNA, four types of bases are A, G, C, and uracil (U). The “base pair that forms a specific pair” means a base pair between A and T or U or a base pair between G and C.
[0013]
The oligonucleotide of the present invention has a specific region and one or two non-specific regions linked to at least one of both ends of the specific region. The oligonucleotide of the present invention may be an oligodeoxyribonucleotide or an oligoribonucleotide.
[0014]
The specific region is the target sequence in the sample nucleic acid.Complementary toIt has a specific base sequence. "Complementary"Is a mismatch of at least one base other than when it is perfectly complementary to the target sequence"ButIt means to include the case where it exists. The length of the specific region is not particularly limited as long as it can function as a probe in hybridization, but a length of about 6 to 50, preferably about 6 to 20 base pairs is appropriate. The base sequence of the hybridization region DNA is not particularly limited, and the sequence can be appropriately determined according to the base sequence of the sample DNA that is symmetrical to the base sequence.
[0015]
The non-specific region is composed of at least one nucleotide having an other base capable of forming a base pair with each of bases constituting a normal nucleic acid or an oligomer thereof. The base as described above is preferably one that normally binds to any of the bases constituting the nucleic acid, and the binding strength is weaker than the binding strength of the base pair that forms the specific pairing. Is more preferable.
[0016]
Specific examples of such a base include hypoxanthine. Specific examples of the nucleotide include deoxyinosine which is a deoxyribonucleotide having hypoxanthine as a base.
[0017]
The number of nucleotides or oligomers constituting the non-specific region is not particularly limited, but is usually at least one, preferably 2 to 20, more preferably 2 to 8.
[0018]
The position at which the non-specific region is linked to the specific region is not particularly limited, and may be either the 5 'end or the 3' end of the specific region. Furthermore, a non-specific region may be linked to both the 5 'end and the 3' end of the specific region. Of these, the latter is preferred.
[0019]
The ratio of the length of the specific region to the length of the non-specific region varies depending on the length of the specific region and the GC content, but usually the length of the specific region is equal to or less than the length of the non-specific region. The above is preferable.
[0020]
The method for synthesizing the oligonucleotide of the present invention is not particularly limited. For example, the β-cyanoethyl phosphoramidite method [Nucleic Acids Res. 12 4539 (1984)], phosphate triester method [Proc. Natl. Acad. Sci. USA 81 5956 (1984)], phosphonic acid ester method [Nucleic Acids Res. 14 5399 (1986). The specific region and the non-specific region may be linked to each other after being synthesized separately, or after the specific region is synthesized, the non-specific region may be sequentially added one nucleotide at a time. Moreover, a specific region and a non-specific region may be synthesized simultaneously.
[0021]
The oligonucleotide of the present invention may be used in a form immobilized on an insoluble carrier when used as a probe for hybridization. Examples of the insoluble carrier include a filter made of nitrocellulose or nylon, a glass plate, porous glass, silica gel, latex and the like. The method for immobilizing the oligonucleotide of the present invention on these insoluble carriers is not particularly limited. For example, when an amino-modified oligonucleotide is used, it is used on a nylon filter having a high-density carboxyl group on the surface. A method in which a carboxyl group is activated with water-soluble carbodiimide to form an amide bond [J. Org. 26 2525 (1961)], and in the case of an oligonucleotide modified with biotin, a method in which avidin is bound to a carrier coated on the surface by a biotin-avidin reaction [Biochemistry 11 2291 (1972)], and an amino-modified oligonucleotide is converted into a tresyl group. And a method of immobilizing on silica gel introduced with [Analytical Chemistry Symposium Series 9 203 (1981)]. In addition, as a method for producing an oligonucleotide immobilized on a filter, a method of simultaneously synthesizing many types of DNA on a solid phase by a technique applying photolithography [Science, 251 767 (1991)] and sealing were performed. Synthesis method by contacting a DNA synthesis reagent on a glass plate [Nucleic Acids Res. 22 1368 (1994)] can also be used.
[0022]
The oligonucleotide of the present invention described in detail above can be used for nucleic acid base sequence analysis. That is, the nucleic acid base sequence analysis method of the present invention comprises a step of hybridizing the oligonucleotide to a sample nucleic acid and a specific region in the oligonucleotide in the sample nucleic acid depending on the hybridization intensity or presence / absence of hybridization. Determining the presence or absence of a target sequence having a sequence complementary to the base sequence. The method of the present invention makes it possible to increase hybridization sensitivity, and makes it easy to distinguish between completely complementary hybrids and hybrids having mismatches, particularly terminal mismatches that were difficult to distinguish by conventional methods. This facilitates DNA base sequence analysis by the hybridization method.
[0023]
Next, in the hybridization using the oligonucleotide of the present invention, it will be explained how hybridization can be accurately and highly sensitively performed when there is a mismatch and when there is no mismatch. . FIG. 1 is a schematic diagram showing a hybrid of a normal oligonucleotide and a sample nucleic acid that is almost complementary to this oligonucleotide but contains a mismatch. In general, when a mismatch exists in the center of the hybrid, it becomes a serious obstacle to hybridization, but when near the end of the hybrid, the obstacle is smaller than when it exists in the center.
[0024]
On the other hand, a hybrid of the oligonucleotide of the present invention having a specific region having the same sequence as the oligonucleotide and the sample nucleic acid is schematically shown in FIG. In FIG. 2, I represents inosine. As shown in this figure, since inosine can base pair with any nucleotide, the length of the hybrid is increased by the non-specific region. That is, the mismatch existing at the end of the hybrid in the normal oligonucleotide is located in the center of the hybrid in the oligonucleotide of the present invention. Also, the hybridization intensity is increased. Therefore, it becomes easy to distinguish between hybridization that does not include a mismatch and hybridization that includes a mismatch. If the non-specific region is linked to the 5 ′ end of the specific region, it becomes easy to detect a mismatch on the 5 ′ end side of the specific region, and the non-specific region is linked to the 3 ′ end of the specific region. If so, it becomes easier to detect a mismatch on the 3 ′ end side of the specific region. Furthermore, if the non-specific region is linked to both the 5 ′ end and the 3 ′ end of the specific region, it is easy to detect both the 5 ′ end and 3 ′ end mismatches of the specific region. It becomes.
[0025]
The conditions for hybridization using the oligonucleotide of the present invention are not particularly limited, and the optimum conditions may vary depending on the type and sequence of the sample nucleic acid and the sequence of the oligonucleotide. For example, when a relatively short oligonucleotide is used, it is desirable to set the conditions loosely, for example, by lowering the hybridization temperature. In addition, when there are many GC residues in the DNA base sequence, the stability of the hybrid is stronger than when there are many AT residues. To offset this phenomenon, quaternary amines such as tetramethylammonium chloride Method using salt [Proc. Natl. Acad. Sci. USA 82, 1585 (1985)] is effective. In addition, a method of adding a surfactant, for example, sodium dodecyl sulfate, sodium N-lauroyl sarcosinate, etc. to the hybridization solution is also effective in improving the hybridization sensitivity (implementation). See Example C).
[0026]
When analyzing the base sequence of a nucleic acid by hybridization, it is preferable that either the sample nucleic acid or the oligonucleotide of the present invention is labeled. The labeling method is not particularly limited, and examples thereof include a method using a radioisotope or a fluorescent dye. The result of hybridization can be measured by a method according to various labeling methods.
[0027]
Examples of application of the base sequence analysis of the present invention include determination of DNA base sequences using hybridization methods [Genomics, 13 1378 (1992)], diagnosis of infectious diseases and genetic diseases, and the like for mapping of large genomic DNA. Applicable. As a method for diagnosing infectious diseases, for example, DNA is extracted from the blood of a subject, DNA probes are prepared from the sequences unique to various pathogens for the DNA by the method of the present invention, hybridization reaction is performed, and pathogens are detected. A method for detecting the presence is mentioned. As a method for diagnosing a genetic disease, an oligonucleotide is prepared by the method of the present invention based on a sequence specific to a gene causing a genetic disease, and hybridization with a chromosomal DNA obtained from a subject is performed. Detect the presence or absence of mutations. Mapping of large genomic DNA is an essential technique for genomic DNA analysis projects, etc., but hybridization is performed on the genome of each clone on the genome bank by using a number of DNA probes prepared by the method of the present invention. The arrangement can be determined [Abstracts of the 16th Annual Meeting of the Molecular Biology Society of Japan 1334 (1993)]. The determination of the DNA base sequence is conventionally performed by a method of chemically degrading DNA [Proc. Natl. Acad. Sci. USA, 74 560 (1977)] and a method using DNA synthase [Proc. Natl. Acad. Sci. USA, 745463 (1977)] is used, but a DNA base sequence analysis method (SBH; Sequencing By Hybridization) using a hybridization method is a technique that has attracted attention in recent years [Science, 260 1649 (1993). ]]. In the SBH method, by setting appropriate hybridization conditions, only an oligo DNA probe that is completely complementary to the target long-chain DNA is selectively hybridized, and specifically hybridized oligo DNA. This is a technique for determining the base sequence of a target long-chain DNA by accumulating and analyzing probe data.
[0028]
Furthermore, the oligonucleotide of the present invention is also expected to be used as a primer for polymerase reaction in PCR method (polymerase chain reaction), base sequence determination and the like.
[0029]
【Example】
EXAMPLES The present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.
[0030]
[Example A]
Measurement of hybridization intensity
(A) Synthesis of oligonucleotide
The oligonucleotides of the present invention having the sequences shown in Table 1 by using a DNA synthesizer (Applied Biosystems; ABI) (apparatus name: 394 DNA / RNA Synthesizer), a comparison target oligonucleotide, and a sample DNA were used. Synthesized. In addition, the following oligonucleotide No. Corresponds to the SEQ ID No. in the sequence listing.
[0031]
[Table 1]

[0032]
The oligonucleotide and sample DNA were synthesized according to a standard protocol, and the synthesis was completed in a cycle in which the trityl group which is a protecting group at the 5 'end was not removed. The sample DNA of (9) was purified using an OPC cartridge (ABI). After concentrating and drying the oligonucleotides (1) to (9) and the sample DNA, 0.5M sodium bicarbonate buffer (pH 8.4) is used for (1) to (8), and (9) is used for (9). It was suspended in TE buffer, quantified by measuring absorbance at 260 nm, and adjusted to 1 nmol / μl.
[0033]
(B) Immobilization of oligonucleotide
Immobilization of the oligonucleotide was carried out as follows by amide bonding the amino group of the amino-modified oligonucleotide to a nylon membrane having a high-density anionic carboxyl group on the surface.
[0034]
A Biodyne C (trademark; manufactured by Pall) membrane was rinsed with 0.1 N HCl, acidified, and then immersed in 20% EDC (1-ethyl-3-dimethylaminopropylcarbodiimide hydrochloride) at room temperature for 15 to 30 minutes. After rinsing lightly with deionized water and 0.5 M sodium bicarbonate buffer (pH 8.4), the sample was set on a dot blot apparatus (Bio-Rad). Amino modified oligonucleotide suspended in 0.5 M sodium bicarbonate buffer (pH 8.4) was allowed to react with the membrane for 15 minutes at room temperature. Membranes rinsed with TBS (Tris-buffered saline) /0.1% Tween-20 were treated with 0.1 N NaOH for 10 minutes, rinsed lightly with deionized water and then air dried.
[0035]
(C) Labeling of sample DNA
The sample DNA was labeled with [γ-32P] ATP by radiolabeling the 5 'end. The reaction was performed with a DNA 5 'end labeling kit (MEGALABELTM; manufactured by Takara Shuzo Co., Ltd.).
[0036]
(D) Hybridization reaction
The oligonucleotide-immobilized filter and the radiolabeled sample DNA were placed in 5 × SSC (750 mM sodium chloride / 75 mM trisodium citrate) /0.1% (sodium dodecyl sulfate) buffer for 4-10. C. Hybridization was performed for 1 hour to overnight. After the hybridization reaction, washing was performed 3 times with 2 × SSC / 0.1% SDS buffer at 4 to 10 ° C. for 5 minutes. After air drying, the radiation dose of each dot was measured by autoradiography, and the hybridization intensity was calculated.
[0037]
The results are shown in Table 2. The results are shown as relative values with the hybridization intensity of the comparative oligonucleotide as a control taken as 1. When the sequence of the specific region is different, the amount of hybridization also changes, but it has been found that the amount of hybridization increases in any case by introducing inosine (non-specific region). When the types and numbers of bonds were exactly the same, it was found that the amount of hybridization was higher when inosine was introduced at both 3 'and 5' ends than when inosine was introduced only at the 5 'end. By introducing inosine at both ends of 3 'and 5', highly sensitive hybridization is realized.
[0038]
[Table 2]

[0039]
Example B
Mismatch determination
(A) Synthesis of oligonucleotide
The oligonucleotides of the present invention having the sequences shown in Table 3 using the DNA synthesizer (Applied Biosystems; ABI) (apparatus name: 394 DNA / RNA Synthesizer) manufactured by Applied Biosystems (ABI), the comparative oligonucleotides and the sample DNA, The compound was synthesized in the same manner as in Example A (A).
[0040]

[0041]
(B) Immobilization of oligonucleotide
It carried out like (B) of Example A.
(C) Labeling of sample DNA
It carried out like (C) of Example A.
(D) Hybridization reaction
It carried out like (D) of Example A.
(E) Calculation of mismatch discrimination ability
The mismatch discrimination ability was calculated from the results obtained by the hybridization reaction. The mismatch discriminating ability is a concept using the following D value (Discrimination value) as an index, and indicates ease of discriminating mismatches.
[0042]
[Expression 1]

[0043]
The amount of hybridization of the sequence completely complementary to the sample DNA refers to the amount of hybridization of the sequence of the control example, and the amount of hybridization of the sequence having a mismatch refers to that of the comparative example or example corresponding to the control example. Refers to the amount of hybridization of the sequence. The D value is the ratio of the amount of hybridization of the comparative example or the example sequence to the control sequence. That is, the D value of the sequence of the control example is 1, and for the comparative example and the example, if the D value is high, the sequence can be easily distinguished from the sequence of the control example (the amount of hybridization is smaller than that of the control example). Represents.
[0044]
Table 4 shows the D value of each sequence. Oligonucleotides with inosine introduced have a higher D value, especially those with inosine introduced at both 3 'and 5' ends. It was found that the mismatch discriminating ability was superior to oligonucleotides introduced with inosine, particularly oligonucleotides introduced with inosine at both ends of 3 'and 5'.
[0045]
[Table 4]

[0046]
Example C
Effect of surfactant on hybridization reaction
(A) DNA synthesis
The oligonucleotides of the present invention having the sequences shown in Table 5 using the DNA synthesizer (Applied Biosystems; ABI) (apparatus name: 394 DNA / RNA Synthesizer), the comparative symmetry oligonucleotide and the sample DNA The compound was synthesized in the same manner as in Example A (A).
[0047]
[Table 5]

[0048]
(B) Immobilization of DNA
It carried out like (B) of Example A.
(C) DNA labeling
It carried out like (C) of Example A.
(D) Hybridization reaction
For the oligonucleotide-immobilized filter, radiolabeled sample DNA was placed in 5 × SSC (750 mM sodium chloride / 75 mM trisodium citrate) / 7% N-lauroyl sarcosinate buffer for 4-10. The hybridization reaction was carried out for 1 hour to overnight. After the hybridization reaction, washing was performed 3 times with 2 × SSC / 0.1% SDS buffer at 4 to 10 ° C. for 5 minutes. Further, as a control experiment, hybridization was performed at 4 to 10 ° C. for 1 hour to overnight in 5 × SSC (750 mM sodium chloride / 75 mM trisodium citrate) buffer with respect to the similarly prepared oligonucleotide-immobilized filter. Reacted. After the hybridization reaction, washing was performed 3 times with 2 × SSC buffer at 4 to 10 ° C. for 5 minutes. After air drying, the radiation dose of each dot was measured by autoradiography, and the hybridization intensity was calculated.
[0049]
The results are shown in Table 6. The results are shown as relative values with a background radiation dose of 1 when a surfactant (sodium N-lauroyl sarcosinate) was used. When sodium N-lauroyl sarcosinate is added to the hybridization reaction, the hybridization intensity increases with the effect of reducing the background signal due to non-specific adsorption to the filter, and as a result, an improvement in hybridization sensitivity is recognized. It was.
[0050]
[Table 6]

[0051]
【The invention's effect】
According to the present invention, it becomes possible to increase the sensitivity of hybridization, and it becomes easy to distinguish a completely complementary hybrid from a hybrid having a mismatch, particularly a mismatch at the end of the hybrid. Facilitates DNA base sequence analysis.
[0052]
[Sequence Listing]
SEQ ID NO: 1
Sequence length: 8
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: Other nucleic acid Synthetic oligonucleotide
Array

[0053]
SEQ ID NO: 2
Sequence length: 8
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: Other nucleic acid Synthetic oligonucleotide
Array

[0054]
SEQ ID NO: 3
Sequence length: 8
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: Other nucleic acid Synthetic oligonucleotide
Array

[0055]
SEQ ID NO: 4
Sequence length: 8
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: Other nucleic acid Synthetic oligonucleotide
Array

[0056]
SEQ ID NO: 5
Sequence length: 12
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: Other nucleic acid Synthetic oligonucleotide
Array

[0057]
SEQ ID NO: 6
Sequence length: 12
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: Other nucleic acid Synthetic oligonucleotide
Array

[0058]
SEQ ID NO: 7
Sequence length: 12
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: Other nucleic acid Synthetic oligonucleotide
Array

[0059]
SEQ ID NO: 8
Sequence length: 12
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: Other nucleic acid Synthetic oligonucleotide
Array

[0060]
SEQ ID NO: 9
Sequence length: 21
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: Other nucleic acid Synthetic oligonucleotide
Array

[0061]
SEQ ID NO: 10
Sequence length: 8
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: Other nucleic acid Synthetic oligonucleotide
Array

[0062]
SEQ ID NO: 11
Sequence length: 12
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: Other nucleic acid Synthetic oligonucleotide
Array

[0063]
SEQ ID NO: 12
Sequence length: 16
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: Other nucleic acid Synthetic oligonucleotide
Array

[0064]
SEQ ID NO: 13
Sequence length: 16
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: Other nucleic acid Synthetic oligonucleotide
Array

[0065]
SEQ ID NO: 14
Sequence length: 24
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: Other nucleic acid Synthetic oligonucleotide
Array

[0066]
SEQ ID NO: 15
Sequence length: 26
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: Other nucleic acid Synthetic oligonucleotide
Array

[0067]
SEQ ID NO: 16
Sequence length: 8
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: Other nucleic acid Synthetic oligonucleotide
Array

[0068]
SEQ ID NO: 17
Sequence length: 8
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: Other nucleic acid Synthetic oligonucleotide
Array

[0069]
SEQ ID NO: 18
Sequence length: 8
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: Other nucleic acid Synthetic oligonucleotide
Array

[0070]
SEQ ID NO: 19
Sequence length: 8
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: Other nucleic acid Synthetic oligonucleotide
Array

[0071]
SEQ ID NO: 20
Sequence length: 12
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: Other nucleic acid Synthetic oligonucleotide
Array

[0072]
SEQ ID NO: 21
Sequence length: 12
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: Other nucleic acid Synthetic oligonucleotide
Array

[0073]
SEQ ID NO: 22
Sequence length: 12
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: Other nucleic acid Synthetic oligonucleotide
Array

[0074]
SEQ ID NO: 23
Sequence length: 12
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: Other nucleic acid Synthetic oligonucleotide
Array

[0075]
SEQ ID NO: 24
Sequence length: 16
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: Other nucleic acid Synthetic oligonucleotide
Array

[0076]
SEQ ID NO: 25
Sequence length: 16
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: Other nucleic acid Synthetic oligonucleotide
Array

[0077]
SEQ ID NO: 26
Sequence length: 16
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: Other nucleic acid Synthetic oligonucleotide
Array

[0078]
SEQ ID NO: 27
Sequence length: 16
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: Other nucleic acid Synthetic oligonucleotide
Array

[0079]
SEQ ID NO: 28
Sequence length: 16
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: Other nucleic acid Synthetic oligonucleotide
Array

[0080]
SEQ ID NO: 29
Sequence length: 16
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: Other nucleic acid Synthetic oligonucleotide
Array

[0081]
SEQ ID NO: 30
Sequence length: 16
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: Other nucleic acid Synthetic oligonucleotide
Array

[0082]
SEQ ID NO: 31
Sequence length: 16
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: Other nucleic acid Synthetic oligonucleotide
Array

[0083]
SEQ ID NO: 32
Sequence length: 24
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: Other nucleic acid Synthetic oligonucleotide
Array

[0084]
SEQ ID NO: 33
Sequence length: 24
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: Other nucleic acid Synthetic oligonucleotide
Array

[0085]
SEQ ID NO: 34
Sequence length: 24
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: Other nucleic acid Synthetic oligonucleotide
Array

[0086]
SEQ ID NO: 35
Sequence length: 24
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: Other nucleic acid Synthetic oligonucleotide
Array

[Brief description of the drawings]
FIG. 1 is a schematic diagram showing a hybrid of a normal oligonucleotide and a nucleic acid.
FIG. 2 is a schematic diagram showing a hybrid of the oligonucleotide of the present invention and a nucleic acid.

Claims (19)

A specific region composed of nucleotides containing a base selected from adenine (A), guanine (G), cytosine (C), thymine (T) or uracil (U) , and the 5 ′ end and 3 of this specific region 'Two non-specific regions composed of nucleotides other than adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U) linked to each of the ends An oligonucleotide having
Specific region has a specific nucleotide sequence complementary for the target sequence in the sample nucleic acid to be hybridized to said oligonucleotide,
Each of the non- specific regions can form a base pair with each of the bases included in the nucleotides constituting the specific region , adenine (A), guanine (G), cytosine (C), thymine (T) And an oligonucleotide comprising at least one nucleotide having a base other than uracil (U) or an oligomer thereof. The oligonucleotide according to claim 1 , wherein the binding force between the base contained in the nucleotide constituting the specific region and another base is weaker than the binding force of the base pair forming the specific pairing. . The oligonucleotide according to claim 2 , wherein the other base is hypoxanthine. The oligonucleotide according to claim 1 , wherein the oligonucleotide is an oligodeoxyribonucleotide. The oligonucleotide according to claim 1 , wherein the length of the specific region is 6 to 50 bases, and the length of the non-specific region is 2 to 20 bases. The oligonucleotide according to claim 5, wherein the length of the specific region is equal to or longer than the length of the non-specific region. The oligonucleotide according to any one of claims 1 to 6 , which is immobilized on an insoluble carrier. The oligonucleotide according to claim 7, which is immobilized on an insoluble carrier in a region on the 5 'end side. A specific region composed of nucleotides containing a base selected from adenine (A), guanine (G), cytosine (C), thymine (T) or uracil (U), and the 5 ′ end and 3 of this specific region 'Nonspecific consisting of nucleotides containing other bases other than adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U) linked to at least one of the ends An oligonucleotide having a region and immobilized on an insoluble carrier,
The specific region has a specific base sequence complementary to a target sequence in a sample nucleic acid to be hybridized with the oligonucleotide,
  The non-specific region can form a base pair with each of the bases included in the nucleotide constituting the specific region, and adenine (A), guanine (G), cytosine (C), thymine (T) and uracil An oligonucleotide comprising at least one nucleotide having a base other than (U) or an oligomer thereof. The oligonucleotide according to claim 9, which is immobilized on an insoluble carrier in a 5 'terminal region. The oligonucleotide according to claim 9 or 10, wherein the other base is hypoxanthine. A specific region composed of nucleotides containing a base selected from adenine (A), guanine (G), cytosine (C), thymine (T) or uracil (U), and the 5 ′ end and 3 of this specific region 'Nonspecific consisting of nucleotides containing other bases other than adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U) linked to at least one of the ends A specific region has a specific base sequence complementary to a target sequence in a sample nucleic acid to be hybridized with the oligonucleotide, and a non-specific region is the specific region may be formed base pairs with each of the bases contained in nucleotides that make up, adenine (a), guanine (G), cytosine (C), thymine A step of hybridizing the nucleic acid sample at least one nucleotide or oligonucleotide consisting oligomer thereof having a T) and uracil (U) other than the other bases,
The presence or absence of hybridization intensities or hybridization, characterized in that it comprises a step of determining presence or absence of a target sequence having a nucleotide sequence complementary to the sequence of a specific region in the oligonucleotide in the sample nucleic acid Nucleic acid base sequence analysis method. The method according to claim 12, wherein the oligonucleotide is immobilized on an insoluble carrier. The method according to claim 12 or 13, wherein the oligonucleotide is immobilized on an insoluble carrier in a region on the 5 'end side. The method according to any one of claims 12 to 14, wherein the other base is hypoxanthine. A specific region composed of nucleotides containing a base selected from adenine (A), guanine (G), cytosine (C), thymine (T) or uracil (U), and the 5 ′ end and 3 of this specific region 'Nonspecific consisting of nucleotides containing other bases other than adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U) linked to at least one of the ends A specific region has a specific base sequence complementary to a target sequence in a sample nucleic acid to be hybridized with the oligonucleotide, and a non-specific region is Which can form a base pair with each of the bases contained in the nucleotides constituting the specific region, adenine (A), guanine (G), cytosine (C), thymine (T) and comprising: at least one nucleotide or oligonucleotide consisting oligomer thereof with other bases other than uracil (U) is hybridized to the sample nucleic acid,
Measuring the hybridization intensity;
A method for discriminating a mismatch of nucleic acid bases, comprising: calculating a mismatch discriminating ability between a target sequence and a sequence in a sample nucleic acid based on the measured hybridization intensity. The method according to claim 16, wherein the oligonucleotide is immobilized on an insoluble carrier. The method according to claim 16 or 17, wherein the oligonucleotide is immobilized on an insoluble carrier in a region on the 5 'end side. The method according to any one of claims 16 to 18, wherein the other base is hypoxanthine.

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