JP2004236655A - Method for simply detecting genetic polymorph and reagent for detecting the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To product a method for clearly detecting genetic polymorphs in several sites, especially the genetic polymorphs of cytochrome 2D6, 2C19, etc., in several sites, with good repeatability, and to provide a reagent for the same. <P>SOLUTION: This method for detecting the genetic polymorphs comprises conducting amplification by using any one of a wild type-detecting oligonucleotide and a variant-type detecting oligonucleotide and a nucleic acid-specific label, wherein a sequence having at least two variation sites different from each other is detected under an identical amplification condition. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

【００６５】
【表２】

【００６６】
実施例２ ＣＹＰ２Ｄ６＊４、ＣＹＰ２Ｄ６＊３６遺伝子多型の同時検出
（１）ＣＹＰ２Ｄ６遺伝子の１８４６番目及びＥｘｏｎ９ ｃｏｎｖｅｒｓｉｏｎの多型を検出するオリゴヌクレオチド
オリゴ７及び８はＣＹＰ２Ｄ６遺伝子の１８４６番目の多型を検出するためのものである。
オリゴ７は３’末端から２番目に野生型（Ｇ）のヌクレオチド配列、および３番目に人為的ミスマッチ（Ｃ→Ａ）を有し、オリゴ８は３’末端から２番目に変異型（Ａ）のヌクレオチド配列、および３番目に人為的ミスマッチ（Ｃ→Ａ）を有し、オリゴ９がアンチセンス鎖であり、オリゴ７、８と組み合わせて増幅反応のオリゴヌクレオチドとして使用される。なお、オリゴ７、８、９は必要により標識して使用される。
【００６７】
オリゴ１０及び１１はＣＹＰ２Ｄ６遺伝子のＥｘｏｎ９ ｃｏｎｖｅｒｓｉｏｎを検出するためのものである。
オリゴ１０は野生型（２Ｄ６ Ｅｘｏｎ９）のヌクレオチド配列を有し、オリゴ１１は変異型（２Ｄ７ Ｅｘｏｎ９）のヌクレオチド配列を有し、オリゴ１２がアンチセンス鎖であり、オリゴ１０、１１と組み合わせて増幅反応のオリゴヌクレオチドとして使用される。なお、オリゴ１０、１１、１２は必要により標識して使用される。
【００６８】
（２）ＰＣＲ法によるＣＹＰ２Ｄ６＊４、ＣＹＰ２Ｄ６＊３６遺伝子多型遺伝子多型の解析
▲１▼ ＰＣＲ法による増幅反応
ヒト白血球からフェノール・クロロフォルム法により抽出したＤＮＡ溶液をサンプルとして使用して、下記オリゴヌクレオチドを含む試薬を添加して、前記条件によりＣＹＰ２Ｄ６＊４、ＣＹＰ２Ｄ６＊３６遺伝子多型を解析した。なお、反応は反応液ａ、ｂ、ｃ、ｄとも同じ条件で同時に行った。
【００６９】
反応液２−ａのオリゴヌクレオチド
オリゴ７およびオリゴ９
反応液２−ｂのオリゴヌクレオチド
オリゴ８およびオリゴ９
反応液２−ｃのオリゴヌクレオチド
オリゴ１０およびオリゴ１２
反応液２−ｄのオリゴヌクレオチド
オリゴ１１およびオリゴ１２
【００７０】
【表３】

【００７１】
【表４】

【００７２】
実施例３ ＣＹＰ２Ｄ６＊４、ＣＹＰ２Ｄ６＊１４遺伝子多型の検出
（１）ＣＹＰ２Ｄ６遺伝子の１８４６番目の多型を検出するオリゴヌクレオチド
オリゴ１３は３’末端から２番目に野生型（Ｇ）に相補的なヌクレオチド配列、および３番目に人為的ミスマッチ（Ｃ→Ａ）を有し、オリゴ１４は３’末端から２番目に変異型（Ａ）に相補的なヌクレオチド配列、および３番目に人為的ミスマッチ（Ｃ→Ａ）を有し、オリゴ１５がアンチセンス鎖であり、オリゴ１３、１４と組み合わせて増幅反応のオリゴヌクレオチドとして使用される。なお、オリゴ１３、１４、１５は必要により標識して使用される。
（２）ＣＹＰ２Ｄ６遺伝子の１７５８番目の多型を検出するオリゴヌクレオチド
オリゴ１６は３’末端から２番目に野生型（Ｇ）に相補的なヌクレオチド配列、および３番目に人為的ミスマッチ（Ｃ→Ａ）を有し、オリゴ１７は３’末端から２番目に変異型（Ａ）に相補的なヌクレオチド配列、および３番目に人為的ミスマッチ（Ｃ→Ａ）を有し、オリゴ１５がアンチセンス鎖であり、オリゴ１６、１７と組み合わせて増幅反応のオリゴヌクレオチドとして使用される。なお、オリゴ１６、１７、１５は必要により標識して使用される。
（３）ＰＣＲ法によるＣＹＰ２Ｄ６＊４、ＣＹＰ２Ｄ６＊１４遺伝子多型の解析
▲１▼ ＰＣＲ法による増幅反応
ヒト白血球からフェノール・クロロフォルム法により抽出したＤＮＡ溶液をサンプルとして使用して、下記オリゴヌクレオチドを含む試薬を添加して、前記条件によりＣＹＰ２Ｄ６＊４、ＣＹＰ２Ｄ６＊１４遺伝子多型を解析した。なお、反応は反応液ａ、ｂ、ｃ、ｄとも同じ条件で同時に行った。
【００７３】
反応液３−ａのオリゴヌクレオチド
オリゴ１３およびオリゴ１５
反応液３−ｂのオリゴヌクレオチド
オリゴ１４およびオリゴ１５
反応液３−ｃのオリゴヌクレオチド
オリゴ１６およびオリゴ１５
反応液３−ｄのオリゴヌクレオチド
オリゴ１７およびオリゴ１５
【００７４】
【表５】

【００７５】
実施例４ ＣＹＰ２Ｃ１９＊２、ＣＹＰ２Ｃ１９＊３遺伝子多型の検出
（１）ＣＹＰ２Ｃ１９遺伝子の６８１番目の多型を検出するオリゴヌクレオチド
オリゴ１８は３’末端から２番目に野生型（Ｇ）に相補的なヌクレオチド配列、および３番目に人為的ミスマッチ（Ｇ→Ａ）を有し、オリゴ１９は３’末端から２番目に変異型（Ａ）に相補的なヌクレオチド配列、および３番目に人為的ミスマッチ（Ｇ→Ａ）を有し、オリゴ２０がアンチセンス鎖であり、オリゴ１８、１９と組み合わせて増幅反応のオリゴヌクレオチドとして使用される。なお、オリゴ１８、１９、２０は必要により標識して使用される。
【００７６】
（２）ＣＹＰ２Ｃ１９遺伝子の６３６番目の多型を検出するオリゴヌクレオチドの合成
オリゴ２１は３’末端から２番目に野生型（Ｇ）に相補的なヌクレオチド配列、および３番目に人為的ミスマッチ（ｃ→ｔ）を有し、オリゴ２２は３’末端から２番目に変異型（Ａ）に相補的なヌクレオチド配列、および３番目に人為的ミスマッチ（ｃ→ｔ）を有し、オリゴ２３がアンチセンス鎖であり、オリゴ２１、２２と組み合わせて増幅反応のオリゴヌクレオチドとして使用される。なお、オリゴ２１、２２、２３は必要により標識して使用される。
【００７７】
（３）ＰＣＲ法によるＣＹＰ２Ｃ１９＊２、ＣＹＰ２Ｃ１９＊３遺伝子多型の解析
▲１▼ ＰＣＲ法による増幅反応
ヒト白血球からフェノール・クロロフォルム法により抽出したＤＮＡ溶液をサンプルとして使用して、下記オリゴヌクレオチドを含む試薬を添加して前記条件によりＣＹＰ２Ｃ１９＊２、ＣＹＰ２Ｃ１９＊３遺伝子多型を解析した。なお、反応は反応液ａ、ｂ、ｃ、ｄとも同じ条件で同時に行った。
【００７８】
反応液４−ａのオリゴヌクレオチド
オリゴ１８およびオリゴ２０
反応液４−ｂのオリゴヌクレオチド
オリゴ１９およびオリゴ２０
反応液４−ｃのオリゴヌクレオチド
オリゴ２１およびオリゴ２３
反応液４−ｄのオリゴヌクレオチド
オリゴ２１およびオリゴ２３
【００７９】
【表６】

【００８０】
２Ｃ１９＊２、ＣＹＰ２Ｃ１９＊３遺伝子多型の同時検出（その２）
（１）ＣＹＰ２Ｃ１９遺伝子の６８１番目及び６３６番目の多型を検出するオリゴヌクレオチド
オリゴ２４及び２５はＣＹＰ２Ｃ１９遺伝子の６８１番目の多型を検出するためのものである。
オリゴ２４は３’末端から２番目に野生型（Ｇ）のヌクレオチド配列、および３番目に人為的ミスマッチ（Ｇ→Ａ）を有し、オリゴ２５は３’末端から２番目に変異型（Ａ）のヌクレオチド配列、および３番目に人為的ミスマッチ（Ｇ→Ａ）を有し、オリゴ３がアンチセンス鎖であり、オリゴ２４、２５と組み合わせて増幅反応のオリゴヌクレオチドとして使用される。なお、オリゴ２４、２５、２６は必要により標識して使用される。
【００８１】
オリゴ２７及び２８はＣＹＰ２Ｃ１９遺伝子の６３６番目の多型を検出するためのものである。
オリゴ２７は３’末端から２番目に野生型（Ｇ）のヌクレオチド配列、および３番目に人為的ミスマッチ（Ｃ→Ａ）を有し、オリゴ２８は３’末端から２番目に変異型（Ａ）のヌクレオチド配列、および３番目に人為的ミスマッチ（Ｃ→Ａ）を有し、オリゴ２９がアンチセンス鎖であり、オリゴ２７、２８と組み合わせて増幅反応のオリゴヌクレオチドとして使用される。なお、オリゴ２７、２８、２９は必要により標識して使用される。
【００８２】
（２）ＰＣＲ法によるＣＹＰ２Ｃ１９＊２、ＣＹＰ２Ｃ１９＊３遺伝子多型の解析
▲１▼ ＰＣＲ法による増幅反応
ヒト白血球からフェノール・クロロフォルム法により抽出したＤＮＡ溶液をサンプルとして使用して、下記オリゴヌクレオチドを含む試薬を添加して前記条件によりＣＹＰ２Ｃ１９＊２、ＣＹＰ２Ｃ１９＊３遺伝子多型を解析した。なお、反応は反応液ａ、ｂ、ｃ、ｄとも同じ条件で同時に行った。
【００８３】
反応液５−ａのオリゴヌクレオチド
オリゴ２４およびオリゴ２６
反応液５−ｂのオリゴヌクレオチド
オリゴ２５およびオリゴ２６
反応液５−ｃのオリゴヌクレオチド
オリゴ２７およびオリゴ２９
反応液５−ｄのオリゴヌクレオチド
オリゴ２８およびオリゴ２９
【００８４】
【表７】

【００８５】
【表８】

【００８６】
実施例６ ＮＡＴ２＊５、ＮＡＴ２＊６、ＮＡＴ２＊７遺伝子多型の検出
（１）ＮＡＴ２遺伝子の４８１番目の多型を検出するオリゴヌクレオチド
オリゴ３０は３’末端から２番目に野生型（Ｃ）に相補的なヌクレオチド配列、および３番目に人為的ミスマッチ（Ａ→Ｃ）を有し、オリゴ３１は３’末端から２番目に変異型（Ｔ）に相補的なヌクレオチド配列、および３番目に人為的ミスマッチ（Ａ→Ｃ）を有し、オリゴ３２がセンス鎖であり、オリゴ３０、３１と組み合わせて増幅反応のオリゴヌクレオチドとして使用される。なお、オリゴ３０、３１、３２は必要により標識して使用される。
【００８７】
（２）ＮＡＴ２遺伝子の５９０番目の多型を検出するオリゴヌクレオチド
オリゴ３３は３’末端から２番目に野生型（Ｇ）に相補的なヌクレオチド配列、および３番目に人為的ミスマッチ（Ｔ→Ａ）を有し、オリゴ３４は３’末端から２番目に変異型（Ａ）に相補的なヌクレオチド配列、および３番目に人為的ミスマッチ（Ｔ→Ａ）を有し、オリゴ３２がセンス鎖であり、オリゴ３３、３４と組み合わせて増幅反応のオリゴヌクレオチドとして使用される。なお、オリゴ３３、３４は必要により標識して使用される。
【００８８】
（３）ＮＡＴ２遺伝子の８５７番目の多型を検出するオリゴヌクレオチド
オリゴ３５は３’末端から２番目に野生型（Ｇ）に相補的なヌクレオチド配列、および３番目に人為的ミスマッチ（Ｔ→Ｇ）を有し、オリゴ３６は３’末端から２番目に変異型（Ａ）に相補的なヌクレオチド配列、および３番目に人為的ミスマッチ（Ｔ→Ｇ）を有し、オリゴ３２がセンス鎖であり、オリゴ３５、３６と組み合わせて増幅反応のオリゴヌクレオチドとして使用される。なお、オリゴ３５、３６は必要により標識して使用される。
【００８９】
（４）ＰＣＲ法によるＮＡＴ２＊５、ＮＡＴ２＊６、ＮＡＴ２＊７遺伝子多型の解析
▲１▼ ＰＣＲ法による増幅反応
ヒト白血球からフェノール・クロロフォルム法により抽出したＤＮＡ溶液をサンプルとして使用して、下記オリゴヌクレオチドを含む試薬を添加して前記条件によりＮＡＴ２＊５、ＮＡＴ２＊６、ＮＡＴ２＊７遺伝子多型を解析した。なお、反応は反応液ａ、ｂ、ｃ、ｄ、ｅ、ｆとも同じ条件で同時に行った。
【００９０】
反応液６−ａのオリゴヌクレオチド
オリゴ３０およびオリゴ３２
反応液６−ｂのオリゴヌクレオチド
オリゴ３１およびオリゴ３２
反応液６−ｃのオリゴヌクレオチド
オリゴ３３およびオリゴ３２
反応液６−ｄのオリゴヌクレオチド
オリゴ３４およびオリゴ３２
反応液６−ｅのオリゴヌクレオチド
オリゴ３５およびオリゴ３２
反応液６−ｆのオリゴヌクレオチド
オリゴ３６およびオリゴ３２
【００９１】
【表９】

【００９２】
以上、何れの実施例でも明らかなように、複数箇所の多型部位を同一の条件で増幅でき、さらに明確にその差を区別することが出来た。
【００９３】
実施例７ ＣＹＰ２Ａ６＊３遺伝子多型の検出
（１）ＣＹＰ２Ａ６＊３遺伝子の多型を検出するオリゴヌクレオチド
オリゴ３７はおよびオリゴ３９はＣＹＰ２ＤＡ６（野生型）に相補的なヌクレオチド配列を有し、オリゴ３８およびオリゴ４０はＣＹＰ２Ａ６＊３（変異型）に相補的なヌクレオチド配列を有する。オリゴ３７と３９、オリゴ３８と４０は組み合わせて増幅反応のオリゴヌクレオチドとして使用される。なお、オリゴ３７、３８、３９、４０は必要により標識して使用される。
【００９４】
（２）ＰＣＲ法によるＣＹＰ２Ａ６＊３遺伝子多型の解析
▲１▼ ＰＣＲ法による増幅反応
ヒト白血球からフェノール・クロロフォルム法により抽出したＤＮＡ溶液をサンプルとして使用して、下記オリゴヌクレオチドを含む試薬を添加し前記条件によりＣＹＰ２Ａ６＊３遺伝子多型を解析した。反応は反応液ａ、ｂとも同じ条件で同時に行った。
【００９５】
反応液７−ａのオリゴヌクレオチド
オリゴ３７およびオリゴ３９
反応液７−ｂのオリゴヌクレオチド
オリゴ３８およびオリゴ４０
【００９６】
【表１０】

【００９７】
ＣＹＰ２Ａ６＊３遺伝子多型の解析は他の実施例と同一の増幅の温度・時間条件で検出可能であることが判り、実施例で示した他の箇所と組み合わせて同時に増幅反応を行っても同時に検出できることが明らかになった。
【００９８】
上記実施例で示した通り、オリゴヌクレオチドを用いて複数箇所を同一温度、時間条件で同時に塩基多型特異的増幅反応を行い、増幅反応物質を核酸特異的結合物質を用いて測定することで、容易にかつ迅速に遺伝子型を明確に判定することができた。
【００９９】
また、実施例では特定の組み合わせしか行っていないが、増幅の温度・時間条件は同じであり、これらの箇所の中で自由な組み合わせを行っても、同時に検出できることは明かである。
【０１００】
また、本実施例での各オリゴヌクレオチドは、この記載のオリゴヌクレオチドに限定されず、前述したように３’末端から１０〜５０塩基の長さであれば同様の結果が得られる。さらに、いくつかのオリゴヌクレオチドは３’末端から３番目に人為的ミスマッチを導入したが、前述のように、３〜７番目の範囲であっても良いし、さらにはこれ以外に数カ所（１〜５箇所程度でプライマーの全体長による（特に５’末端））のミスマッチが導入されていても良い。
さらに、リバース側は伸長反応の有無を検出するのに適当な長さ等を基準に決めたものであって、対象となる遺伝子の適当な部分に対応するものであれば、本実施例の配列に限定されるものではない。
【０１０１】
【発明の効果】
上述したように、本発明により、試料核酸中の遺伝子多型を明確にまた簡便に検出できる方法が提供される。本発明の方法では、これまでの方法のように煩雑な操作を必要としないので、迅速で容易に再現性の良い結果が得られた。
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[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for simultaneously detecting two or more gene polymorphisms present in a sample, and more particularly to a method for detecting two or more cytochrome 2D6 (may be abbreviated as CYP2D6) gene polymorphisms present in a sample. Related to the simultaneous detection method.
In addition, the present invention particularly relates to a method for simultaneously detecting two or more types of cytochrome 2C19 (CYP2C19) present in a sample) gene polymorphism.
Furthermore, the present invention relates to a method for simultaneously detecting gene polymorphisms of cytochrome 2D6 and cytochrome 2C19.
The present invention also relates to a method for simultaneously detecting two or more gene polymorphisms of N-acetyltransferase 2 (may be abbreviated as NAT2).
[0002]
[Prior art]
In the present invention, a gene polymorphism means having a nucleotide sequence different from that of a wild type. Gene polymorphism plays an important role in drug metabolism as a cause of inter-individual variation in the occurrence of side effects and treatment failures, and is also known as a cause of individual differences such as basal metabolism known as a constitution. Moreover, they also serve as genetic markers for many diseases. Therefore, the elucidation of these mutations is clinically important, and routine phenotyping is particularly recommended for psychiatric patients and volunteers in clinical studies (see Non-Patent Documents 1 and 2). Also desired is a nucleic acid sequence analysis method for the detection of each genotype following the identification of the causative mutant gene.
[0003]
As a conventional nucleic acid sequence analysis technique, there is, for example, a nucleic acid sequencing method (sequencing method). Although the nucleic acid sequencing method can detect and identify nucleotide polymorphisms contained in a nucleic acid sequence, it requires a large amount of nucleic acid sequence analysis such as preparation of a template nucleic acid, DNA polymerase reaction, polyacrylamide gel electrophoresis, and the like. Requires labor and time. In addition, although labor saving can be achieved by using a recent automatic sequencer, there is a problem that an expensive apparatus is required.
On the other hand, various genetic diseases caused by point mutations in genes are known, and some of them are known where and how point mutations in genes cause genetic diseases. Not a few.
[0004]
In particular, the polymorphism of human cytochrome P4502D6 is involved in the metabolism of drugs such as antidepressants and remedies for hypertension, and is highly significant when effective administration is performed while suppressing side effects of the drugs.
In addition, the polymorphism of human cytochrome P4502C19 is clinically significant, for example, it is known to be related to eradication treatment of Helicobacter pylori.
It is said that the NAT2 polymorphism is mainly related to the metabolism of isoniazid which is a therapeutic agent for tuberculosis, and is highly clinically significant in this regard.
[0005]
As a method for detecting such an expected point mutation, detection of a point mutation of a gene using a gene amplification method such as a PCR (polymerase chain reaction) method (see Patent Literatures 1 and 2) has hitherto been known. Methods are known. In this method, as one of a pair of oligonucleotides used in the gene amplification method, an oligonucleotide for a wild-type completely complementary to an end region of an amplification region of a wild-type gene and an oligonucleotide for a mutant gene are used. A mutant oligonucleotide completely complementary to the end region of the region is used. The mutant oligonucleotide has a nucleotide whose 3 'end is complementary to the nucleotide having the expected point mutation. The sample gene is subjected to a gene amplification method using the oligonucleotides for wild type and mutant type separately.
[0006]
If the sample gene is wild-type, nucleic acid amplification occurs when using the wild-type oligonucleotide, but when using the mutant-type oligonucleotide, the 3 ′ end of the oligonucleotide corresponds to the sample gene. Since it is not complementary to the nucleotide (mismatch), no elongation reaction occurs and no amplification of the nucleic acid occurs. On the other hand, if the sample gene is mutant, amplification does not occur when the wild-type oligonucleotide is used, and amplification occurs when the mutant oligonucleotide is used. Therefore, by examining whether or not amplification occurs when each oligonucleotide is used, it is possible to determine whether the sample gene is a wild type or a mutant type, thereby identifying a point mutation in the sample gene. Can be. As a method to check whether amplification has occurred at this time, the amplified product is subjected to agarose gel electrophoresis, stained with a nucleic acid-specific binding fluorescent reagent such as ethidium bromide, and then irradiated with UV to detect the presence of amplified nucleic acid. it can. In another mode, the amplified nucleic acid is immobilized on a nylon membrane and detected using a labeled probe.Southern blotting is used.Sandwich hybridization is performed using a detection probe after capturing with a supplementary probe immobilized on an individual carrier. Laws and the like have been developed.
[0007]
[Non-patent document 1]
Gram and Brsen, European Consensus Conference on Pharmacogenetics. Commission of the European Communities, Luxembourg, 1990, pp. 87-96.
[Non-patent document 2]
Balant et al., Eur. J. Clin. Pharmacol. Vol. 36 (1989), pp. 551-554
[Patent Document 1]
Japanese Patent Publication No. 4-67960
[Patent Document 2]
Japanese Patent Publication No. 4-67957
[0008]
[Problems to be solved by the invention]
It seems that the amplified nucleic acid can be easily detected and the polymorphism can be identified by the method as described above. However, in practice, the operation is complicated and it is difficult to quantify the detected value. Analysis such as taking the ratio between the type signal and the polymorphic signal became difficult, and a great deal of work was required to identify the accurate polymorphism.
[0009]
For example, according to the electrophoresis method, it is necessary to separately detect the wild type and the mutant type, and it is difficult to accurately quantify the nucleic acid amount from the electrophoresis image. In the Southern blot method and the sandwich hybridization method, a hybridization reaction with a probe is required, and the conditions must be strictly adjusted. Furthermore, a step of removing excess probe is required, and the operation is very complicated. In particular, in order to detect a gene polymorphism of a drug metabolizing enzyme gene, particularly a cytochrome gene, and among them, a cytochrome 2D6 gene polymorphism having a large number of polymorphism sites, it is necessary to examine several mutation sites. For this reason, in the above-mentioned method, many steps must be individually performed for several places, and a large amount of labor is required for the detection work, and it is still necessary to simultaneously measure a plurality of mutation sites of the gene polymorphism of cytochrome 2D6. The simple and short detection method described above has not been proposed.
[0010]
Similarly, in order to detect a gene polymorphism of cytochrome 2C19 having a large number of polymorphisms, it is necessary to examine several mutations. For this reason, in the above method, many steps must be individually performed for several places, and a large amount of labor is required for the detection work, and multiple mutation sites of the gene polymorphism of cytochrome 2C19 are still measured simultaneously. In this case, a simple and short-time detection method without using a special dedicated device has not been proposed.
[0011]
An object of the present invention is to solve the above-mentioned problems and to improve several gene polymorphisms, particularly several gene polymorphisms of cytochrome 2D6, several gene polymorphisms of cytochrome 2C19, and several gene polymorphisms of NAT2. An object of the present invention is to provide a method that can be detected clearly and with good reproducibility, and a reagent therefor.
[0012]
[Means for Solving the Problems]
In view of the above circumstances, the present inventors have conducted intensive studies and found that, compared to the conventional method described above, a nucleic acid sequence containing a specific nucleotide polymorphism site has a wild-type detection oligonucleotide and / or one or two types thereof. After reacting the mutant-type detection oligonucleotides simultaneously or separately, by measuring the amount of the amplified oligonucleotide, data that is easily quantified without the need for a complicated detection operation can be obtained. The present inventors have found a method that enables clear polymorphism identification, and have completed the present invention.
[0013]
That is, the present invention has the following configuration.
(1) In a method for detecting a cytochrome 2D6 gene polymorphism by performing amplification using a wild-type detection oligonucleotide and / or a mutant-type detection oligonucleotide, at least two different mutations are performed under the same amplification conditions. A method for detecting a polymorphism in a cytochrome 2D6 gene, comprising detecting a sequence at a site.
(2) The method for detecting a polymorphism in the cytochrome 2D6 gene according to (1), wherein an oligonucleotide having at least one mismatch other than the mutation site is used.
(3) The oligonucleotide is designed such that the 3 'end of the oligonucleotide is complementary to the target gene, and the second nucleotide from the 3' end is complementary to the nucleotide of the wild-type or mutant sequence. The method for detecting a polymorphism in the cytochrome 2D6 gene according to (1) or (2), wherein
(4) The cytochrome 2D6 according to any one of (1) to (3), wherein the amplification reaction is any method selected from the group consisting of PCR, NASBA, LCR, SDA, RCR, and TMA. A method for detecting a gene polymorphism.
(5) The method for detecting a polymorphism in the cytochrome 2D6 gene according to any one of (1) to (4), wherein the oligonucleotide is labeled in advance.
(6) The method for detecting a polymorphism in the cytochrome 2D6 gene according to any one of (1) to (5), wherein the method is performed by detecting with a nucleic acid specific label.
(7) By performing amplification using at least one of a wild-type detection oligonucleotide and a mutant-type detection oligonucleotide, a reagent for detecting a cytochrome 2D6 gene polymorphism can be used at least under the same amplification conditions. A reagent for detecting a polymorphism in a cytochrome 2D6 gene, which is prepared so that sequences at two different mutation sites can be detected.
(8) The oligonucleotide is designed such that the 3 'end of the oligonucleotide is complementary to the target gene and the second nucleotide from the 3' end is complementary to the nucleotide of the wild-type or mutant sequence. (7) The reagent for detecting a polymorphism in the cytochrome 2D6 gene according to (7).
(9) The polymorphism of the cytochrome 2D6 gene polymorphism according to (7) or (8), wherein the amplification reaction is any method selected from the group consisting of PCR, NASBA, LCR, SDA, RCR and TMA. Detection reagent.
(10) The reagent for detecting a polymorphism in the cytochrome 2D6 gene according to any one of (7) to (9), which is detected by a nucleic acid-specific label.
(11) In a method for detecting a gene polymorphism by performing amplification using at least one of a wild-type detection oligonucleotide and a mutant-type detection oligonucleotide and a nucleic acid-specific label, at least under the same amplification conditions, A method for detecting a gene polymorphism, comprising detecting sequences at two different mutation sites.
(12) A method for detecting a genetic polymorphism, wherein the 3 'end of the oligonucleotide for detecting wild type and the oligonucleotide for detecting mutant type according to (11) is complementary to a target gene.
(13) A method for detecting a genetic polymorphism, wherein the nucleic acid-specific label according to (11) or (12) is a fluorescent aromatic reagent that specifically recognizes a double-stranded polynucleotide.
(14) The method for detecting a gene polymorphism according to any of (11) to (13), wherein the gene polymorphism is a drug metabolism-related enzyme gene polymorphism.
(15) The method according to any one of (11) to (14), wherein the amplification reaction is any method selected from the group consisting of PCR, NASBA, LCR, SDA, RCR and TMA. Type detection method.
(16) The method for detecting a genetic polymorphism according to any one of (11) to (15), wherein the oligonucleotide for detecting wild type and the oligonucleotide for detecting mutant type are labeled in advance.
(17) The method according to any one of (11) to (14), wherein the detection is performed by an interaction between the amplification product obtained by the method according to (15) and a nucleic acid-specific label.
[0014]
Further, the present invention has the following configuration.
(18) Amplification using at least one of the wild-type detection oligonucleotide and the mutant-type detection oligonucleotide and a nucleic acid-specific label is performed to obtain the same amplification conditions for a reagent for detecting a gene polymorphism. 2. A reagent for detecting a gene polymorphism, which has been prepared so as to be able to detect the sequences of at least two different mutation sites.
(19) A reagent for detecting a gene polymorphism, wherein the 3 ′ end of the oligonucleotide according to (18) is complementary to a target gene.
(20) The reagent for detecting a genetic polymorphism according to (18) or (19), wherein the nucleic acid-specific label is a fluorescent aromatic reagent that specifically recognizes a double-stranded polynucleotide.
(21) The gene polymorphism detection reagent according to any of (18) to (20), wherein the gene polymorphism is a drug metabolism-related enzyme gene polymorphism.
(22) The detection reagent according to any one of (18) to (21), wherein the amplification reaction is any method selected from the group consisting of PCR, NASBA, LCR, SDA, RCR, and TMA. .
[0015]
Further, the present invention has the following configuration.
(23) In a method for detecting a cytochrome 2C19 gene polymorphism by performing amplification using a wild-type detection oligonucleotide and / or a mutant-type detection oligonucleotide, at least two different mutations are obtained under the same amplification conditions. A method for detecting a polymorphism in a cytochrome 2C19 gene, comprising detecting a sequence at a site.
(24) The method for detecting a polymorphism in the cytochrome 2C19 gene according to (23), wherein an oligonucleotide having at least one mismatch other than the mutation site is used.
(25) The oligonucleotide is designed so that the 3 'end of the oligonucleotide is complementary to the target gene and the second nucleotide from the 3' end is complementary to the nucleotide of the wild-type or mutant sequence. The method for detecting a polymorphism in the cytochrome 2C19 gene according to (23) or (24), which is characterized in that:
(26) The method according to any one of (23) to (25), wherein the amplification reaction is any method selected from the group consisting of PCR, NASBA, LCR, SDA, RCR, and TMA. A method for detecting a cytochrome 2C19 gene polymorphism.
(27) The method for detecting a polymorphism in the cytochrome 2C19 gene according to any one of (23) to (26), wherein the oligonucleotide is labeled in advance.
(28) The method for detecting a polymorphism in the cytochrome 2C19 gene according to any one of (23) to (27), wherein the method is detected by a nucleic acid specific label.
(29) By performing amplification using at least one of a wild-type detection oligonucleotide and a mutant-type detection oligonucleotide, a reagent for detecting a cytochrome 2C19 gene polymorphism can be used at least under the same amplification conditions. A reagent for detecting a polymorphism in the cytochrome 2C19 gene, which is prepared so that sequences at two different mutation sites can be detected.
(30) An oligonucleotide designed so that the 3 'end of the oligonucleotide is complementary to the target gene and the second nucleotide from the 3' end is complementary to the nucleotide of the wild-type or mutant sequence (29) The reagent for detecting a polymorphism in the cytochrome 2C19 gene according to (29).
(31) The polymorphism of the cytochrome 2C19 gene polymorphism according to (29) or (30), wherein the amplification reaction is any method selected from the group consisting of PCR, NASBA, LCR, SDA, RCR and TMA. Detection reagent.
(32) The reagent for detecting a polymorphism in the cytochrome 2C19 gene according to any of (29) to (31), which is detected by a nucleic acid-specific label.
(33) In a method for detecting an N-acetyltransferase 2 gene polymorphism by performing amplification using an oligonucleotide for detecting a wild type and / or an oligonucleotide for detecting a mutant type, at least two sites are detected under the same amplification conditions. A method for detecting an N-acetyltransferase 2 gene polymorphism, comprising detecting sequences at different mutation sites.
[0016]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in detail. The chromosome containing the gene polymorphism site or a fragment thereof contained in the sample is not particularly limited as long as it is a target nucleic acid containing the gene polymorphism site that carries information on the target gene. Examples of the target nucleic acid include an Alu sequence, exons and introns of a gene encoding a protein, and a promoter.
[0017]
In the present invention, a nucleic acid sequence may be simply referred to as a nucleic acid. A mutant nucleic acid is a nucleic acid in which at least one, preferably one nucleotide, of wild-type nucleic acids is point-mutated and replaced with another nucleotide, or an insertion or deletion sequence in a part of a wild-type nucleic acid. The nucleic acid containing which has been clarified at which site the nucleotide is mutated. For example, it has been elucidated that the occurrence of several such gene polymorphisms results in different metabolic activities of drug metabolizing enzymes. This is a method for examining whether or not the gene is present, and detecting the type of the gene that the subject has.
[0018]
In the present invention, the reaction between the wild-type detection oligonucleotide and the mutant-type detection oligonucleotide generally means that the target nucleic acid denatured to a single strand is treated with an oligonucleotide and four kinds of deoxynucleoside triphosphates (dNTPs). And a DNA polymerase, an oligonucleotide extension reaction using a target nucleic acid as a template occurs, and a reaction in which a complementary strand of a nucleic acid sequence is synthesized.
[0019]
In the present invention, a wild-type detection oligonucleotide is an oligonucleotide having a sequence complementary to a chromosome containing a polymorphic site having a normal phenotype or a fragment thereof, and a mutant-type detection oligonucleotide. The nucleotide is an oligonucleotide having a sequence complementary to a chromosome or a fragment thereof including a polymorphic site having a sequence different from that of the wild type.
[0020]
In order to achieve the present invention, the vicinity of a gene polymorphism site of an oligonucleotide which is an important element for detection, a mutant sequence in the case of a wild-type detection oligonucleotide, and a mutant sequence in the case of a mutant detection oligonucleotide It is preferable to select an oligonucleotide for wild-type detection and a mutant-type detection that clearly dissociates without forming a double strand with the wild-type sequence. By clearly dissociating in this manner, the range of conditions in which a difference between a complementary oligonucleotide and a non-complementary oligonucleotide occurs, such as the presence or absence of an extension reaction or hybridization, is widened, which is suitable for achieving the present invention. It is.
[0021]
As an example of such an oligonucleotide, an oligonucleotide for detecting a wild type and an oligonucleotide for detecting a mutant having at least one mismatch other than the mutation is also preferable. The mismatch site other than the mutation site is preferably within 5 bases from the mutation site, more preferably within 4 bases, particularly preferably within 3 bases. Most preferably it is within 2 bases.
[0022]
Conventionally, the presence or absence of the formation of a double strand was sometimes unclear because a difference was made only at the mutation site. However, by adopting such a configuration as the oligonucleotide, the mutation site and the other mismatch site could be used at two sites. This prevents double-strand formation and facilitates detection of multiple mutation sites under the same conditions.
[0023]
As an example of such an oligonucleotide, when an oligonucleotide for wild-type detection and a mutant-type detection is used as a forward primer for amplification, preferably, from the 3 ′ end of the oligonucleotide for wild-type detection and the mutant-type detection oligonucleotide Oligonucleotides designed such that the second nucleotide is complementary to the nucleotide of the wild-type or mutant sequence. More preferably, the second nucleotide from the 3 ′ end of the oligonucleotide for wild-type detection and the mutant-type detection complementarily corresponds to the nucleotide of the wild-type or mutant-type sequence, and is complementary to both the wild-type and the mutant-type. The oligonucleotide is designed to have a non-base at least at the third to seventh positions (particularly preferably at the third position) from the 3 ′ end.
[0024]
In the case of such a design, in the combination of the oligonucleotide for wild type / wild type nucleic acid and the oligonucleotide for mutant type / mutant type nucleic acid, since each oligonucleotide matches at least from the 3 ′ end to the second sequence, the extension reaction is not performed. Occur. On the other hand, in the combination of the oligonucleotide for wild-type / mutated nucleic acid and the oligonucleotide for mutant / wild-type nucleic acid, an artificial mismatch of the 3 ′ end of the oligonucleotide or the second base from the 3 ′ end, and further another base is caused. Therefore, no elongation reaction occurs.
[0025]
The lower limit of the length of the oligonucleotide in the present invention is preferably 10, more preferably 13, and more preferably 16 bases, and the upper limit is preferably 50, more preferably 35 bases, and still more preferably 30 bases. It is preferable that at least one mismatch site is present in the oligonucleotide. The position is not particularly limited as long as it is any position from the 3 'end of the 3' end to the 5 'end, but is preferably a position close to the 3' end of the 3 'end, more preferably from the 3' end. The third is preferred.
Third, when an artificial mismatch is used, when a nucleic acid sequence that is not expected to undergo an extension reaction is used as a template, a mismatch of two bases is formed together with the nucleotide polymorphism site, and the extension reaction is strongly inhibited.
[0026]
In the above description, an example is shown in which the 3'-terminal portion of the extension reaction start point is used for the forward primer to clearly dissociate. However, when the reverse primer is used, the dissociation can be similarly performed. Furthermore, based on the above-mentioned concept, it is also possible to make it difficult to hybridize a non-complementary primer itself.
[0027]
In the present invention, the above-mentioned wild-type oligonucleotide and one or two mutant-type detection oligonucleotides are allowed to act on a sample separately or simultaneously.
In the present invention, the method for extending an oligonucleotide can be basically performed using a conventional method. Usually, a single-stranded denatured chromosome or a fragment thereof containing a specific nucleotide polymorphism site is combined with four types of deoxynucleoside triphosphates (dNTPs) and a DNA polymerase together with a wild-type detection oligonucleotide and one or more types. The oligonucleotides are extended using the target nucleic acid as a template by simultaneously or separately using the two mutant-type detection oligonucleotides.
[0028]
The extension reaction can be performed according to the method described in Molecular Cloning, A Laboratory Manual (Sambrook et al., 1989). In the present invention, a method for amplifying a chromosome or a fragment containing a specific nucleotide polymorphism site can also be basically performed using a conventional method, for example, a specific nucleotide polymorphism denatured into a single strand. On the chromosome containing the site or its fragment, together with four types of deoxynucleoside triphosphates (dNTPs), a DNA polymerase and a reverse primer, an oligonucleotide for detecting wild type and one or two types of oligonucleotides for detecting mutant type (Corresponding to a word primer) simultaneously or separately, and the amplification is performed between the forward oligonucleotide and the reverse oligonucleotide using the target nucleic acid as a template.
[0029]
Examples of the nucleic acid amplification method include PCR, NASBA (Nucleic acid sequence-based amplification method; Nature, vol. 350, p. 91 (1991)), LCR (WO 89/12696, JP-A-2-2934), SDA (Strand Displacement Amplification: Nucleic Acid Research, Vol. 20, p. 1691 (1992)), RCR (International Publication No. WO 90/1069), TMA (Transscription mediated adaptation, J.Med. 3270 (1993)).
[0030]
The LCR method is a method in which two kinds of oligonucleotides, deoxynucleoside triphosphate (dNTP) and ligase are allowed to act on a target nucleic acid, and a reaction in which the two kinds of oligonucleotides are bound on the target nucleic acid is repeated to amplify the target nucleic acid. is there.
SDA is a method of amplifying a nucleic acid by repeating an extension reaction and a treatment with a restriction enzyme using an oligonucleotide that is complementary to a template having a restriction enzyme site in its sequence.
RCA is a method in which a newly generated oligonucleotide is continuously amplified from a template by causing a complementary oligonucleotide and a DNA polymerase to act on a circular target nucleic acid while being separated from a template.
[0031]
The invader method uses an invader oligo having a sequence complementary to a DNA target fragment and a complementary oligo (signal probe) having a 5 'flap structure and detecting SNP. First, an invader oligo and a signal probe are hybridized to a target DNA. At this time, the invader oligo and the probe have a structure in which one base overlaps (invasive structure). Cleavase acts on this portion, and when the base of the signal probe at the SNP site and the base of the target are complementary (no SNP), the 5 'flip of the signal probe is cleaved. The cut 5 'flips hybridize to the FRET Probe (Fluorescence resonance energy transfer probe). The fluorescent dye and the quencher (Quencher) are close to each other on the FRET probe, and the fluorescence is suppressed. However, when the 5 'flip DNA is bound, the fluorescent dye portion is cleaved by Cleavase, and the fluorescent signal is detected. It is possible.
[0032]
Above all, in the PCR method, in the presence of a sample nucleic acid, four kinds of deoxynucleoside triphosphates, a pair of oligonucleotides and a thermostable DNA polymerase, a cycle consisting of three steps of denaturation, annealing, and extension is repeated, thereby Is a method of exponentially amplifying a region of a sample nucleic acid sandwiched between the oligonucleotides. That is, the nucleic acid of the sample is denatured in the denaturation step, each oligonucleotide is hybridized with a region on the single-stranded sample nucleic acid complementary thereto in the subsequent annealing step, and each oligonucleotide is used as a starting point in the subsequent extension step. As a result, a DNA strand complementary to each single-stranded sample nucleic acid serving as a template is extended by the action of a DNA polymerase to obtain a double-stranded DNA. By this one cycle, one double-stranded DNA is amplified into two double-stranded DNAs. Therefore, if this cycle is repeated n times, the region of the sample DNA sandwiched between the pair of oligonucleotides theoretically becomes 2ⁿIt is amplified twice. Since the amplified DNA region exists in a large amount, it can be easily detected by a method such as electrophoresis. Therefore, by using the gene amplification method, it is possible to detect a very small amount (even a single molecule) of a sample nucleic acid, which was not detectable in the past, and is a technique which has been widely used recently. .
[0033]
In the method utilizing the nucleic acid amplification method as described above, a gene amplification method using a wild-type detection oligonucleotide capable of amplifying a wild-type nucleic acid and a mutation-type detection oligonucleotide capable of amplifying a mutant nucleic acid is used separately or simultaneously. Do.
[0034]
When a sample nucleic acid is extended or amplified using a wild-type detection oligonucleotide, a reaction occurs when the sample nucleic acid is wild-type, but does not occur when a mutant nucleic acid is used. Conversely, when the sample nucleic acid is extended or amplified using the mutant-type detection oligonucleotide, the reaction occurs if the sample nucleic acid is a mutant type, but does not occur if the sample nucleic acid is a wild-type. Therefore, one sample is divided into two, and one performs the reaction using the wild-type detection oligonucleotide, and the other performs the reaction using the mutant-type detection oligonucleotide, and examines whether the reaction has occurred. This makes it possible to clearly know whether the sample nucleic acid is a wild type or a mutant type.
[0035]
In addition, by performing a reaction using both the oligonucleotide for wild-type detection and the oligonucleotide for mutant-type detection in one sample, and examining which oligonucleotide caused the reaction, whether the sample nucleic acid is wild-type It is also possible to know clearly whether it is a mutant type.
[0036]
In particular, higher organisms, including humans, have one father-derived gene and one maternal-derived gene for one type of gene. It is also possible to distinguish between homozygous mutants or both heterozygotes. That is, in the case of heterozygous reaction, since both the wild-type gene and the mutant gene are present, a reaction occurs both when the wild-type detection oligonucleotide is used and when the mutant-type detection oligonucleotide is used.
[0037]
The target gene polymorphism site is not particularly limited as long as the sequence of at least two different mutation sites can be detected under the same amplification conditions. However, it is preferably used for simultaneous detection of gene polymorphisms containing at least one, and preferably two or more, cytochromes 2D6 and 2C19. Furthermore, simultaneous detection of a gene polymorphism containing at least one, preferably two or more sites of cytochrome 2D6, simultaneous detection of a gene polymorphism containing at least one site of cytochrome 2C19, preferably two or more sites, and simultaneous detection of a gene polymorphism containing at least two sites of cytochrome 2D6 It is preferably used for simultaneous detection of a gene polymorphism including a site and at least one site of cytochrome 2C19.
[0038]
It is also preferably used for the simultaneous detection of gene polymorphisms containing at least one, and preferably two or more, sites of NAT2, and a gene polymorphism containing at least one site of NAT2 and at least one site of cytochrome 2D6 and cytochrome 2C19. Is also preferably used for simultaneous detection of
Furthermore, in addition to the above-mentioned sites, simultaneous detection of at least one gene polymorphism in cytochrome 2A6 may be performed.
[0039]
Above all, among cytochromes 2D6, simultaneous gene polymorphisms containing at least one, and preferably two or more, 2D6 * 2, 2D6 * 4, 2D6 * 10, 2D6 * 14, 2D6 * 18, 2D6 * 21, 2D6 * 36 It is preferably used for detection, and is preferably used for simultaneous detection of a gene polymorphism containing at least one of 2C19 * 2 and 2C19 * 3 among cytochromes 2C19, and NAT2 * 5 and NAT2 * 6 among NAT2. , NAT2 * 7 are preferably used for simultaneous detection of gene polymorphisms containing at least one, and preferably two or more.
[0040]
In addition, combinations including different genes include cytochromes 2D6 * 2, 2D6 * 4, 2D6 * 10, 2D6 * 14, 2D6 * 18, 2D6 * 21, 2D6 * 36, 2C19 * 2, 2C19 * 3, and 2A6 *. 3, NAT2 * 5, NAT2 * 6, and NAT2 * 7, which are preferably used for simultaneous detection of gene polymorphisms containing at least one, and preferably two or more.
[0041]
As an example of a method for detecting whether or not the oligonucleotide has reacted, a nucleic acid sample containing a chromosome or a fragment containing a specific nucleotide polymorphism site, a certain amount of a wild-type detection oligonucleotide and one or The reaction is carried out simultaneously or separately using two types of oligonucleotides for detecting a mutant type (corresponding to forward primers), and then the nucleic acid is labeled with a nucleic acid-specific label, particularly a double-stranded nucleic acid-specific binding substance. The amount of oligonucleotide generated by the reaction can be detected.
In addition, any method that can detect the presence or absence of an amplification reaction, such as a method by electrophoresis and a method by hybridization, can be used.
[0042]
When a nucleic acid-specific label is used, it is not necessary to use a labeled oligonucleotide because it is only necessary to determine whether an extension reaction has occurred depending on the type of the forward primer without performing post-treatment such as electrophoresis. Although it was advantageous, it was still considered difficult to carry out the extension reaction under the same conditions. However, for example, by using the above-mentioned primers, it was possible to carry out the extension reaction under the same conditions.
[0043]
As the above detection method, a labeled oligonucleotide can be used.
For example, for the detection, the respective oligonucleotides are labeled in advance with an enzyme, biotin, a fluorescent substance, a hapten, an antigen, an antibody, a radioactive substance, a luminophore, or the like, and after the extension or amplification reaction, the labeled oligonucleotides reacted. By detecting, a gene polymorphism can be detected. Examples of the enzyme include alkaline phosphatase, peroxidase and the like. Examples of the fluorescent substance include FITC, 6-FAM, HEX, TET, TAMRA, Texas Red, Cy3, Cy5, and the like. Examples of the double-stranded nucleic acid-specific binding substance include ethylene bromide and SYBR Green I. Haptens include biotin, digoxigenin and the like. As radioactive materials,³²P,³⁵S and the like.
Examples of the luminophore include ruthenium. The label may be attached to any position of the oligonucleotide as long as it does not affect the extension reaction of the oligonucleotide. When used as a forward primer, it is preferably at the 5 'site.
[0044]
When different labels were used for the wild-type detection oligonucleotide and the mutant-type detection oligonucleotide, both the wild-type detection oligonucleotide and the mutation-type detection oligonucleotide were added to one sample, and one reaction was performed. Can be detected in the tank.
[0045]
When performing amplification in the present invention, a reaction solution suitable for the amplification method is used as a solution. A known reaction solution can be used.
[0046]
For example, in the case of the PCR method, DNA polymerase, four kinds of deoxynucleoside triphosphates (dNTP), buffer, MgCl₂, MgSO₄It is a solution to which various salts and the like are added. In order to simplify the operation of the present invention and reduce human error, it is preferable to use a reaction solution other than the oligonucleotide prepared at a predetermined concentration in advance, and furthermore, make the concentration of the sample the same. However, in order to detect sequences of at least two different mutation sites under the same amplification conditions according to the present invention, the concentration of salts and the like can be appropriately changed.
[0047]
As a method for adjusting the reaction solution, specifically, when both the wild-type detection oligonucleotide and the mutant-type detection oligonucleotide are amplified with respect to the sample, the oligonucleotide, the salts, and the DNA are used. It is preferable to lower the concentration of polymerase and the like. It is also preferable to lower only the concentration that should not be amplified.
Conversely, when amplification is not recognized in both the wild-type detection oligonucleotide and the mutant-type detection oligonucleotide, it is preferable to increase the concentration of the oligonucleotide, salts, DNA polymerase, and the like. It is also preferable to increase only the concentration to be amplified.
The concentrations of the buffer and dNTP are preferably the same irrespective of the wild-type detection oligonucleotide, the mutant-type detection oligonucleotide and the target mutation site.
[0048]
These reaction solutions were: 0.5 to 50 pmol of oligonucleotide, 0.5 to 50 μl of × 10 buffer solution, 0.5 to 50 μl of 2 mM dNTP, and 0 to 50 μl of salt at 25 mM concentration per 25 μl of reaction solution. It is preferable that the amount of DNA polymerase is about 0.1 to 30 ng.
[0049]
In the present invention, amplification reactions targeting at least two or more different mutation sites are performed under the same temperature and time cycle conditions.
In the method of detecting a mutation site using a conventionally known oligonucleotide, since the nucleotide sequence around each mutation site is not exactly the same, it is necessary to perform amplification reaction under different conditions, In order to specifically amplify a nucleic acid site, the number of bases of an amplification product was different depending on each mutation site, so it was thought that amplification could not be performed on a plurality of mutation sites under the same temperature and time cycle conditions. In the present invention, conventional problems were overcome by using a specific oligonucleotide for each mutation site and by appropriately adjusting the reaction solution, and the present invention was completed.
[0050]
As the amplification reaction, the first heat deformation step is 80 to 100 ° C for 1 to 15 minutes, the repeated heat deformation step is 80 to 100 ° C for 2 to 300 seconds, and the annealing link is 40 to 80 ° C for 2 to 300 seconds, It is preferable that the extension reaction step is performed at 60 to 85 ° C. for about 2 to 300 seconds, and this repetition is repeated 20 to 50 times.
[0051]
According to the method of the present invention, conventionally, mutation types are individually examined for mutation sites, and the data are integrated to determine and detect gene polymorphisms. Since the detection of the pattern can be completed in a short time and the complexity of the condition setting has been eliminated, human errors such as simple setting errors can be reduced.
[0052]
kit
In the present invention, the kit includes a gene type oligonucleotide containing a wild-type detection oligonucleotide and one or two mutant-type detection oligonucleotides, a reverse oligonucleotide, a DNA polymerase, and four types of deoxynucleoside triphosphates (dNTPs). The kit includes a type detection reagent kit.
Each of the oligonucleotides may be labeled in advance with an enzyme, biotin, a fluorescent substance, a hapten, an antigen, an antibody, a radioactive substance, a luminophore, or the like as described above.
[0053]
In addition, it is preferable that the kit is packaged as one set of each of the oligonucleotides for detecting wild type and / or the oligonucleotides for detecting mutant type corresponding to a plurality of mutation sites and the reverse primer.
The kit includes, in a reaction vessel, the respective oligonucleotides for detecting wild-type and / or mutant-type, reverse primer, DNA polymerase, four types of deoxynucleoside triphosphate (dNTP), salts, etc., other than the sample. It is preferable that all of the components necessary for amplification are dissolved, or the reaction solution is prepared by adding only a fixed amount of water and then adding a sample.
In addition, it is preferable that the composition and the content are the same except for the type of the oligonucleotide of the kit. However, in order to detect the sequence of at least two different mutation sites under the same amplification conditions according to the present invention, salts such as salts Can be appropriately changed as described above.
[0054]
【Example】
Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.
Oligonucleotide synthesis
Each oligonucleotide in the examples was synthesized by a phosphoramidite method using a DNA synthesizer 392 manufactured by PerkinElmer. The synthesis was performed according to the manual, and deprotection of various oligonucleotides was performed overnight at 55 ° C. with aqueous ammonia. Oligonucleotide purification was carried out using an OPC column from PerkinElmer.
Alternatively, a request was made to a DNA synthesis contract company (Japan Bioservice Co., Ltd., Sawasdee Co., Ltd., GENSET KK, Amersham Pharmacia Biotech Co., Ltd.).
[0055]
Reagent adjustment
A 25 μl solution containing the following reagents was prepared.
Taq DNA polymerase reaction solution a
Oligonucleotide (forward primer) 5 pmol
Oligonucleotide (reverse primer) 5 pmol
× 10 buffer 2.5 μl
2.5 μl of 2 mM dNTP
25 mM MgCl₂                                                      1.5 μl
Taq DNA polymerase 1.3 U
Extracted DNA solution 100 ng
[0056]
Amplification conditions
95 ° C for 5 minutes
95 ° C for 30 seconds, 65 ° C for 30 seconds, 72 ° C for 30 seconds (35 cycles)
72 ° C for 2 minutes.
[0057]
(2) Detection with nucleic acid-specific binding substance
3 μl of each amplification reaction solution was added to 100 μl of a 10000-fold solution of CyberGreen I (manufactured by Molecular Probe), stirred at room temperature, and the fluorescence intensity was measured with a fluorescence plate reader (Dainippon Pharmaceutical Co., Ltd.) in a dark room. . The time required was about 5 minutes.
From the obtained fluorescence intensity, the fluorescence intensity of the sample was calculated using the following calculation formula.
FL (fluorescence intensity of sample) = FLs−FLb (Equation 1)
FLb: Fluorescence intensity of Negative Control (no sample added)
FLs: fluorescence intensity of each sample
[0058]
Example 1 Simultaneous detection of CYP2D6 * 2 and CYP2D6 * 10 gene polymorphisms
(1) Oligonucleotides for detecting polymorphisms at positions 100 and 2850 of the CYP2D6 gene
[0059]
Oligos 1 and 2 are for detecting the polymorphism at position 2850 of the CYP2D6 gene.
Oligo 1 has the wild-type (C) nucleotide sequence second from the 3 'end and an artificial mismatch (C → A) third, and oligo 2 has the mutant (T) second from the 3' end. And an artificial mismatch (C → A) at the third position, and oligo 3 is an antisense strand, and is used as an oligonucleotide in an amplification reaction in combination with oligos 1 and 2. Oligos 1, 2, and 3 are used after being labeled as necessary.
[0060]
Oligos 4 and 5 are for detecting the 100th polymorphism of the CYP2D6 gene.
Oligo 4 has the nucleotide sequence of wild type (C) second from the 3 'end and an artificial mismatch (C → A) third, and oligo 5 has the mutated (T) second from the 3' end. , And a third artificial mismatch (C → A), oligo 6 is an antisense strand, and is used in combination with oligos 4 and 5 as an oligonucleotide for an amplification reaction. Oligos 4, 5, and 6 are used after being labeled as necessary.
[0061]
Further, the oligonucleotides of oligo 1, oligo 2, oligo 4, and oligo 5 in the present example are not limited to the oligonucleotides described in this example, and have a length of 10 to 50 bases from the 3 ′ end as described above. If so, similar results can be obtained. Further, each oligonucleotide introduced an artificial mismatch at the third position from the 3 ′ end, but may be in the third to seventh positions as described above, or may be located at several other positions (1 to 5 positions). To the extent, mismatches due to the overall length of the primer (especially at the 5 'end) may be introduced.
Furthermore, Oligo 3 and Oligo 6 are determined based on an appropriate length or the like for detecting the presence or absence of an extension reaction, and if they correspond to an appropriate portion of the gene of interest, It is not limited to the example arrangement.
[0062]
(2) Analysis of CYP2D6 * 2 and CYP2D6 * 10 gene polymorphisms by PCR
(1) Amplification reaction by PCR method
Using a DNA solution extracted from human leukocytes by the phenol-chloroform method as a sample, a reagent containing the following oligonucleotide was added, and CYP2D6 * 2 and CYP2D6 * 10 gene polymorphisms were analyzed under the above conditions. The reaction was performed simultaneously under the same conditions for the reaction solutions a, b, c, and d.
[0063]
Oligonucleotide of reaction solution 1-a
Oligo 1 and Oligo 3
Oligonucleotide of reaction solution 1-b
Oligo 2 and Oligo 3
Oligonucleotide of reaction solution 1-c
Oligo 4 and Oligo 6
Oligonucleotide of reaction solution 1-d
Oligo 5 and Oligo 6
[0064]
[Table 1]

[0065]
[Table 2]

[0066]
Example 2 Simultaneous detection of CYP2D6 * 4 and CYP2D6 * 36 gene polymorphisms
(1) Oligonucleotide for detecting polymorphism at position 1846 of CYP2D6 gene and Exon9 conversion
Oligos 7 and 8 are for detecting the polymorphism at position 1846 of the CYP2D6 gene.
Oligo 7 has the nucleotide sequence of the wild type (G) second from the 3 ′ end and an artificial mismatch (C → A) third, and oligo 8 has the mutated (A) second from the 3 ′ end. And the third has an artificial mismatch (C → A), oligo 9 is an antisense strand, and is used as an oligonucleotide in an amplification reaction in combination with oligos 7 and 8. Oligos 7, 8, and 9 are used after being labeled as necessary.
[0067]
Oligos 10 and 11 are for detecting Exon9 conversion of the CYP2D6 gene.
Oligo 10 has a nucleotide sequence of wild type (2D6 Exon9), oligo 11 has a nucleotide sequence of mutant type (2D7 Exon9), oligo 12 is an antisense strand, and is used in combination with oligos 10 and 11 for amplification reaction. Used as oligonucleotides. Oligos 10, 11, and 12 are used after being labeled as necessary.
[0068]
(2) Analysis of CYP2D6 * 4 and CYP2D6 * 36 gene polymorphisms by PCR
(1) Amplification reaction by PCR method
Using a DNA solution extracted from human leukocytes by the phenol-chloroform method as a sample, reagents containing the following oligonucleotides were added, and CYP2D6 * 4 and CYP2D6 * 36 gene polymorphisms were analyzed under the above conditions. The reaction was performed simultaneously under the same conditions for the reaction solutions a, b, c, and d.
[0069]
Oligonucleotide of reaction solution 2-a
Oligo 7 and Oligo 9
Oligonucleotide of reaction solution 2-b
Oligo 8 and Oligo 9
Oligonucleotide of reaction solution 2-c
Oligo 10 and Oligo 12
Oligonucleotide of reaction solution 2-d
Oligo 11 and Oligo 12
[0070]
[Table 3]

[0071]
[Table 4]

[0072]
Example 3 Detection of CYP2D6 * 4 and CYP2D6 * 14 gene polymorphisms
(1) Oligonucleotide for detecting polymorphism at position 1846 of CYP2D6 gene
Oligo 13 has a nucleotide sequence complementary to the wild type (G) second from the 3 ′ end, and an artificial mismatch (C → A) third, and oligo 14 is the second variant from the 3 ′ end. It has a nucleotide sequence complementary to (A) and an artificial mismatch (C → A) at the third position, and oligo 15 is an antisense strand, and is used as an oligonucleotide in an amplification reaction in combination with oligos 13 and 14. You. Oligos 13, 14, and 15 are used after being labeled as necessary.
(2) an oligonucleotide for detecting the 1758th polymorphism of the CYP2D6 gene
Oligo 16 has a nucleotide sequence complementary to the wild type (G) second from the 3 ′ end, and an artificial mismatch (C → A) third, and oligo 17 is the second variant from the 3 ′ end. It has a nucleotide sequence complementary to (A) and a third artificial mismatch (C → A), and oligo 15 is an antisense strand, and is used as an oligonucleotide in an amplification reaction in combination with oligos 16 and 17. You. Oligos 16, 17, and 15 are used after being labeled as necessary.
(3) Analysis of CYP2D6 * 4 and CYP2D6 * 14 gene polymorphisms by PCR
(1) Amplification reaction by PCR method
Using a DNA solution extracted from human leukocytes by the phenol-chloroform method as a sample, a reagent containing the following oligonucleotide was added, and CYP2D6 * 4 and CYP2D6 * 14 gene polymorphisms were analyzed under the above conditions. The reaction was performed simultaneously under the same conditions for the reaction solutions a, b, c, and d.
[0073]
Oligonucleotide of reaction solution 3-a
Oligo 13 and Oligo 15
Oligonucleotide of reaction solution 3-b
Oligo 14 and Oligo 15
Oligonucleotide of reaction solution 3-c
Oligo 16 and Oligo 15
Oligonucleotide of reaction solution 3-d
Oligo 17 and Oligo 15
[0074]
[Table 5]

[0075]
Example 4 Detection of CYP2C19 * 2 and CYP2C19 * 3 gene polymorphisms
(1) Oligonucleotide for detecting polymorphism at position 681 of CYP2C19 gene
Oligo 18 has a nucleotide sequence complementary to the wild type (G) second from the 3 ′ end, and an artificial mismatch (G → A) third, and oligo 19 is the second variant from the 3 ′ end. It has a nucleotide sequence complementary to (A) and a third artificial mismatch (G → A), oligo 20 is an antisense strand, and is used as an oligonucleotide in an amplification reaction in combination with oligos 18 and 19. You. Oligos 18, 19 and 20 are used after being labeled as necessary.
[0076]
(2) Synthesis of oligonucleotide for detecting polymorphism at position 636 of CYP2C19 gene
Oligo 21 has a nucleotide sequence complementary to the wild type (G) second from the 3 ′ end, and an artificial mismatch (c → t) third, and oligo 22 is the second variant from the 3 ′ end. It has a nucleotide sequence complementary to (A) and a third artificial mismatch (c → t), oligo 23 is an antisense strand, and is used as an oligonucleotide in an amplification reaction in combination with oligos 21 and 22. You. The oligos 21, 22, and 23 are used after being labeled as necessary.
[0077]
(3) Analysis of CYP2C19 * 2 and CYP2C19 * 3 gene polymorphisms by PCR
(1) Amplification reaction by PCR method
Using a DNA solution extracted from human leukocytes by the phenol-chloroform method as a sample, the following CYP2C19 * 2 and CYP2C19 * 3 gene polymorphisms were analyzed under the above conditions by adding a reagent containing the following oligonucleotide. The reaction was performed simultaneously under the same conditions for the reaction solutions a, b, c, and d.
[0078]
Oligonucleotide of reaction solution 4-a
Oligo 18 and Oligo 20
Oligonucleotide of reaction solution 4-b
Oligo 19 and Oligo 20
Oligonucleotide of reaction solution 4-c
Oligo 21 and Oligo 23
Oligonucleotide of reaction solution 4-d
Oligo 21 and Oligo 23
[0079]
[Table 6]

[0080]
Simultaneous detection of 2C19 * 2 and CYP2C19 * 3 gene polymorphisms (Part 2)
(1) Oligonucleotides for detecting polymorphisms at positions 681 and 636 of the CYP2C19 gene
Oligos 24 and 25 are for detecting the polymorphism at position 681 of the CYP2C19 gene.
Oligo 24 has the nucleotide sequence of the wild type (G) second from the 3 ′ end and an artificial mismatch (G → A) third, and oligo 25 has the mutant type (A) second from the 3 ′ end. And an artificial mismatch (G → A) at the third position, and oligo 3 is an antisense strand, and is used as an oligonucleotide in an amplification reaction in combination with oligos 24 and 25. The oligos 24, 25 and 26 are used after being labeled as necessary.
[0081]
Oligos 27 and 28 are for detecting the polymorphism at position 636 of the CYP2C19 gene.
Oligo 27 has the nucleotide sequence of wild type (G) second from the 3 ′ end and an artificial mismatch (C → A) third, and oligo 28 has the second variant (A) from the 3 ′ end. And the third has an artificial mismatch (C → A), oligo 29 is an antisense strand, and is used as an oligonucleotide in an amplification reaction in combination with oligos 27 and 28. The oligos 27, 28 and 29 are used after being labeled as necessary.
[0082]
(2) Analysis of CYP2C19 * 2 and CYP2C19 * 3 gene polymorphisms by PCR
(1) Amplification reaction by PCR method
Using a DNA solution extracted from human leukocytes by the phenol-chloroform method as a sample, the following CYP2C19 * 2 and CYP2C19 * 3 gene polymorphisms were analyzed under the above conditions by adding a reagent containing the following oligonucleotide. The reaction was performed simultaneously under the same conditions for the reaction solutions a, b, c, and d.
[0083]
Oligonucleotide of reaction solution 5-a
Oligo 24 and Oligo 26
Oligonucleotide of reaction solution 5-b
Oligo 25 and Oligo 26
Oligonucleotide of reaction solution 5-c
Oligo 27 and Oligo 29
Oligonucleotide of reaction solution 5-d
Oligo 28 and Oligo 29
[0084]
[Table 7]

[0085]
[Table 8]

[0086]
Example 6 Detection of NAT2 * 5, NAT2 * 6, NAT2 * 7 gene polymorphism
(1) Oligonucleotide for detecting the 481st polymorphism of NAT2 gene
Oligo 30 has a nucleotide sequence complementary to wild type (C) second from the 3 'end, and an artificial mismatch (A → C) third, and oligo 31 has the second variant from the 3' end. It has a nucleotide sequence complementary to (T) and an artificial mismatch (A → C) at the third position, and oligo 32 is a sense strand, and is used as an oligonucleotide in an amplification reaction in combination with oligos 30 and 31. . The oligos 30, 31, and 32 are used after being labeled as necessary.
[0087]
(2) Oligonucleotide for detecting polymorphism at position 590 of NAT2 gene
Oligo 33 has a nucleotide sequence complementary to the wild type (G) second from the 3 'end, and an artificial mismatch (T → A) third, and oligo 34 has the second variant from the 3' end. It has a nucleotide sequence complementary to (A) and a third artificial mismatch (T → A), oligo 32 is the sense strand, and is used as an oligonucleotide in an amplification reaction in combination with oligos 33 and 34 . The oligos 33 and 34 are used after being labeled as necessary.
[0088]
(3) Oligonucleotide for detecting polymorphism at position 857 of NAT2 gene
Oligo 35 has a nucleotide sequence complementary to the wild type (G) second from the 3 'end and an artificial mismatch (T → G) third, and oligo 36 has the second variant from the 3' end. It has a nucleotide sequence complementary to (A) and a third artificial mismatch (T → G), oligo 32 is the sense strand, and is used as an oligonucleotide in an amplification reaction in combination with oligos 35 and 36 . The oligos 35 and 36 are used after being labeled as necessary.
[0089]
(4) Analysis of NAT2 * 5, NAT2 * 6, NAT2 * 7 gene polymorphism by PCR method
(1) Amplification reaction by PCR method
Using a DNA solution extracted from human leukocytes by the phenol-chloroform method as a sample, NAT2 * 5, NAT2 * 6, and NAT2 * 7 gene polymorphisms were analyzed under the above conditions by adding a reagent containing the following oligonucleotide. The reaction was performed simultaneously under the same conditions for the reaction solutions a, b, c, d, e, and f.
[0090]
Oligonucleotide of reaction solution 6-a
Oligo 30 and Oligo 32
Oligonucleotide of reaction solution 6-b
Oligo 31 and Oligo 32
Oligonucleotide of reaction solution 6-c
Oligo 33 and Oligo 32
Oligonucleotide of reaction solution 6-d
Oligo 34 and Oligo 32
Oligonucleotide of reaction solution 6-e
Oligo 35 and Oligo 32
Oligonucleotide of reaction solution 6-f
Oligo 36 and Oligo 32
[0091]
[Table 9]

[0092]
As described above, as is clear from any of the examples, a plurality of polymorphic sites could be amplified under the same conditions, and the differences could be more clearly distinguished.
[0093]
Example 7 Detection of CYP2A6 * 3 gene polymorphism
(1) Oligonucleotide for detecting polymorphism of CYP2A6 * 3 gene
Oligo 37 and oligo 39 have nucleotide sequences complementary to CYP2DA6 (wild type), and oligo 38 and oligo 40 have nucleotide sequences complementary to CYP2A6 * 3 (mutant). Oligos 37 and 39 and oligos 38 and 40 are used in combination as oligonucleotides in an amplification reaction. The oligos 37, 38, 39, and 40 are used after being labeled as necessary.
[0094]
(2) Analysis of CYP2A6 * 3 gene polymorphism by PCR method
(1) Amplification reaction by PCR method
Using a DNA solution extracted from human leukocytes by the phenol-chloroform method as a sample, a reagent containing the following oligonucleotide was added, and the CYP2A6 * 3 gene polymorphism was analyzed under the above conditions. The reaction was carried out simultaneously with the reaction solutions a and b under the same conditions.
[0095]
Oligonucleotide of reaction solution 7-a
Oligo 37 and Oligo 39
Oligonucleotide of reaction solution 7-b
Oligo 38 and Oligo 40
[0096]
[Table 10]

[0097]
The analysis of the CYP2A6 * 3 gene polymorphism was found to be detectable under the same amplification temperature and time conditions as in the other examples. It became clear that it could be detected.
[0098]
As shown in the above examples, a plurality of sites are simultaneously subjected to a base polymorphism-specific amplification reaction under the same temperature and time conditions using an oligonucleotide, and the amplification reaction substance is measured using a nucleic acid-specific binding substance. The genotype could be clearly and easily determined quickly.
[0099]
In addition, although only specific combinations are performed in the embodiments, the temperature and time conditions for amplification are the same, and it is clear that detection can be performed simultaneously even if free combinations are performed in these locations.
[0100]
Further, each oligonucleotide in the present example is not limited to the oligonucleotide described above, and similar results can be obtained as long as the length is 10 to 50 bases from the 3 'end as described above. Furthermore, some oligonucleotides introduced an artificial mismatch at the third position from the 3 'end, but may be in the third to seventh positions, as described above, or at several other positions (1 to 7). In about 5 places, mismatches due to the entire length of the primer (particularly at the 5 'end) may be introduced.
Furthermore, the reverse side is determined on the basis of an appropriate length or the like for detecting the presence or absence of an extension reaction, and if the reverse side corresponds to an appropriate portion of the gene of interest, the sequence of this example However, the present invention is not limited to this.
[0101]
【The invention's effect】
As described above, the present invention provides a method for clearly and easily detecting a gene polymorphism in a sample nucleic acid. The method of the present invention does not require complicated operations as in the conventional methods, so that quick and easy reproducible results were obtained.
[Sequence list]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146]

[0147]

[0148]

[0149]

[0150]

Claims (33)

In a method for detecting a cytochrome 2D6 gene polymorphism by performing amplification using a wild-type detection oligonucleotide and / or a mutant-type detection oligonucleotide, the sequences of at least two different mutation sites under the same amplification conditions A method for detecting a polymorphism in the cytochrome 2D6 gene, characterized by detecting The method for detecting a polymorphism in the cytochrome 2D6 gene according to claim 1, wherein an oligonucleotide having at least one mismatch other than the mutation site is used. The oligonucleotide is designed such that the 3 ′ end of the oligonucleotide is complementary to the target gene and the second nucleotide from the 3 ′ end is complementary to the nucleotide of the wild-type or mutant sequence. The method for detecting a polymorphism in the cytochrome 2D6 gene according to claim 1 or 2. The detection of a cytochrome 2D6 gene polymorphism according to any one of claims 1 to 3, wherein the amplification reaction is any method selected from the group consisting of PCR, NASBA, LCR, SDA, RCR and TMA. Method. The method for detecting a polymorphism in the cytochrome 2D6 gene according to any one of claims 1 to 4, wherein the oligonucleotide is labeled in advance. The method for detecting a polymorphism in the cytochrome 2D6 gene according to any one of claims 1 to 5, wherein the detection is performed by using a nucleic acid specific label. By performing amplification using at least one of the wild-type detection oligonucleotide and the mutation-type detection oligonucleotide, in a reagent for detecting a polymorphism in the cytochrome 2D6 gene, at least two sites are used under the same amplification conditions. A reagent for detecting a polymorphism in a cytochrome 2D6 gene, which is prepared so as to detect a sequence at a different mutation site. The oligonucleotide is designed such that the 3 ′ end of the oligonucleotide is complementary to the target gene and the second nucleotide from the 3 ′ end is complementary to the nucleotide of the wild-type or mutant sequence. The detection reagent of the cytochrome 2D6 gene polymorphism according to claim 7, wherein 9. The detection reagent for a cytochrome 2D6 gene polymorphism according to claim 7 or 8, wherein the amplification reaction is any method selected from the group consisting of PCR, NASBA, LCR, SDA, RCR and TMA. The reagent for detecting a polymorphism in the cytochrome 2D6 gene according to any one of claims 7 to 9, wherein the reagent is detected by a nucleic acid specific label. By performing amplification using at least one of a wild-type detection oligonucleotide and a mutant-type detection oligonucleotide and a nucleic acid-specific label, in a method for detecting a gene polymorphism, at least two sites are used under the same amplification conditions. A method for detecting a gene polymorphism, comprising detecting sequences at different mutation sites. A method for detecting a genetic polymorphism, wherein the 3 'end of the wild-type detection oligonucleotide and the mutant-type detection oligonucleotide according to claim 11 is complementary to a target gene. A method for detecting a genetic polymorphism, wherein the nucleic acid-specific label according to claim 11 or 12 is a fluorescent aromatic reagent that specifically recognizes a double-stranded polynucleotide. The method for detecting a gene polymorphism according to any one of claims 11 to 13, wherein the gene polymorphism is a drug metabolism-related enzyme gene polymorphism. The method for detecting a gene polymorphism according to any one of claims 11 to 14, wherein the amplification reaction is any method selected from the group consisting of PCR, NASBA, LCR, SDA, RCR and TMA. The method for detecting a genetic polymorphism according to any one of claims 11 to 15, wherein the oligonucleotide for detecting wild type and the oligonucleotide for detecting mutant type are labeled in advance. The method according to any one of claims 11 to 14, wherein the detection is performed by an interaction between an amplification product obtained by the method according to claim 15 and a nucleic acid-specific label. By performing amplification using at least one of the wild-type detection oligonucleotide and the mutant-type detection oligonucleotide and a nucleic acid-specific label, the reagent for detecting a gene polymorphism has at least 2 A reagent for detecting a gene polymorphism, which is prepared so as to be able to detect a sequence of a different mutation site. A reagent for detecting a genetic polymorphism, wherein the 3 'end of the oligonucleotide according to claim 18 is complementary to a target gene. 20. The reagent for detecting a genetic polymorphism according to claim 18 or 19, wherein the nucleic acid-specific label is a fluorescent aromatic reagent that specifically recognizes a double-stranded polynucleotide. 21. The reagent for detecting a gene polymorphism according to claim 18, wherein the gene polymorphism is a drug metabolism-related enzyme gene polymorphism. The detection reagent according to any one of claims 18 to 21, wherein the amplification reaction is any method selected from the group consisting of PCR, NASBA, LCR, SDA, RCR, and TMA. In a method for detecting a cytochrome 2C19 gene polymorphism by performing amplification using a wild-type detection oligonucleotide and / or a mutation-type detection oligonucleotide, the sequences of at least two different mutation sites under the same amplification conditions A method for detecting a polymorphism in the cytochrome 2C19 gene, comprising detecting The method for detecting a polymorphism in the cytochrome 2C19 gene according to claim 23, wherein an oligonucleotide having at least one mismatch other than the mutation site is used. The oligonucleotide is designed such that the 3 ′ end of the oligonucleotide is complementary to the target gene and the second nucleotide from the 3 ′ end is complementary to the nucleotide of the wild-type or mutant sequence. 25. The method for detecting a polymorphism in the cytochrome 2C19 gene according to claim 23 or 24. The detection of the cytochrome 2C19 gene polymorphism according to any one of claims 23 to 25, wherein the amplification reaction is any method selected from the group consisting of PCR, NASBA, LCR, SDA, RCR, and TMA. Method. The method for detecting a polymorphism in the cytochrome 2C19 gene according to any one of claims 23 to 26, wherein the oligonucleotide is labeled in advance. The method for detecting a polymorphism in the cytochrome 2C19 gene according to any one of claims 23 to 27, wherein the detection is performed by using a nucleic acid-specific label. By performing amplification using at least one of the wild-type detection oligonucleotide and the mutant-type detection oligonucleotide, at least two sites are detected under the same amplification conditions in the reagent for detecting the cytochrome 2C19 gene polymorphism. A reagent for detecting a polymorphism in a cytochrome 2C19 gene, which is adjusted so that sequences at different mutation sites can be detected. The oligonucleotide is designed such that the 3 ′ end of the oligonucleotide is complementary to the target gene and the second nucleotide from the 3 ′ end is complementary to the nucleotide of the wild-type or mutant sequence. The reagent for detecting a polymorphism in the cytochrome 2C19 gene according to claim 29. 31. The reagent for detecting a polymorphism in the cytochrome 2C19 gene according to claim 29 or 30, wherein the amplification reaction is any method selected from the group consisting of PCR, NASBA, LCR, SDA, RCR and TMA. The detection reagent for a polymorphism in the cytochrome 2C19 gene according to any one of claims 29 to 31, which is detected by a nucleic acid specific label. In a method for detecting an N-acetyltransferase 2 gene polymorphism by performing amplification using a wild-type detection oligonucleotide and / or a mutant-type detection oligonucleotide, at least two different mutations are obtained under the same amplification conditions. A method for detecting an N-acetyltransferase 2 gene polymorphism, comprising detecting a sequence at a site.