JP6644297B2 - ハイブリダイゼーション用バッファー組成物及びハイブリダイゼーション方法 - Google Patents
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- JP6644297B2 JP6644297B2 JP2015539042A JP2015539042A JP6644297B2 JP 6644297 B2 JP6644297 B2 JP 6644297B2 JP 2015539042 A JP2015539042 A JP 2015539042A JP 2015539042 A JP2015539042 A JP 2015539042A JP 6644297 B2 JP6644297 B2 JP 6644297B2
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Description
プライマー1:5'- gtgtgacatgttctaatatagtcac -3'(配列番号1)及び
プライマー2:5'- gaatggtcctgcaccagtaa -3'(配列番号2)
からなるプライマーのセットが挙げられる。
本実施例では、K-rasにおけるコドン12及びコドン13の野生型の塩基配列(GGTGGC)を非検出対象塩基とした。野生型の検体としては、RKO株ゲノムDNA(野生型検体、コドン12,13の配列:GGTGGC)を使用した。また、検出対象塩基としては、LoVo株ゲノムDNA (G13D変異型検体、コドン12,13の配列:GGTGAC)を用いた。これら細胞株から抽出したDNAを用いて、G13D変異型検体の割合を0%、0.625%、1.25%、2.5%、5%とした5種類の検体DNAを調整した。
本実施例では、ブロッキング用核酸に含まれる、非検出対象塩基に相補的な塩基配列の位置及び、ブロッキング用核酸に含まれるミスマッチ塩基について検討した。
本実施例では、ターゲット核酸を検出するための核酸プローブの長さと、ブロッキング用核酸の長さについて検討した。
Claims (8)
- 遺伝子多型における一方のアレルを含むターゲット核酸と、当該ターゲット核酸に対して相補的な塩基配列からなり、基板上に固定されてなる核酸プローブとのハイブリダイゼーションに使用するバッファー組成物であって、
前記遺伝子多型における上記アレル以外のアレルを含み、上記ターゲット核酸を核酸増幅法により増幅する際に同時に増幅される非ターゲット核酸に対して相補的な塩基配列からなり、上記核酸プローブの長さの60%以上100%未満の長さであって15塩基以上の長さのブロッキング用核酸を含有する、ハイブリダイゼーション用バッファー組成物。 - 上記核酸プローブは15〜25塩基長であり、上記ブロッキング用核酸の塩基長が15〜24塩基長であることを特徴とする請求項1記載のハイブリダイゼーション用バッファー組成物。
- クエン酸ナトリウム二水和物(SSC)及びドデシル硫酸ナトリウム(SDS)を更に含むことを特徴とする請求項1記載のハイブリダイゼーション用バッファー組成物。
- 上記ターゲット核酸を増幅する核酸増幅反応の後の反応液と混合されることを特徴とする請求項1記載のハイブリダイゼーション用バッファー組成物。
- 遺伝子多型における一方のアレルを含むターゲット核酸と、当該ターゲット核酸に対して相補的な塩基配列からなり、基板上に固定されてなる核酸プローブとのハイブリダイゼーション方法であって、
前記遺伝子多型における上記アレル以外のアレルを含み、上記ターゲット核酸を核酸増幅法により増幅する際に同時に増幅される非ターゲット核酸に対して相補的な塩基配列からなり、上記核酸プローブの長さの60%以上100%未満の長さであって15塩基以上の長さのブロッキング用核酸を含有するハイブリダイゼーション用バッファー組成物と、上記ターゲット核酸を含有する溶液とを混合し、その後、上記核酸プローブと上記ターゲット核酸とのハイブリダイズを行う、ハイブリダイゼーション方法。 - 上記核酸プローブは15〜25塩基長であり、上記ブロッキング用核酸の塩基長が15〜24塩基長であることを特徴とする請求項5記載のハイブリダイゼーション方法。
- 上記ハイブリダイゼーション用バッファー組成物は、クエン酸ナトリウム二水和物(SSC)及びドデシル硫酸ナトリウム(SDS)を更に含むことを特徴とする請求項5記載のハイブリダイゼーション方法。
- 上記ターゲット核酸を含有する溶液は、上記ターゲット核酸を増幅する核酸増幅反応の後の反応液であり、当該反応液と上記ハイブリダイゼーション用バッファー組成物と混合することを特徴とする請求項5記載のハイブリダイゼーション方法。
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