JP5849107B2 - 核酸合成反応の向上方法 - Google Patents
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Description
[1]核酸合成反応の反応性の向上方法であって、ω−アミノ酸を反応液に添加する工程を含む方法、
[2]ω−アミノ酸が、下記の式1で表される化合物である、[1]に記載の方法、
[3]式中nが2以上、7以下の整数である、[2]に記載の方法。
[4]核酸合成反応がポリメラーゼ連鎖反応である、[1]に記載の方法、
[5]ω−アミノ酸、DNAポリメラーゼ、少なくとも1種のプライマー、及び少なくとも1種のデオキシリボヌクレオチド三リン酸を含有する反応液を調製する工程を含む、[1]に記載の方法、
[6]さらに、ベタインを反応液に添加する工程を含む、[1]に記載の方法、
[7]核酸合成反応用の組成物であって、DNAポリメラーゼ、反応緩衝剤、少なくとも1種のプライマー、少なくとも1種のデオキシリボヌクレオチド三リン酸、及びω−アミノ酸を含有する組成物、
[8]ω−アミノ酸が、[2]に示される式1で表される化合物である、[7]に記載の組成物、
[9]さらに、ベタインを含有する、[7]に記載の組成物、
[10]ω−アミノ酸を含有する核酸合成反応用の反応緩衝液、
[11]ω−アミノ酸が、[2]に示される式1で表される化合物である、[10]に記載の反応緩衝液、
[12]さらに、ベタインを含有する、[10]に記載の反応緩衝液、
[13]核酸合成反応のためのキットであって、以下の構成品を含むキット:DNAポリメラーゼ、反応緩衝剤、少なくとも1種のデオキシリボヌクレオチド三リン酸、及びω−アミノ酸、
[14]ω−アミノ酸が、[2]に示される式1で表される化合物である、[13]に記載のキット、
[15]さらに、ベタインを含有する、[13]に記載のキット
に関する。
で表わされる化合物が例示され、なかでも前記の式1で表わされる化合物においてnが2以上、7以下の整数である化合物がより好適に例示される。
β―アラニン(SIGMA−ALDRICH社)を超純水(ミリQ水)に溶解した後、トリス酢酸緩衝液(pH8.9)を滴下することでpHを8.5に調整した5Mのβ−アラニン溶液を調製した。同様にして、6−アミノヘキサン酸(SIGMA−ALDRICH社)をミリQ水に溶解した後にpHを8.5に調整した2Mの6−アミノヘキサン酸溶液、及び8−アミノオクタン酸(SIGMA−ALDRICH社)をミリQ水に溶解した後にpHを8.5に調整した1Mの8−アミノオクタン酸溶液を調製した。また、ベタイン(トリメチルグリシン;SIGMA−ALDRICH社、B2629)をミリQ水に溶解し、5Mのベタイン溶液を調製した。これらを下記の反応液の調製に用いた。
TaKaRa Ex Taq(登録商標)(タカラバイオ社)に代えてPyrobest(登録商標) DNA Polymerase(タカラバイオ社)を用いる点、及びベタインを含む反応液についてベタインの終濃度が1MとなるようにPCR反応液を調製する点以外は実施例1と同様の方法で、ω−アミノ酸の効果を検討した。なお、Pyrobest(登録商標) DNA Polymeraseは、Pyrococcus sp.由来DNAポリメラーゼを含む製品である。
TGFβ1遺伝子領域を増幅するプライマー対に代えてHomo sapiens DNA, translocation breakpoint sequences on 22q11: Type C(GenBank:AB261999.1)領域の965bp(GC比率35.4%)を増幅するプライマー対(配列番号3の塩基配列からなるプライマー、及び配列番号4の塩基配列からなるプライマー)を使用する以外は、実施例1と同様の方法で、ω−アミノ酸の効果を確認した。
核酸合成反応の反応性の向上に有効な6−アミノヘキサン酸の濃度を検証した。各種のω−アミノ酸の代わりに終濃度2mM、5mM、10mM、20mM、50mM、100mM、200mM、又は300mMの6−アミノヘキサン酸を含む以外は実施例1と同様の反応液、及びω−アミノ酸を含まない対照としての20μLの反応液、の合わせて9種類の反応液を調製した。次に、実施例1と同様の方法でPCRを行った後、各反応液のうち4μLをアガロースゲル電気泳動に供して増幅産物の鎖長と増幅量を確認した。アガロースゲル電気泳動の結果を図4に示す。図中Mは、1kb DNA Ladder(タカラバイオ社)マーカー200ngの電気泳動を行ったレーンであることを示す。
TGFβ1遺伝子領域を増幅するプライマー対に代えてHomo sapiens DNA, translocation breakpoint sequences on 22q11: Type C(GenBank:AB261999.1)領域のプライマー対(配列番号3の塩基配列からなるプライマー、及び配列番号4の塩基配列からなるプライマー)を使用する以外は実施例4と同様の方法で、核酸合成反応の反応性の向上に有効な6−アミノヘキサン酸の有効濃度を検討した。
SEQ ID NO:2 ; Synthetic primer for PCR to amplify of TGF-beta 1 gene.
SEQ ID NO:3 ; Synthetic primer for PCR to amplify of translocation breakpoint sequence region.
SEQ ID NO:4 ; Synthetic primer for PCR to amplify of translocation breakpoint sequence region.
Claims (11)
- 式中nが2以上、7以下の整数である、請求項1に記載の方法。
- 核酸合成反応がポリメラーゼ連鎖反応である、請求項1に記載の方法。
- ω−アミノ酸、DNAポリメラーゼ、少なくとも1種のプライマー、及び少なくとも1種のデオキシリボヌクレオチド三リン酸を含有する反応液を調製する工程を含む、請求項1に記載の方法。
- さらに、ベタインを反応液に添加する工程を含む、請求項1に記載の方法。
- さらに、ベタインを含有する、請求項6に記載の組成物。
- さらに、ベタインを含有する、請求項8に記載の反応緩衝液。
- さらに、ベタインを含有する、請求項10に記載のキット。
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