JP6572268B2 - 重合反応のための合成核酸 - Google Patents
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1241—Nucleotidyltransferases (2.7.7)
- C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
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Description
後続のPCRアッセイにおいて用いられる温度より低い温度でポリメラーゼ活性の抑制を測定した。
Claims (27)
- 熱安定性古細菌DNAポリメラーゼ、
3’末端および5’末端を有し、ヘアピン構造を形成する合成DNAオリゴヌクレオチドであって、該オリゴヌクレオチドはヘアピン構造を形成したときに3’末端から開始する二本鎖領域と5’一本鎖DNA突出部を有し、該5’一本鎖DNA突出部の一本鎖部分は、3’末端と対を形成している塩基に隣接する塩基から数えて4番目の位置にウラシル又はイノシンを含み、
調製物が25℃の温度であるときにおいて該ポリメラーゼを阻害し、72℃以上の温度において該ポリメラーゼを阻害しない、オリゴヌクレオチド、
およびバッファーを含む調製物。 - さらにプライマーを含み、少なくとも1つの二本鎖領域が、増幅反応におけるプライマーの融解温度より少なくとも10℃低い融解温度を有する、請求項1記載の調製物。
- 少なくとも1つの二本鎖領域が、90℃未満の融解温度を有する、請求項1または請求項2記載の調製物。
- 合成核酸が、複数の一本鎖領域を形成しうる、請求項1〜3のいずれか1項記載の調製物。
- 第2一本鎖領域が、スペーサーである、請求項4記載の調製物。
- 第3一本鎖領域が、合成核酸内の内部位置で一本鎖ループを形成する、請求項4又は5記載の調製物。
- ポリメラーゼ、dNTP、またはプライマーの少なくとも1つを更に含む、請求項1〜6のいずれか1項記載の調製物。
- スペーサーが、ヘキサ−エチレングリコールまたは1’,2’−ジデオキシリボースを含む、請求項1〜7のいずれか1項記載の調製物。
- 合成核酸が、3’末端の配列内に少なくとも1つの誘導体ヌクレオチドおよび/またはヌクレオチド結合を含有する、請求項1〜8のいずれか1項記載の調製物。
- 少なくとも1つの誘導体ヌクレオチドが、1以上の逆方向ヌクレオチド、ジ−デオキシヌクレオチドまたはアミノ修飾ヌクレオチドから選択される、請求項9記載の調製物。
- 少なくとも1つのヌクレオチド結合が、ホスホロチオアート結合である、請求項1〜9のいずれか1項記載の調製物。
- 合成核酸および熱安定性ポリメラーゼが、0.5:1〜10:1のモル比で存在する、請求項1〜11のいずれか1項記載の調製物。
- 野生型ポリメラーゼの変異体を更に含む、請求項1〜12のいずれか1項記載の調製物。
- 野生型ポリメラーゼの変異体が、配列番号25に対して少なくとも93%の配列同一性を含み、更に、配列番号25における278、307および/または402に対応するアミノ酸位置に少なくとも1つの突然変異を含む、野生型ポリメラーゼの変異体である請求項13記載の調製物。
- 野生型ポリメラーゼの変異体が、DNA結合ドメインに融合している、請求項14記載の調製物。
- 野生型ポリメラーゼの変異体のDNA結合ドメインが、Sso7dである、請求項14又は15記載の調製物。
- 野生型ポリメラーゼの変異体における、278、307および/または402に対応する位置の1以上におけるアミノ酸が、ヒスチジンではなく、場合により、DNA結合タンパク質に融合していてもよい、請求項14〜16のいずれか1項記載の調製物。
- 野生型ポリメラーゼの変異体が、H278Q、H307R、H402Qに対応する突然変異の群から選択される1以上の突然変異を更に含み、場合により、DNA結合タンパク質に融合していてもよい、請求項14〜17のいずれか1項記載の調製物。
- (a)少なくとも1つのポリメラーゼ、標的DNAおよびdNTPを含有する混合物に、請求項1〜13のいずれか1項記載の調製物を加え、
(b)標的DNAの伸長または増幅の前に、合成DNAオリゴヌクレオチドの二本鎖部分の融解温度より低い温度で混合物を維持することを含む、ポリメラーゼ伸長反応を抑制する方法であって、
ウラシルまたはイノシンが、合成DNAオリゴヌクレオチドの5’一本鎖DNA突出部において、3’末端と対形成している塩基に隣接する塩基から数えて4番目の位置に位置する、方法。 - 合成核酸の二本鎖部分の融解温度が、90℃未満である、請求項19記載の方法。
- 合成核酸が、複数の一本鎖領域を形成しうる、請求項19または20記載の方法。
- 第2一本鎖領域が、スペーサーである、請求項21記載の方法。
- 第3一本鎖領域が、合成核酸内の内部位置でループを形成する、請求項21又は22記載の方法。
- スペーサーが、ヘキサ−エチレングリコールまたは1’,2’−ジデオキシリボースを含む、請求項22記載の方法。
- 3’末端が、誘導体ヌクレオチドおよび/またはヌクレオチド結合を含有する、請求項19〜24のいずれか1項記載の方法。
- 誘導体ヌクレオチドが、1以上の逆方向ヌクレオチド、ジ−デオキシヌクレオチドまたはアミノ修飾ヌクレオチドから選択される、請求項25記載の方法。
- ヌクレオチド結合が、ホスホロチオアート結合である、請求項25記載の方法。
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US5830714A (en) * | 1996-04-17 | 1998-11-03 | Molecular Biology Resources, Inc. | Biologically active fragment of bacillus stearothermophilus DNA polymerase |
US6238865B1 (en) * | 1997-10-17 | 2001-05-29 | Guangtian Chen | Simple and efficient method to label and modify 3′-termini of RNA using DNA polymerase and a synthetic template with defined overhang nucleotides |
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