JP2009507478A - ポリメラーゼ反応のために核酸を活性化する方法 - Google Patents
ポリメラーゼ反応のために核酸を活性化する方法 Download PDFInfo
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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
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- 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
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Abstract
Description
(a)核酸を温度55℃から80℃へ加熱、
(b)核酸をポリメラーゼが活性に実質的低下を示さない温度へ冷却、および
(c)ポリメラーゼの核酸への添加によってポリメラーゼ反応を開始
:の段階を含むポリメラーゼ反応のために核酸を活性化する方法によってこの課題を達成する。
(a)核酸を熱安定性ポリメラーゼと共に温度55℃から80℃へ加熱
:の段階を含みうる。
本実施例は、単純な温度活性化段階によって、全血から開始するSDR(ここでは多置換増幅、MDA)が可能になり、DNA収量およびDNA品質に関しては先行技術に記載の反応(対照反応)と同等であるのを示す。
(プライマー配列:
Sat1.1 TCTTTCCACTCCATTGCAT および
Sat1.2 GGAATGGAATCAACCCAA
プライマー1 GTCTCAAGTCAGTGTACAGG
プライマー2 GTGATAGCATTGCTTTCGTG
(試験: TGATGGCATTACTGGCACTTTGAGTTTTAC,
プライマー1: GTCTTTAGCTGCTGAGGAAATG,
プライマー2: AGCAGAATTCTGCACATGACG) および
(試験: TGAACTGCTCCTTGGCAGGGATTT,
プライマー1: TGCTCCCTGTCCCATCTG,
プライマー2: AGACAGTATGCCTTTATTTCACCC)。
この実施例は、温度活性化段階中の低すぎる温度はSDR中に生じるDNAの品質を損ないうることを示す役割を果たす。
(試験: TGATGGCATTACTGGCACTTTGAGTTTTAC,
プライマー1: GTCTTTAGCTGCTGAGGAAATG および
プライマー2: AGCAGAATTCTGCACATGACG)、および
(試験: TGAACTGCTCCTTGGCAGGGATTT,
プライマー1: TGCTCCCTGTCCCATCTG,
プライマー2: AGACAGTATGCCTTTATTTCACCC)。
この実施例では、特定の温度限界内でSDR前の単純な温度活性化は、先行技術に記載の反応(対照反応)と比較して、SDR中に生じるDNAの品質に影響しないことが示される。
(試験: TGATGGCATTACTGGCACTTTGAGTTTTAC,
プライマー1: GTCTTTAGCTGCTGAGGAAATG,
プライマー2: AGCAGAATTCTGCACATGACG) および
(試験: TGAACTGCTCCTTGGCAGGGATTT,
プライマー1: TGCTCCCTGTCCCATCTG,
プライマー2: AGACAGTATGCCTTTATTTCACCC)。
(プライマー配列:
Sat1.1 TCTTTCCACTCCATTGCAT および
Sat1.2 GGAATGGAATCAACCCAA
プライマー1 GTCTCAAGTCAGTGTACAGG
プライマー2 GTGATAGCATTGCTTTCGTG
本実施例は、単純な温度活性化段階によって、単離されたゲノムDNAから開始するSDR(ここでは多置換増幅、MDA)が可能になり、DNA収量およびDNA品質に関しては先行技術に記載の反応(対照反応)と同等であるのを示す役割を果たす。
(試験: TGATGGCATTACTGGCACTTTGAGTTTTAC,
プライマー1: GTCTTTAGCTGCTGAGGAAATG,
プライマー2: AGCAGAATTCTGCACATGACG)。
この実施例では、漸増温度での単純な変性段階によって、特定配列がSDR(ここでは多置換反応)でもう増幅され得ないことを示す。
(試験: TGATGGCATTACTGGCACTTTGAGTTTTAC,
プライマー1: GTCTTTAGCTGCTGAGGAAATG,
プライマー2: AGCAGAATTCTGCACATGACG) および
(試験: TGAACTGCTCCTTGGCAGGGATTT,
プライマー1: TGCTCCCTGTCCCATCTG,
プライマー2: AGACAGTATGCCTTTATTTCACCC)。
(1)DNAの熱処理での温度漸増によって、漸増的に高いCT値が測定される。
(2)両方の遺伝子座は熱処理に際して異なる挙動を示す:遺伝子座699の場合、75℃または95℃での処理の値を比較すると、9.6サイクルのCtシフトが測定できた。いずれにしろ、遺伝子座1004の場合、別の5サイクルのCtシフトが測定される。すなわちリアルタイムPCRのために統一量の増幅産物(10ng)に導入されたにもかかわらず、DNAの熱処理での温度漸増によって、MDA増幅産物中で測定される遺伝子座はますます悪化した。
この実施例は、SDR(ここでは多置換増幅)において可能な限り良好な配列提示を達成するため、どの温度で最適な温度活性化段階が起こるかを示す。
(a)11/12という遺伝子座から生じる配列
(プライマー1: TTTCTGTAACAGCTAAGGAC,
プライマー2: TAGGGTGCTTAGCTGTTAAC) および
(b)665という遺伝子座から生じる配列
(プライマー1: CTCTTGCTCAGCCTATATAC,
プライマー2: GTAGAAAATGTAGCCCATTAC.
(1)DNAの熱処理での温度漸増によって、漸増的に高いCT値が測定される。
(2)CT最小値(つまりここで調べた遺伝子座の最良の提示)は温度65℃から85℃での熱処理で見られる。
(3)これらの遺伝子座では、KOHによる参照処理よりも良好なCt値が温度65〜85℃にて測定された。
(4)KOHによる参照処理よりも悪いCT値が85℃にて既に測定された比較実施例5から、最適処理温度は細胞内のゲノムの遺伝子座に依存すると推論できる。
Claims (8)
- (a)核酸を温度55℃から80℃へ加熱、
(b)温度4から45℃、好ましくは15℃から42℃、特に25から37℃へ冷却、および
(c)ポリメラーゼの核酸への添加によってポリメラーゼ反応を開始
:の段階を含むポリメラーゼ反応のために核酸を活性化する方法。 - 段階(c)でのポリメラーゼが熱不安定性ポリメラーゼである点で特徴づけられる、請求項1に記載の方法。
- ポリメラーゼ反応が鎖置換反応である点で特徴づけられる、請求項1または2に記載の方法。
- 鎖置換反応が多置換反応である点で特徴づけられる、請求項3に記載の方法。
- 核酸がDNAである点で特徴づけられる、前記請求項のうちの一つに記載の方法。
- 段階(a)での核酸が温度60℃から70℃、特に65℃へ加熱される点で特徴づけられる、前記請求項のうちの一つに記載の方法。
- 核酸が水溶液中に精製された形で存在する点で特徴づけられる、前記請求項のうちの一つに記載の方法。
- 核酸が細胞内に存在する点で特徴づけられる、請求項1から6のうちの一つに記載の方法。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP05019665.8 | 2005-09-09 | ||
EP05019665A EP1762627A1 (de) | 2005-09-09 | 2005-09-09 | Verfahren zur Aktivierung einer Nukleinsäure für eine Polymerase-Reaktion |
PCT/EP2006/066223 WO2007028833A2 (de) | 2005-09-09 | 2006-09-11 | Verfahren zur aktivierung einer nukleinsäure für eine polymerase-reaktion |
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JP2009507478A true JP2009507478A (ja) | 2009-02-26 |
JP2009507478A5 JP2009507478A5 (ja) | 2009-09-10 |
JP5330830B2 JP5330830B2 (ja) | 2013-10-30 |
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JP2008529643A Active JP5330830B2 (ja) | 2005-09-09 | 2006-09-11 | ポリメラーゼ反応のために核酸を活性化する方法 |
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US (1) | US9683255B2 (ja) |
EP (2) | EP1762627A1 (ja) |
JP (1) | JP5330830B2 (ja) |
AT (1) | ATE513928T1 (ja) |
WO (1) | WO2007028833A2 (ja) |
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GB201611469D0 (en) * | 2016-06-30 | 2016-08-17 | Lumiradx Tech Ltd | Improvements in or relating to nucleic acid amplification processes |
GB2569965A (en) | 2018-01-04 | 2019-07-10 | Lumiradx Uk Ltd | Improvements in or relating to amplification of nucleic acids |
CA3226451A1 (en) | 2021-07-21 | 2023-01-26 | Sam Reed | Method and system comprising a cartridge for sequencing target polynucleotides |
GB202110485D0 (en) | 2021-07-21 | 2021-09-01 | Dnae Diagnostics Ltd | Compositions, kits and methods for sequencing target polynucleotides |
GB202110479D0 (en) | 2021-07-21 | 2021-09-01 | Dnae Diagnostics Ltd | Compositions, kits and methods for sequencing target polynucleotides |
Citations (2)
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WO2007028833A3 (de) | 2007-08-02 |
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ATE513928T1 (de) | 2011-07-15 |
JP5330830B2 (ja) | 2013-10-30 |
US20090299047A1 (en) | 2009-12-03 |
US9683255B2 (en) | 2017-06-20 |
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