JP5456240B2 - 配列要素の核酸への挿入 - Google Patents
配列要素の核酸への挿入 Download PDFInfo
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- JP5456240B2 JP5456240B2 JP2007121557A JP2007121557A JP5456240B2 JP 5456240 B2 JP5456240 B2 JP 5456240B2 JP 2007121557 A JP2007121557 A JP 2007121557A JP 2007121557 A JP2007121557 A JP 2007121557A JP 5456240 B2 JP5456240 B2 JP 5456240B2
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Classifications
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- C—CHEMISTRY; METALLURGY
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
-
- 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
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
Description
(a)鋳型核酸を準備する工程;
(b)少なくとも一つのアンカーオリゴヌクレオチドの少なくとも一つのアンカー配列を、鋳型核酸の少なくとも一つの配列断片とハイブリダイズする工程;および、
(c)鋳型核酸に対し部分的に相補的であり、かつ、アンカーオリゴヌクレオチドの非ハイブリダイズ部分、すなわち、少なくとも一つの鋳型タグ配列に対して相補的である配列をその3'末端に含む、新規核酸鎖を合成する工程を含み、それによって核酸の2本鎖領域が形成されることを特徴とする方法。
2)核酸の自由末端の連結であって、この過程は、天然の1本鎖または2本鎖特異的リガーゼ、またはその他のリガーゼ、例えばリボザイム、化学的連結、例えば、チオフォスフェートによるもの(米国特許第6,635,425号参照)、または、連結に好適な他の酵素、例えばトポイソメラーゼによる連結によって実施される。
3)核酸を配列特異的、または配列非特異的に切断/結合するヌクレアーゼ、例えば、エンドヌクレアーゼまたはエキソヌクレアーゼによる処理。
4)得られたRNAのその後の翻訳を伴う、または伴わないRNA合成。
5)DNA合成、例えば、新規に合成される核酸鎖の少なくとも一部に対して相補的な1本鎖核酸の合成、または、鋳型核酸の少なくとも一部の増幅。
6)核酸-核酸相互作用、例えば、新規に合成された核酸とアプタマーとの相互作用、または、新規に合成された核酸と、別の核酸、例えば、1個以上の特異的プライマーとのハイブリダイゼーション。
7)得られた核酸鎖に対する、例えば、タンパク結合によるある核酸分子の精製または濃縮による、ただしこれらに限定されないが、1種以上のタンパクの結合。
8)新規に合成された核酸鎖、またはその増幅産物の標識。
ポリメラーゼ:ポリメラーゼとは、ある核酸鎖内部の個別のヌクレオチド間においてフォスフォジエステル結合の形成を触媒する酵素である(例えば、DNAおよりRNAポリメラーゼ)。選択された実験条件の下で鎖置換活性をほとんど、または全く持たないポリメラーゼは全て、本発明の方法による工程(c)における使用にとって好適である。鎖置換活性が高すぎると、アンカーオリゴヌクレオチドが分裂される可能性があり、その場合、タグ配列の挿入が妨げられ、極端な場合、完全に阻止される恐れがある。従って、本発明の方法によれば、使用されるポリメラーゼは、重合工程において鎖置換性を全く持たないことが特に好ましい。ごく僅かな鎖置換活性を持つ他のポリメラーゼの外に、DNAポリメラーゼIのクレノウ断片、T4 DNAポリメラーゼ、T7 DNAポリメラーゼ、DNAポリメラーゼI、および、DNAおよびRNA依存性逆転写酵素が、この好ましいポリメラーゼに属する。ウィルス、細菌、古細菌、および真核細胞の酵素も後者に属し、これはさらに、イントロン、レトロトランスポゾン、およびレトロウィルス、例えば、MMLV、AMV、HIV由来の酵素も含む。しかしながら、本発明の方法によって合成核酸鎖の中にタグ配列を挿入するには、ごく僅かな鎖置換性しか持たない他のポリメラーゼも好適である。本発明による方法のいくつかの特異的実施態様では、僅かな鎖置換性しか持たないポリメラーゼと組み合わされるとさらに効果的に工夫することが可能である(下記参照)。本発明の意味における中等度の鎖置換活性とは、ポリメラーゼによる鎖置換の確率が、アンカーオリゴヌクレオチドの30%未満、好ましくは50%未満、特に好ましくは80%未満であることを指す。当業者にはよく知られるように、鎖置換確率は、反応温度、バッファー条件、それぞれのポリメラーゼ、およびアンカーオリゴヌクレオチドのハイブリダイズ割合の関数として変動する可能性がある。
(実施態様1)
(実施態様2)
(実施態様3)
(実施態様4)
(実施態様5)
(実施態様6)
(実施態様7)
(実施態様8)
(実施態様9)
(実施態様10)
(実施態様11)
を用いた。リアルタイムPRCは、下記のプライマーを用いて実行した。すなわち、
T7P-N8: CAATTCTAATACGACTCACTA TAGGGAGAAGGNNNNNNNNG
T7P: AATTCTAATACGACTCACTA TAGGGAGAAGG
β-actin: GTCTCAAGTCAGTGTACAGG
s-actin primer: gtctcaagtcagtgtacagg
gtga tagcattgctttcgtg
T7N8: gga tga cga cgc agt att g nnn nnn nn
T7N10: gga tga cga cgc agt att g nnn nnn nnn n
T7N12: gga tga cga cgc agt att g nnn nnn nnn nnn
T7 N6 T4: gga tta cga ctc agt att g nnnnnn tttt
s-actin primer: gtctcaagtcagtgtacagg
gtga tagcattgctttcgtg
T3: gga tga cga cgc agt att g nnnnnn ttt
T2: gga tga cga cgc agt att g nnnnnn tt
T1: gga tga cga cgc agt att g nnnnnn t
T0: gga tga cga cgc agt att g nnnnnn
s-actin primer: gtctcaagtcagtgtacagg
gtga tagcattgctttcgtg
N8: gga tga cga cgc agt att g nnn nnn nn
N10: gga tga cga cgc agt att g nnn nnn nnn n
N12: gga tga cga cgc agt att g nnn nnn nnn nnn
G2 N6: gga tga cga cgc agt att ggg nnn nnn
G4 N6: gga tga cga cgc agt att ggg gg nnn nnn
CAA TTC TAA TAC GAC TCA CTA TAG GGA GAA GGN NNN NNN N
T7P N8 block:
CAA TTC TAA TAC GAC TCA CTA TAG GGA GAA GGN NNN NNN N -dG-ref Q.
N8:
NNN NNN NN
CAA TTC TAA TAC GAC TCA CTA TAG GGA GAA GG
Claims (8)
- (a)鋳型核酸を準備する工程;
(b)少なくとも一つのアンカーオリゴヌクレオチドを、前記鋳型核酸の少なくとも一つの配列セクションとハイブリダイズさせる工程であって、
前記アンカーオリゴヌクレオチドは、前記少なくとも一つの配列セクションにハイブリダイズ可能なアンカー配列を3’末端領域に含み、かつ
前記アンカーオリゴヌクレオチドは、特定の配列を有する少なくとも1つの鋳型タグ配列を5’末端領域に含み、
該鋳型タグ配列は、前記鋳型核酸に対してアンカーオリゴヌクレオチドの非ハイブリダイズ部分である、工程;
(c)前記鋳型核酸に対し部分的に相補的であり、かつ、前記少なくとも一つの鋳型タグ配列に対して相補的な配列を3'末端領域に含む、新規核酸鎖を合成することにより、二本鎖核酸領域を含む核酸構造体を形成する工程であって、前記新規核酸鎖の合成は、僅かしか鎖置換活性を有しないポリメラーゼ又は全く鎖置換活性を有しないポリメラーゼの作用により、前記アンカーオリゴヌクレオチドの鋳型タグ配列と同一であるか、或いは部分的に同一である配列を5’末端領域に有するプライマーの伸長を介して実施されるものである、工程;並びに、
(d)工程(c)において形成した核酸構造体をさらに処理する工程であって、
当該核酸構造体の処理は、工程(c)において合成された新規核酸鎖を鋳型核酸から分離し、分離した新規核酸鎖の5’末端領域及び3’末端領域を自己ハイブリダイズさせることによりヘアピン構造体を形成させることを含む、工程
を含む、核酸の中に1個以上のタグ配列を挿入する方法。 - 前記アンカーオリゴヌクレオチドの鋳型タグ配列は、ハイブリダイゼーション部位、プライマー結合部位、プローブ結合部位、転写開始及び/又は翻訳開始のためのプロモーター又はシグナル配列、制限エンドヌクレアーゼ認識切断部位、リボソーム結合部位、タンパク結合部位、抗体認識部位、並びにそれらに相補的な配列から成る群から選択される、少なくとも1個の機能配列を含む、請求項1に記載の方法。
- 工程(d)において、工程(c)において合成された新規核酸鎖を鋳型核酸から分離する前に、前記核酸構造体を配列特異的エンドヌクレアーゼを用いてさらに処理する、請求項1又は2に記載の方法。
- 前記配列特定的エンドヌクレアーゼが、メチル化感受性制限エンドヌクレアーゼである、請求項3に記載の方法。
- 工程(d)において、さらに、前記ヘアピン構造体2つを互いに連結する、請求項1から4の何れか1項に記載の方法。
- 工程(d)において、前記ヘアピン構造体2つを互いに連結した結果、ダンベル型環状核酸分子が形成され、前記ダンベル型環状核酸分子をローリングサークル増幅法により増幅する、請求項5に記載の方法。
- 前記少なくとも1個の機能配列がRNAポリメラーゼプロモーターである、請求項2から6に記載の方法。
- 工程(d)において、さらに、インビトロ転写が実施される、請求項7に記載の方法。
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US20080057543A1 (en) | 2008-03-06 |
JP2007300921A (ja) | 2007-11-22 |
US8932831B2 (en) | 2015-01-13 |
EP1889925B1 (de) | 2012-08-22 |
EP1889925A1 (de) | 2008-02-20 |
DE102006020885A1 (de) | 2007-11-08 |
EP2316965A1 (de) | 2011-05-04 |
EP2316965B1 (de) | 2014-04-30 |
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