JP6491458B2 - 核酸増幅方法 - Google Patents
核酸増幅方法 Download PDFInfo
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- JP6491458B2 JP6491458B2 JP2014223027A JP2014223027A JP6491458B2 JP 6491458 B2 JP6491458 B2 JP 6491458B2 JP 2014223027 A JP2014223027 A JP 2014223027A JP 2014223027 A JP2014223027 A JP 2014223027A JP 6491458 B2 JP6491458 B2 JP 6491458B2
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Classifications
-
- 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
- C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
-
- 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
-
- 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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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biophysics (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
光応答性核酸を該第1の核酸に相補的に会合させることにより、該第1の核酸から該第2の核酸を解離させる工程と、
解離した第2の核酸に第1プライマーを相補的に会合させる工程と、
DNA合成酵素によって第1プライマーを起点として相補鎖を伸長させる工程と
を含み、実質的に等温条件下で二重鎖核酸の増幅を行う、核酸増幅方法を提供する。
光応答性核酸を該第1の核酸に相補的に会合させることにより、該第1の核酸から該第2の核酸を解離させる工程と、
該第1の波長とは異なる波長の第2の波長の光の照射によって光応答性核酸を該第1の核酸に会合できない形態にする工程と、
光応答性核酸と該第1の核酸との会合を解離させる工程と、
解離した第1の核酸に第2プライマーを相補的に会合させる工程と、
DNA合成酵素によって第2プライマーを起点として相補鎖を伸長させる工程と
を含み、実質的に等温条件下で二重鎖核酸の増幅を行う、核酸増幅方法を提供する。
本実施例では、光応答性核酸を用いて、光照射に依存する核酸増幅反応を行うことができるか否かについて評価した。
光応答性核酸として、2', 6'-ジメチルアゾベンゼンを2塩基ごとに1つ結合させた一本鎖DNAを、つくばオリゴサービス株式会社に委託して合成した。この光応答性核酸の配列を以下に示す。
5’- CT(Z)TT(Z)AA(Z)GA(Z)AG(Z)GA(Z)GA(Z)TA(Z)TA(Z)CC(Z)TG(Z)AG(Z)TG(Z)AT(Z)CT(Z)
AG(Z)TG(Z)TA(Z)CT(Z)TA -3’(配列番号1)
5’- CTTTAAGAAGGAGATATACCTGAGTGATCTAGTGTACTTAGTATGCTTCC -3’(配列番号2)
第1プライマー:5'- GGAAGCATACTAAGTACACT -3'(配列番号3)
第2プライマー:5’-TexasRed- CTTTAAGAAGGAGATATACC -3'(配列番号4)
以下の4種の試料を2つずつ調製した。
・鋳型DNA(終濃度10 nM)および光応答性核酸(終濃度500 nM)を含む試料
・鋳型DNA(終濃度10 nM)を含むが、光応答性核酸を含まない試料
・光応答性核酸(終濃度500 nM)を含むが、鋳型DNAを含まない試料
・鋳型DNAおよび光応答性核酸を含まない試料
i) チューブを、60℃に加熱したステンレス製チューブラックに移し、水銀ランプ(超高圧UVランプUSH-1030L、オリンパス株式会社)を光源として、可視光フィルター(U-MNIBA3、オリンパス株式会社)を通過させた光(波長470〜495 nm)をチューブの上部から1分間照射した。
ii) チューブを、60℃に加熱したサーマルサークラーに戻して、1分間インキュベートした。
iii) チューブを、60℃に加熱したステンレス製チューブラックに移し、水銀ランプ(超高圧UVランプUSH-1030L、オリンパス株式会社)を光源として、紫外光フィルター(U-MWU2、オリンパス株式会社)を通過させた光(波長330〜385 nm)をチューブの上部から1分間照射した。
iv) チューブを、60℃に加熱したサーマルサークラーに戻して、1分間インキュベートした。
図8に示されるように、光応答性核酸が存在しないか、または、光照射がない条件下では、鋳型DNAが存在していても、DNAの増幅を示すシグナルは検出できなかった。これに対して、光応答性核酸の存在下で光照射時において、鋳型DNAに特異的なDNA合成が起こることを確認した(図8中の矢印部参照)。よって、光照射に依存する核酸増幅反応を行うことができること示された。
本実施例では、光応答性核酸を用いた核酸増幅反応が、光照射の回数に依存して進行するか否かを検証した。
本実施例では、上記の実施例1と同じ光応答性核酸、鋳型DNA、第1プライマーおよび第2プライマーを用いた。また、実施例1と同様に、鎖交換促進物質としてPLL-g-Dex(PLLの分子量8000、グラフト率90%)を用い、DNA合成酵素として、Bst DNAポリメラーゼ(ニューイングランドバイオラボ社)を用いた。なお、反応溶液には、添付の10xThermoPolバッファー(ニューイングランドバイオラボ社)を希釈して用いた。
1xThermoPolバッファー溶液、鋳型DNA(終濃度10 nM)、光応答性核酸(終濃度500 nM)、第1プライマー(終濃度500 nM)、第2プライマー(終濃度500 nM)、PLL-g-Dex(終濃度78μM)およびBst DNAポリメラーゼ(終濃度0.4 unit/μL)を含む試料を4つ調製した。
Claims (9)
- 第1の波長の光の照射によって光応答性核酸を、一本鎖である第1の核酸と、一本鎖であり且つ前記第1の核酸と相補的な配列を有する第2の核酸とから構成される二重鎖核酸の前記第1の核酸に会合可能な形態にする工程と、
光応答性核酸を前記第1の核酸に相補的に会合させることにより、前記第1の核酸から前記第2の核酸を解離させる工程と、
解離した第2の核酸に第1プライマーを相補的に会合させる工程と、
DNA合成酵素によって第1プライマーを起点として相補鎖を伸長させる工程と、
前記第1の波長とは異なる波長の第2の波長の光の照射によって光応答性核酸を前記第1の核酸に会合できない形態にする工程と、
光応答性核酸と前記第1の核酸との会合を解離させる工程と、
解離した第1の核酸に第2プライマーを相補的に会合させる工程と、
DNA合成酵素によって第2プライマーを起点として相補鎖を伸長させる工程と
を含み、
実質的に等温条件下且つ鎖交換反応促進物質の存在下で、前記全ての工程を繰り返すことにより二重鎖核酸の増幅を行う、核酸増幅方法。 - 光応答性核酸が、アゾベンゼンおよびジメチルアゾベンゼンから選択される少なくとも1種を1つ以上結合させた核酸である請求項1に記載の核酸増幅方法。
- 前記第1の波長の光を照射し、前記アゾベンゼンおよびジメチルアゾベンゼンから選択される少なくとも1種を、シス体からトランス体に変形させることにより、前記光応答性核酸を第1の核酸に会合可能な形態にする、請求項2に記載の方法。
- 前記第2の波長の光を照射し、前記アゾベンゼンおよびジメチルアゾベンゼンから選択される少なくとも1種を、トランス体からシス体に変形させることにより、前記光応答性核酸を第1の核酸に会合できない形態にする、請求項2又は3に記載の方法。
- 鎖交換反応促進物質が、カチオン性ホモポリマーおよびカチオン性コポリマーから選択される少なくとも1種である請求項1〜4のいずれか1項に記載の核酸増幅方法。
- カチオン性コポリマーが、ポリLリジン−デキストラン共重合体(PLL-g-Dex)である請求項5に記載の核酸増幅方法。
- 光応答性核酸がジメチルアゾベンゼンを結合させた核酸であり、鎖交換反応促進物質がPLL-g-Dexである請求項1〜6のいずれか1項に記載の核酸増幅方法。
- DNA合成酵素が、鎖置換型DNAポリメラーゼである請求項1〜7のいずれか1項に記載の核酸増幅方法。
- 鎖置換型DNAポリメラーゼが、Bst DNAポリメラーゼある請求項8に記載の核酸増幅方法。
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