JP7563679B2 - Gene amplification method and gene amplification kit - Google Patents
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
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- 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
- C12Q1/686—Polymerase chain reaction [PCR]
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Description
本発明は、遺伝子増幅法及び遺伝子増幅用キット等に関する。 The present invention relates to a gene amplification method and a gene amplification kit, etc.
遺伝子を増幅する技術は、医療分野において病原菌の検査、がん診断などに、ライフサイエンス分野においては、食中毒菌の検査、水質検査、遺伝子組換え作物検査などに広く産業利用されている。遺伝子増幅は、目的遺伝子をテンプレートとしＤＮＡポリメラーゼと作用させ、生成された目的遺伝子を電気泳動と染色で検出する方法が知られている。また、簡便な遺伝子増幅を確認する方法として、反応副産物であるピロリン酸を測定することで目的遺伝子の増幅を判別する方法が使用されている。 Gene amplification technology is widely used industrially in the medical field for testing for pathogens and cancer diagnosis, and in the life science field for testing for food poisoning bacteria, water quality testing, and genetically modified crop testing. A known method of gene amplification is to use the target gene as a template and react it with DNA polymerase, and then detect the generated target gene by electrophoresis and staining. Another simple method of confirming gene amplification is to determine the amplification of the target gene by measuring the reaction by-product pyrophosphate.
反応副産物であるピロリン酸の測定法として蛍光試薬で検出する方法があり、リアルタイムＰＣＲなどの検出系として実用化されている。しかし、該方法は、蛍光試薬を使用するため高価となること、蛍光物質の影響を受けやすいことが知られている。他のピロリン酸の測定方法として集積化トランジスタを用いた方法がある。該方法は、目的遺伝子増幅反応で副生したピロリン酸から生じるプロトンをｐＨセンサで捉え検出することで遺伝子増幅を評価する方法であり、ＤＮＡシーケンサの検出系としても実用化されている。しかしながら、該方法では、遺伝子増幅反応のプロトンを捉える必要があるため、反応液中の緩衝能を極力抑える必要があり、その為、酵素反応を繰り返すと遺伝子増幅反応で産生したプロトンが蓄積することで反応液が酸性に傾き酵素の至適ｐＨから乖離し、酵素活性が低下、遺伝子増幅が低下することが知られている（非特許文献１）。 As a method for measuring pyrophosphate, which is a by-product of the reaction, there is a method for detecting it using a fluorescent reagent, which has been put to practical use as a detection system for real-time PCR and the like. However, this method is known to be expensive because it uses a fluorescent reagent, and to be easily affected by fluorescent substances. Another method for measuring pyrophosphate is a method using an integrated transistor. This method is a method for evaluating gene amplification by capturing and detecting protons generated from pyrophosphate by-products in the target gene amplification reaction with a pH sensor, and has also been put to practical use as a detection system for DNA sequencers. However, in this method, since it is necessary to capture protons from the gene amplification reaction, it is necessary to suppress the buffer capacity in the reaction solution as much as possible, and therefore, it is known that when the enzyme reaction is repeated, the protons produced in the gene amplification reaction accumulate, causing the reaction solution to become acidic and deviating from the optimal pH for the enzyme, resulting in a decrease in enzyme activity and a decrease in gene amplification (Non-Patent Document 1).
そこでこれら課題を解決する方法として、ピロリン酸を特異的に認識するピリジルボロン酸を用いた方法がある。該方法では、蛍光試薬を使用する必要がなく安価であり、また、酸性条件下でピロリン酸とピリジルボロン酸が作用するため、遺伝子増幅反応で産生したプロトンが蓄積しても問題なく検出できる。しかし、ピリジルボロン酸がピロリン酸と結合できるｐＨの範囲が５～７であり、一般的な遺伝子増幅反応が、アルカリ側で行われていること、また、酸性条件下では、遺伝子の脱プリン化が起こりやすいことが知られており（非特許文献２）、脱プリン化が起こった遺伝子では、ＤＮＡポリメラーゼが作用できず、その為、遺伝子増幅が起こらない。このことから、酸性条件下で遺伝子増幅を行える有効な方法が未だないため、本方法は活用されていない状況である。 To solve these problems, there is a method using pyridylboronic acid that specifically recognizes pyrophosphate. This method is inexpensive because it does not require the use of fluorescent reagents, and since pyrophosphate and pyridylboronic acid act under acidic conditions, the accumulation of protons produced in the gene amplification reaction can be detected without any problems. However, it is known that the pH range in which pyridylboronic acid can bind to pyrophosphate is 5 to 7, and general gene amplification reactions are carried out on the alkaline side, and that depurination of genes is likely to occur under acidic conditions (Non-Patent Document 2), and DNA polymerase cannot act on genes that have undergone depurination, and therefore gene amplification does not occur. For this reason, there is still no effective method for gene amplification under acidic conditions, and this method has not been utilized.
その他にも、遺伝子増幅については多く検討されており、これらの方法の多くは、アルカリ側での反応であるが、その中でｐＨが７以下における遺伝子増幅に関する記載のある公知文献がある（特許文献１、２、３）。しかし、該公知文献では、酸性条件下で遺伝子増幅を行うことが目的ではないため、酸性条件下でＤＮＡポリメラーゼを反応させる具体的な組成や条件が示されてなく、実際に酸性条件下で遺伝子増幅を行った例については全く記載されていない。Many other methods for gene amplification have been studied, and while most of these methods involve reactions on the alkaline side, there are known documents that describe gene amplification at a pH of 7 or less (Patent Documents 1, 2, and 3). However, since the purpose of these known documents is not to perform gene amplification under acidic conditions, they do not disclose specific compositions or conditions for reacting DNA polymerase under acidic conditions, and there is no description of any examples of gene amplification actually being performed under acidic conditions.
また、酸性条件に至適ｐＨが６であるＤＮＡポリメラーゼを用いて遺伝子増幅の検討が行なわれている（非特許文献３）。しかしながら、該方法では、遺伝子増幅の最適ｐＨは８.２であり、ｐＨ７.４では遺伝子増幅が認められなかった。酵素の至適ｐＨと遺伝子増幅の至適ｐＨは異なっており、酸性条件に至適ｐＨを有するＤＮＡポリメラーゼであれば、酸性条件で遺伝子増幅ができるわけではない。以上のように、酸性条件下でピロリン酸を検出するのに有用なピリジルボロン酸を用いた方法等を活用できるような、実際に酸性条件下でのＤＮＡポリメラーゼを用いた遺伝子増幅法は未だない状況である。 In addition, gene amplification has been studied using a DNA polymerase with an optimal pH of 6 under acidic conditions (Non-Patent Document 3). However, in this method, the optimal pH for gene amplification is 8.2, and gene amplification was not observed at pH 7.4. The optimal pH of the enzyme and the optimal pH for gene amplification are different, and a DNA polymerase with an optimal pH under acidic conditions does not necessarily enable gene amplification under acidic conditions. As described above, there is still no gene amplification method using DNA polymerase under actual acidic conditions that can utilize methods using pyridylboronic acid, which are useful for detecting pyrophosphate under acidic conditions.
本発明は、上記の遺伝子増幅に関する従来技術に於ける様々な問題点を解決し、酸性条件下でＤＮＡポリメラーゼを用いて、測定対象の遺伝子を選択的且つ簡便に増幅する方法等を提供することを目的とする。The present invention aims to solve the various problems in the conventional techniques related to gene amplification described above, and to provide a method for selectively and easily amplifying the gene to be measured using DNA polymerase under acidic conditions.
発明者らは、ＤＮＡポリメラーゼを用いて、測定対象の遺伝子を増幅する方法を詳細に検討した結果、予想外にも、酸性条件下での遺伝子増幅反応を有意に行うことを可能とする、緩衝液の種類及び各種塩濃度等の遺伝子増幅反応条件が存在することを見出し、本発明を完成させた。The inventors conducted detailed studies on methods for amplifying the genes to be measured using DNA polymerase, and unexpectedly discovered that there exist gene amplification reaction conditions, such as types of buffers and various salt concentrations, that enable significant gene amplification reactions under acidic conditions, and thus completed the present invention.
本発明は、以下の［１］～［８］の態様に関する。
［１］ｐＨ４.２～６.９の酸性反応液中でのＤＮＡポリメラーゼ反応で対象遺伝子を増幅させる工程（Ｉ）を含む、遺伝子増幅法。
［２］工程（Ｉ）で用いる酸性反応液をｐＨ４.０～６．９の緩衝液を用いて調製することを特徴とする、態様［１］に記載の遺伝子増幅法。
［３］工程（Ｉ）で用いる酸性反応液が、５ｍＭ～１２５ｍＭの塩化カリウムを含むことを特徴とする、態様［２］に記載の遺伝子増幅法。
［４］工程（Ｉ）で用いるＤＮＡポリメラーゼが、Ｔｈｅｒｍｕｓ属、Ｔｈｅｒｍｏｃｏｃｃｕｓ属、又は、Ｂａｃｉｌｌｕｓ属バクテリオファージ由来のＤＮＡポリメラーゼであることを特徴とする、態様［１］に記載の遺伝子増幅法。
［５］工程（Ｉ）で用いる酸性反応液が糖類を含むことを特徴とする、態様［１］に記載の遺伝子増幅法。
［６］態様［１］～［５］のいずれか一項に記載の工程（Ｉ）、及び、該工程（Ｉ）で得られた増幅産物又は反応副産物に基づき前記対象遺伝子を検出する工程（ＩＩ）を含む、遺伝子検出法。
［７］工程（ＩＩ）に於いて、該工程（Ｉ）で生じた反応副産物の量を測定し、該反応副産物の測定量に基づき前記対象遺伝子の量を決定することを特徴とする、遺伝子検出法。
［８］反応副産物がピロリン酸である、態様［７］に記載の遺伝子検出法。
［９］工程（ＩＩ）に於いて、ピリジルボロン酸との反応による電位変化を測定することによって工程（Ｉ）で生じた反応ピロリン酸の量を測定する、態様［８］に記載の遺伝子検出法。
［１０］工程（ＩＩ）に於いて、吸光度法により工程（Ｉ）で生じたピロリン酸の量を測定する、態様［８］に記載の遺伝子検出法。
［１１］態様［１］～［５］のいずれか一項に記載の遺伝子増幅法を実施するための遺伝子増幅用キットであって、ＤＮＡポリメラーゼ、緩衝剤、及び反応試薬を含む、前記遺伝子増幅用キット。
［１２］態様［６］～［１０］のいずれか一項に記載の遺伝子検出法を実施するための遺伝子検出装置またはシステムであって、態様［１１］に記載の遺伝子増幅用キットを含む、前記遺伝子検出装置またはシステム。
The present invention relates to the following aspects [1] to [8].
[1] A gene amplification method comprising the step (I) of amplifying a target gene by a DNA polymerase reaction in an acidic reaction solution having a pH of 4.2 to 6.9.
[2] The gene amplification method according to aspect [1], wherein the acidic reaction solution used in step (I) is prepared using a buffer solution having a pH of 4.0 to 6.9.
[3] The gene amplification method according to aspect [2], wherein the acidic reaction solution used in the step (I) contains 5 mM to 125 mM potassium chloride.
[4] The gene amplification method according to aspect [1], wherein the DNA polymerase used in step (I) is derived from a bacteriophage belonging to the genus Thermus, Thermococcus, or Bacillus.
[5] The gene amplification method according to aspect [1], wherein the acidic reaction solution used in step (I) contains a saccharide.
[6] A gene detection method comprising the step (I) according to any one of aspects [1] to [5], and a step (II) of detecting the target gene based on the amplification product or reaction by-product obtained in the step (I).
[7] A gene detection method, comprising: in step (II), measuring the amount of a reaction by-product produced in step (I); and determining the amount of the target gene based on the measured amount of the reaction by-product.
[8] The gene detection method according to embodiment [7], wherein the reaction by-product is pyrophosphate.
[9] The gene detection method according to embodiment [8], wherein in step (II), the amount of reacted pyrophosphate produced in step (I) is measured by measuring a change in potential due to the reaction with pyridylboronic acid.
[10] The gene detection method according to embodiment [8], wherein in step (II), the amount of pyrophosphate produced in step (I) is measured by absorbance spectrometry.
[11] A gene amplification kit for carrying out the gene amplification method according to any one of aspects [1] to [5], comprising a DNA polymerase, a buffer, and a reaction reagent.
[12] A gene detection device or system for carrying out the gene detection method according to any one of aspects [6] to [10], comprising the gene amplification kit according to aspect [11].
従来技術では、酸性条件下で測定対象の遺伝子を選択的に増幅させ、該遺伝子を検出することが不可能であったが、本発明に係る遺伝子増幅法に於いては、従来技術で見られたような遺伝子の脱プリン化も見られず、酸性条件下でもＤＮＡポリメラーゼの作用により測定対象遺伝子を有意に増幅させることが出来る。更に、引き続き、増幅反応に使用した酸性反応液を用いて、該遺伝子を選択的且つ簡便に検出又は測定することが可能となる。 In conventional techniques, it was impossible to selectively amplify and detect the gene to be measured under acidic conditions, but in the gene amplification method of the present invention, depurination of the gene, as seen in conventional techniques, is not observed, and the gene to be measured can be significantly amplified by the action of DNA polymerase even under acidic conditions. Furthermore, it is possible to subsequently selectively and easily detect or measure the gene using the acidic reaction solution used in the amplification reaction.
本発明は、第一に、遺伝子増幅法に係る。本発明の遺伝子増幅法の工程（Ｉ）では、ｐＨ４.２～６.９、例えば、Ｔｈｅｒｍｕｓ属及びＴｈｅｒｍｏｃｏｃｃｕｓ属由来のＤＮＡポリメラーゼの場合、好ましくは、ｐＨ５．７～６．９又は、Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓバクテリオファージ由来のＤＮＡポリメラーゼの場合、好ましくは、ｐＨ５.０～６.５の範囲にある（酸性）反応液中でのＤＮＡポリメラーゼ反応において、測定の対象となる遺伝子（ＤＮＡ）を増幅させる。尚、上記の酸性反応液のｐＨは遺伝子増幅反応が進むにつれて多少変動するので、遺伝子増幅反応開始時のｐＨで規定する。本発明方法に使用するＤＮＡポリメラーゼは、原核生物、真核生物及びウィルス由来の何れのＤＮＡポリメラーゼでも良く、例えば、Ｂａｃｉｌｌｕｓ属、Ｔｈｅｒｍｕｓ属、Ｐｙｒｏｃｏｃｃｕｓ属、Ｔｈｅｒｍｏｃｏｃｃｕｓ属、Ｓｕｌｆｏｌｏｂｕｓ属などの微生物由来やＢａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓバクテリオファージなどのウィルス由来のＤＮＡポリメラーゼが好ましい。また、組換え型ＤＮＡポリメラーゼでも良く、合成したＤＮＡポリメラーゼでも良い。可溶性酵素が好ましいが、不溶性酵素に界面活性剤を組み合わせても良く、可溶化タンパクとの融合又は膜結合部分の削除等により不溶性酵素を可溶化させた酵素でも良い。ＤＮＡポリメラーゼの公知のアミノ酸配列を利用でき、組換え型のＤＮＡポリメラーゼとしては、公知のＤＮＡポリメラーゼと６０％、６５％、７０％、７５％、８０％、８５％、９０％又は９５％以上の同一性を有するアミノ配列を有し、ＤＮＡポリメラーゼ活性を有する蛋白質を使用しても良い。尚、本発明の遺伝子増幅法に於いては、特定のｐＨ範囲の酸性反応液中でのＤＮＡポリメラーゼ反応において、測定対象の遺伝子の増幅を繰り返し行うことによって、ピロリン酸等の反応産生物(反応副産物)を増加させ、その後、該反応副産物を係る酸性ｐＨ条件下で測定することが可能となる。従って、このような反応液のｐＨの範囲に於いて有意な遺伝子増幅活性を有するＤＮＡポリメラーゼを使用することが好ましい。尚、既に述べたように、ＤＮＡポリメラーゼ自体の至適ｐＨと遺伝子増幅活性の至適ｐＨとは必ずしも一致していない。First, the present invention relates to a gene amplification method. In step (I) of the gene amplification method of the present invention, the genera (DNA) to be measured are amplified in a DNA polymerase reaction in an (acidic) reaction solution having a pH of 4.2 to 6.9, for example, in the case of DNA polymerase derived from the genera Thermus and Thermococcus, preferably a pH of 5.7 to 6.9, or in the case of DNA polymerase derived from Bacillus subtilis bacteriophage, preferably a pH of 5.0 to 6.5. Note that the pH of the acidic reaction solution varies somewhat as the gene amplification reaction proceeds, and is therefore defined as the pH at the start of the gene amplification reaction. The DNA polymerase used in the method of the present invention may be any DNA polymerase derived from a prokaryote, eukaryote or virus, and is preferably a DNA polymerase derived from a microorganism such as the genera Bacillus, Thermus, Pyrococcus, Thermococcus, or Sulfolobus, or a virus such as the bacteriophage Bacillus subtilis. It may also be a recombinant DNA polymerase or a synthetic DNA polymerase. Soluble enzymes are preferred, but insoluble enzymes may be combined with a surfactant, or insoluble enzymes may be solubilized by fusion with a solubilizing protein or deletion of the membrane-binding portion. A known amino acid sequence of DNA polymerase can be used, and as a recombinant DNA polymerase, a protein having an amino acid sequence with 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% identity with a known DNA polymerase and having DNA polymerase activity may be used. In the gene amplification method of the present invention, the gene to be measured is amplified repeatedly in a DNA polymerase reaction in an acidic reaction solution of a specific pH range, thereby increasing reaction products (reaction by-products) such as pyrophosphate, and then the reaction by-products can be measured under the acidic pH conditions. Therefore, it is preferable to use a DNA polymerase that has significant gene amplification activity in the pH range of such a reaction solution. As already mentioned, the optimal pH of the DNA polymerase itself does not necessarily coincide with the optimal pH of the gene amplification activity.
本発明の遺伝子増幅法に使用するＤＮＡポリメラーゼの調製方法としては、当業者に公知の任意の方法・手段、例えば、ＤＮＡポリメラーゼを含む対象物に加水し、粉砕機、超音波破砕機などで粉砕後、破砕した破砕物を遠心分離、濾過などで固形物を取り除いた抽出物、さらに当該抽出物をカラムクロマトグラフィーなどにより精製又は単離する方法があり、その精製又は単離したＤＮＡポリメラーゼなどを用いることができる。The DNA polymerase used in the gene amplification method of the present invention can be prepared by any method or means known to those skilled in the art, such as adding water to a target material containing DNA polymerase, crushing it using a grinder, ultrasonic crusher, or the like, and then extracting the crushed material by centrifuging, filtration, or the like to remove solid matter, and further purifying or isolating the extract by column chromatography or the like, and the purified or isolated DNA polymerase can be used.
当該遺伝子増幅反応に使用される酸性反応液中のＤＮＡポリメラーゼ濃度は、試料の種類、推定される試料中の遺伝子濃度及び、反応時間・温度等の各種反応条件に応じて、当業者が適宜決められる。例えば、遺伝子増幅反応液中のＢａｃｉｌｌｕｓ属、Ｔｈｅｒｍｕｓ属、Ｐｙｒｏｃｏｃｃｕｓ属、Ｔｈｅｒｍｏｃｏｃｃｕｓ属、Ｓｕｌｆｏｌｏｂｕｓ属などの微生物由来やＢａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓバクテリオファージなどのウィルス由来のＤＮＡポリメラーゼの濃度は、０.５μｇ/ｍＬ以上、より好ましくは１μｇ/ｍＬ以上、さらに好ましくは３μｇ/ｍＬ以上とすることができる。いずれにしても、本発明方法では、ＤＮＡポリメラーゼを繰り返し使用できるので、予想される試料中の遺伝子に対して、過剰量のＤＮＡポリメラーゼを添加する必要はない、という利点を有する。従って、ＤＮＡポリメラーゼ濃度の上限は、経済性なども考慮して当業者が適宜設定することが出来る。The concentration of DNA polymerase in the acidic reaction solution used in the gene amplification reaction can be appropriately determined by a person skilled in the art depending on the type of sample, the estimated gene concentration in the sample, and various reaction conditions such as reaction time and temperature. For example, the concentration of DNA polymerase derived from microorganisms such as Bacillus, Thermus, Pyrococcus, Thermococcus, and Sulfolobus, or derived from viruses such as Bacillus subtilis bacteriophage, in the gene amplification reaction solution can be 0.5 μg/mL or more, more preferably 1 μg/mL or more, and even more preferably 3 μg/mL or more. In any case, the method of the present invention has the advantage that since DNA polymerase can be used repeatedly, it is not necessary to add an excessive amount of DNA polymerase to the expected genes in the sample. Therefore, the upper limit of the DNA polymerase concentration can be appropriately set by a person skilled in the art, taking into account economic efficiency, etc.
本発明の遺伝子増幅法における対象遺伝子（ＤＮＡ）は、当業者に公知の任意の方法で・手段で取得することが出来る。例えば、血液、生鮮食品、加工食品及び飲料など検査の目的に応じた試料から適宜調製することが出来る。各試料中からのＤＮＡ調製方法としては、当業者に公知の任意の方法・手段、例えば、試料をドデシル硫酸ナトリウム、酵素、ビーズ破壊などで溶解させ、得られた溶解液中のＤＮＡをシリカ膜などに結合後、溶出液でＤＮＡを溶出させるなどにより調製したＤＮＡを遺伝子増幅用のＤＮＡ試料として使用することができる。更に、増幅反応の種類などに応じて、ＤＮＡはｃＤＮＡであっても良い。The target gene (DNA) in the gene amplification method of the present invention can be obtained by any method or means known to those skilled in the art. For example, it can be prepared appropriately from samples such as blood, fresh food, processed food, and beverages depending on the purpose of the test. The method for preparing DNA from each sample can be any method or means known to those skilled in the art, for example, dissolving the sample with sodium dodecyl sulfate, enzymes, bead destruction, etc., binding the DNA in the resulting lysate to a silica membrane, etc., and then eluting the DNA with an elution solution, etc., to prepare DNA that can be used as a DNA sample for gene amplification. Furthermore, depending on the type of amplification reaction, etc., the DNA may be cDNA.
本発明の遺伝子増幅法で使用する特定のｐＨ範囲の酸性反応液は、ＤＮＡポリメラーゼの種類や反応条件等に応じて、例えば、ｐＨ４.０～６．９、好ましくはｐＨ５.０～６.５、より好ましくはｐＨ５．５～６．５等の適当なｐＨ範囲の緩衝液を用いて当業者が適宜調製することが出来る。該緩衝液としては、当業者に公知の任意の緩衝剤、好ましくは、ＮａＨ２ＰＯ４－Ｎａ２ＨＰＯ４、ＮａＨ２ＰＯ４－ＮａＯＨ、ＮａＨ２ＰＯ４－ＫＯＨなどのリン酸塩緩衝剤を用いて調製するリン酸塩緩衝液やクエン酸－クエン酸ナトリウム、クエン酸－ＮａＯＨなどのクエン酸緩衝剤を用いて調製するクエン酸緩衝液などを使用することが出来る。酸性反応液中の緩衝剤の（終）濃度に関しては、例えば、Ｔｈｅｒｍｏｃｏｃｃｕｓ属由来のＤＮＡポリメラーゼをｐＨ５.５の緩衝液を用いて調製した反応液中で反応させる場合、該反応液中のリン酸塩の濃度は、０.１～２００ｍＭがよく、より好ましくは０.５～１００ｍＭ、さらに好ましくは５～５０ｍＭがよい。このような緩衝剤の濃度の上限は、経済性なども考慮して当業者が適宜設定することが出来る。尚、酸性反応液の最終容量に対して、通常、１～１０倍濃度の緩衝剤を１～２５容量％程度を使用することによって上記のｐＨ範囲の酸性反応液を調製することが出来る。 The acidic reaction solution having a specific pH range used in the gene amplification method of the present invention can be prepared by a person skilled in the art using a buffer having an appropriate pH range, for example, pH 4.0 to 6.9, preferably pH 5.0 to 6.5, more preferably pH 5.5 to 6.5, depending on the type of DNA polymerase, reaction conditions, etc. As the buffer, any buffer known to a person skilled in the art can be used, preferably a phosphate buffer prepared using a phosphate buffer such as NaH 2 PO 4 -Na 2 HPO 4 , NaH 2 PO 4 -NaOH, or NaH 2 PO 4 -KOH, or a citrate buffer prepared using a citrate buffer such as citric acid-sodium citrate or citric acid-NaOH. Regarding the (final) concentration of the buffer in the acidic reaction solution, for example, when a DNA polymerase derived from the genus Thermococcus is reacted in a reaction solution prepared using a buffer solution of pH 5.5, the concentration of phosphate in the reaction solution is preferably 0.1 to 200 mM, more preferably 0.5 to 100 mM, and even more preferably 5 to 50 mM. The upper limit of the concentration of such a buffer can be appropriately set by a person skilled in the art, taking into consideration economic efficiency, etc. An acidic reaction solution in the above pH range can usually be prepared by using about 1 to 25 volume % of a buffer having a concentration of 1 to 10 times the final volume of the acidic reaction solution.
本発明の遺伝子増幅法で使用する酸性反応液には塩化カリウムが含まれていることが好ましい。該反応液中の塩化カリウムの濃度は、ＤＮＡポリメラーゼの種類や反応条件に応じて、当業者が適宜決められる。アルカリ側ｐＨで反応させる従来の一般的な遺伝子増幅反応においては、塩化カリウム濃度が７５ｍＭ以上になると遺伝子増幅反応が阻害されることが知られているが、本発明方法に於ける酸性条件下での遺伝子増幅反応においては、塩化カリウム濃度は、使用する反応液のｐＨが酸性になるほど、過剰となるように添加するのが好ましい。例えば、Ｔｈｅｒｍｕｓ属由来のＤＮＡポリメラーゼをｐＨ６.０の緩衝液を用いて調製した酸性反応液中で反応させる場合、該反応液中の塩化カリウムの濃度は、５～１１５ｍＭがよく、より好ましくは３０～１１５ｍＭがよい。例えば、Ｔｈｅｒｍｏｃｏｃｃｕｓ属由来のＤＮＡポリメラーゼをｐＨ５.５の緩衝液を用いて調製した酸性反応液中で反応させる場合、該反応液中の塩化カリウムの終濃度は、５～１１５ｍＭがよく、より好ましくは３０～１１５ｍＭがよい。更に、例えば、Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓバクテリオファージ由来のＤＮＡポリメラーゼをｐＨ４.０の緩衝液を用いて調製した酸性反応液中で反応させる場合、該反応液中の塩化カリウムの終濃度は、５～１２５ｍＭがよく、より好ましくは３０～１２５ｍＭがよい。塩化カリウムの濃度は、緩衝剤の濃度を考慮し、緩衝剤が高濃度の場合、塩化カリウム濃度は低濃度とし、緩衝剤が低濃度の場合、塩化カリウム濃度は高濃度とするのがよい。また、塩化カリウム濃度の上限は、経済性なども考慮して当業者が適宜設定することが出来る。The acidic reaction solution used in the gene amplification method of the present invention preferably contains potassium chloride. The concentration of potassium chloride in the reaction solution can be appropriately determined by those skilled in the art depending on the type of DNA polymerase and the reaction conditions. In conventional general gene amplification reactions that are carried out at alkaline pH, it is known that gene amplification reactions are inhibited when the potassium chloride concentration is 75 mM or more. However, in gene amplification reactions under acidic conditions in the method of the present invention, the more acidic the pH of the reaction solution used, the more potassium chloride is preferably added in excess. For example, when a DNA polymerase derived from the genus Thermus is reacted in an acidic reaction solution prepared using a buffer solution of pH 6.0, the concentration of potassium chloride in the reaction solution is preferably 5 to 115 mM, more preferably 30 to 115 mM. For example, when a DNA polymerase derived from the genus Thermococcus is reacted in an acidic reaction solution prepared using a buffer solution of pH 5.5, the final concentration of potassium chloride in the reaction solution is preferably 5 to 115 mM, more preferably 30 to 115 mM. Furthermore, for example, when a DNA polymerase derived from a Bacillus subtilis bacteriophage is reacted in an acidic reaction solution prepared using a buffer solution of pH 4.0, the final concentration of potassium chloride in the reaction solution is preferably 5 to 125 mM, more preferably 30 to 125 mM. The concentration of potassium chloride should be determined taking into consideration the concentration of the buffering agent, and when the buffering agent is at a high concentration, the potassium chloride concentration should be low, and when the buffering agent is at a low concentration, the potassium chloride concentration should be high. The upper limit of the potassium chloride concentration can be appropriately set by those skilled in the art, taking into consideration economical efficiency and the like.
本発明の遺伝子増幅法で使用する酸性反応液には更に二価イオンが含まれる。該二価イオンとしては、マグネシウム、マンガン、コバルトなどが使用できるが、マグネシウムを使用するのが好ましい。当該反応に使用される反応液中のマグネシウムの濃度は、適宜決められる。例えば、Ｔｈｅｒｍｏｃｏｃｃｕｓ属由来のＤＮＡポリメラーゼをｐＨ５.５の緩衝液で反応させる場合、該反応液中のマグネシウム濃度は、１.５～９ｍＭがよく、より好ましくは２～６ｍＭがよい。例えば、Ｔｈｅｒｍｕｓ属由来のＤＮＡポリメラーゼをｐＨ６.０の緩衝液を用いて調製した酸性反応液中で反応させる場合、該反応液中のマグネシウム濃度は、２～６ｍＭがよく、より好ましくは２～４ｍＭがよい。例えば、Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓバクテリオファージなどのウィルス由来のＤＮＡポリメラーゼをｐＨ４.０の緩衝液を用いて調製した酸性反応液中で反応させる場合、該反応液中のマグネシウム濃度は、１～３０ｍＭがよく、より好ましくは１０～１４ｍＭがよい。The acidic reaction solution used in the gene amplification method of the present invention further contains divalent ions. Magnesium, manganese, cobalt, etc. can be used as the divalent ion, but magnesium is preferably used. The concentration of magnesium in the reaction solution used in the reaction can be appropriately determined. For example, when a DNA polymerase derived from the genus Thermococcus is reacted in a buffer solution of pH 5.5, the magnesium concentration in the reaction solution is preferably 1.5 to 9 mM, more preferably 2 to 6 mM. For example, when a DNA polymerase derived from the genus Thermus is reacted in an acidic reaction solution prepared using a buffer solution of pH 6.0, the magnesium concentration in the reaction solution is preferably 2 to 6 mM, more preferably 2 to 4 mM. For example, when a DNA polymerase derived from a virus such as Bacillus subtilis bacteriophage is reacted in an acidic reaction solution prepared using a buffer solution of pH 4.0, the magnesium concentration in the reaction solution is preferably 1 to 30 mM, more preferably 10 to 14 mM.
本発明の遺伝子増幅法では、遺伝子を増幅させるため、デオキシアデノシン三リン酸（ｄＡＴＰ）、デオキシシチジン三リン酸（ｄＣＴＰ）、デオキシチミジン三リン酸（ｄＴＴＰ）、デオキシグアノシン三リン酸（ｄＧＴＰ）の４種が混合されたデオキシリボヌクレオチド三リン酸（ｄＮＴＰ）を用いる。当該反応に使用される反応液中のｄＮＴＰの濃度は、各種反応条件に応じて、当業者が適宜決められるが、例えば、ｄＮＴＰ濃度は、０.０１ｍＭ以上がよく、より好ましくは０.１ｍＭ以上、さらに好ましくは０.２ｍＭ以上とすることができる。In the gene amplification method of the present invention, deoxyribonucleotide triphosphates (dNTPs) containing a mixture of four types of deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxythymidine triphosphate (dTTP), and deoxyguanosine triphosphate (dGTP) are used to amplify genes. The concentration of dNTPs in the reaction solution used in the reaction can be appropriately determined by those skilled in the art depending on various reaction conditions, but for example, the dNTP concentration is preferably 0.01 mM or more, more preferably 0.1 mM or more, and even more preferably 0.2 mM or more.
更に、本発明の遺伝子増幅法では、遺伝子を増幅させるために各種のプライマーを用いる。該プライマーは増幅の対象とする遺伝子の具体的なヌクレオチド配列に基づき、当業者が適宜、設計・調製することが出来る。当該反応に使用される反応液中のプライマーの濃度は、各種反応条件に応じて、当業者が適宜決められるが、例えば、プライマー濃度は、０.０１ｍＭ以上、より好ましくは０.１ｍＭ以上、さらに好ましくは０.２ｍＭ以上とすることができる。Furthermore, in the gene amplification method of the present invention, various primers are used to amplify genes. The primers can be appropriately designed and prepared by those skilled in the art based on the specific nucleotide sequence of the gene to be amplified. The concentration of the primers in the reaction solution used in the reaction can be appropriately determined by those skilled in the art depending on various reaction conditions, but for example, the primer concentration can be 0.01 mM or more, more preferably 0.1 mM or more, and even more preferably 0.2 mM or more.
本発明の遺伝子増幅法の工程（Ｉ）における遺伝子増幅反応は、当業者に公知の任意の方法、例えば、各種のポリメラーゼ連鎖反応（ＰＣＲ）等で行われ、該反応の各サイクルに於ける温度や時間は、遺伝子増幅反応が生じるような任意の温度で良い。例えば、Ｔｈｅｒｍｕｓ属及びＴｈｅｒｍｏｃｏｃｃｕｓ属のＤＮＡポリメラーゼを用いる場合では、９８℃・１０ｓｅｃ→５５℃・３０ｓｅｃ→７２℃・１ｍｉｎを繰り返すことや、９４℃・３ｍｉｎ→９４℃・３０ｓｅｃ→６２℃・３０ｓｅｃ→７２℃・３０ｓｅｃを繰り返した後、７２℃・１０ｍｉｎとすることなどできる。Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓバクテリオファージ由来のＤＮＡポリメラーゼを用いる場合では、９５℃・１ｍｉｎ加熱、３０℃まで冷却した後、３０℃・１６ｈｒ→６５℃・１０ｍｉｎ処理することなどできる。酸性条件下では、高温ほど遺伝子の脱プリン化が起こりやすいことから低温で遺伝子増幅を行うことが好ましい。The gene amplification reaction in step (I) of the gene amplification method of the present invention can be carried out by any method known to those skilled in the art, such as various polymerase chain reactions (PCR), and the temperature and time in each cycle of the reaction can be any temperature at which the gene amplification reaction occurs. For example, when using DNA polymerases of the genus Thermus and Thermococcus, the reaction can be repeated from 98°C for 10 sec to 55°C for 30 sec to 72°C for 1 min, or from 94°C for 3 min to 94°C for 30 sec to 62°C for 30 sec to 72°C for 30 sec, followed by 72°C for 10 min. When using DNA polymerase derived from Bacillus subtilis bacteriophage, the reaction can be heated at 95°C for 1 min, cooled to 30°C, and then treated at 30°C for 16 hr to 65°C for 10 min. Under acidic conditions, gene amplification is preferably carried out at low temperatures because the higher the temperature, the more likely depurination of the gene occurs.
また、本発明の遺伝子増幅法で使用する酸性反応液に糖類を含有させる（共存させる）ことによって、更に、反応産生物の量を増加させることが出来る。糖類としては、ショ糖、トレハロースなど当業者に公知の糖類が使用できる。当該反応に使用される反応液中の糖類の濃度は、各種反応条件に応じて、当業者が適宜決められる。例えば、Ｔｈｅｒｍｏｃｏｃｃｕｓ属由来のＤＮＡポリメラーゼをｐＨ５.５の緩衝液で反応させる場合、該反応液中の糖類濃度は、１０％以下がよく、より好ましくは５％以下がよい。例えば 、Ｔｈｅｒｍｕｓ属由来のＤＮＡポリメラーゼをｐＨ６.０の緩衝液を用いて調製した酸性反応液中で反応させる場合、該反応液中の糖類濃度は、１％以上がよく、より好ましくは５％以上がよい。例えば、Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓバクテリオファージなどのウィルス由来のＤＮＡポリメラーゼをｐＨ４.０の緩衝液を用いて調製した酸性反応液中で反応させる場合、該反応液中の糖類濃度は、０.１％以上がよく、より好ましくは１％以上がよい。 In addition, the amount of reaction product can be further increased by adding (coexisting) sugars to the acidic reaction solution used in the gene amplification method of the present invention. As the sugar, sugars known to those skilled in the art, such as sucrose and trehalose, can be used. The concentration of sugars in the reaction solution used in the reaction can be appropriately determined by those skilled in the art depending on various reaction conditions. For example, when a DNA polymerase derived from the genus Thermococcus is reacted in a buffer solution of pH 5.5, the sugar concentration in the reaction solution is preferably 10% or less, more preferably 5% or less. For example, when a DNA polymerase derived from the genus Thermus is reacted in an acidic reaction solution prepared using a buffer solution of pH 6.0, the sugar concentration in the reaction solution is preferably 1% or more, more preferably 5% or more. For example, when a DNA polymerase derived from a virus such as Bacillus subtilis bacteriophage is reacted in an acidic reaction solution prepared using a buffer solution of pH 4.0, the sugar concentration in the reaction solution is preferably 0.1% or more, more preferably 1% or more.
さらに、本発明の遺伝子増幅法で使用する酸性反応液に牛血清アルブミン（ＢＳＡ）、非イオン製剤、アンモニウムイオンを添加する（共存させる）ことによって、酵素やＤＮＡの安定性を増し、反応産生物の量を増加させることが出来る。ＢＳＡの濃度は、各種反応条件に応じて、当業者が適宜決められる。例えば、Ｔｈｅｒｍｏｃｏｃｃｕｓ属由来のＤＮＡポリメラーゼをｐＨ５.５の緩衝液で反応させる場合、該反応液中のＢＳＡ濃度は、０.００１％以上がよく、より好ましくは０.００５％以上、さらに好ましくは０.０１％以上がよい。非イオン製剤としては、ＮＰ-４０、ＴｒｉｔｏｎＸ-１００など当業者に公知の非イオン製剤が使用できる。当該反応に使用される反応液中の非イオン製剤の濃度は、各種反応条件に応じて、当業者が適宜決められる。例えば、Ｔｈｅｒｍｏｃｏｃｃｕｓ属由来のＤＮＡポリメラーゼをｐＨ５.５の緩衝液で反応させる場合、該反応液中の非イオン製剤濃度は、０.０００５％以上がよく、より好ましくは０.００１％以上、さらに好ましくは０.０１％以上がよい。また、アンモニウムイオンとしては、硫酸アンモニウムなど当業者に公知のアンモニウムイオンが使用できる。当該反応に使用される反応液中のアンモニウムイオンの濃度は、各種反応条件に応じて、当業者が適宜決められる。例えば、Ｔｈｅｒｍｏｃｏｃｃｕｓ属由来のＤＮＡポリメラーゼをｐＨ５.５の緩衝液で反応させる場合、反応液中のアンモニウムイオン濃度は、１２０ｍＭ以下がよく、より好ましくは１００ｍＭ以下、さらに好ましくは８０ｍＭ以下がよい。Furthermore, by adding (coexisting) bovine serum albumin (BSA), a non-ionic preparation, and ammonium ions to the acidic reaction solution used in the gene amplification method of the present invention, the stability of the enzyme and DNA can be increased and the amount of the reaction product can be increased. The concentration of BSA can be appropriately determined by those skilled in the art depending on various reaction conditions. For example, when a DNA polymerase derived from the genus Thermococcus is reacted in a buffer solution of pH 5.5, the concentration of BSA in the reaction solution is preferably 0.001% or more, more preferably 0.005% or more, and even more preferably 0.01% or more. As the non-ionic preparation, non-ionic preparations known to those skilled in the art, such as NP-40 and TritonX-100, can be used. The concentration of the non-ionic preparation in the reaction solution used in the reaction can be appropriately determined by those skilled in the art depending on various reaction conditions. For example, when a DNA polymerase derived from the genus Thermococcus is reacted in a buffer solution at pH 5.5, the concentration of the non-ionic preparation in the reaction solution is preferably 0.0005% or more, more preferably 0.001% or more, and even more preferably 0.01% or more. In addition, ammonium ions known to those skilled in the art, such as ammonium sulfate, can be used as the ammonium ion. The concentration of ammonium ions in the reaction solution used in the reaction can be appropriately determined by those skilled in the art according to various reaction conditions. For example, when a DNA polymerase derived from the genus Thermococcus is reacted in a buffer solution at pH 5.5, the concentration of ammonium ions in the reaction solution is preferably 120 mM or less, more preferably 100 mM or less, and even more preferably 80 mM or less.
本発明方法の工程（Ｉ）で使用する反応試薬・酵素等の各反応成分は、所定の酸性条件下で遺伝子増幅反応が生じる添加方法である限り、当業者に公知の任意の手段・手順等で反応系に添加することができる。例えば、各成分を反応開始前に一度に反応液に予め添加するか、又は、ＤＮＡポリメラーゼ又は遺伝子を含む試料を最後に添加し反応させても良い。従って、例えば、本願明細書の実施例にあるように、塩化カリウム、二価イオン、及び、糖類等を予めＰＣＲ用緩衝液に含有させておくことが出来る。 The reaction components such as reaction reagents and enzymes used in step (I) of the method of the present invention can be added to the reaction system by any means or procedure known to those skilled in the art, as long as the method of addition allows a gene amplification reaction to occur under predetermined acidic conditions. For example, each component can be added to the reaction solution all at once before the start of the reaction, or a sample containing DNA polymerase or a gene can be added last and reacted. Therefore, for example, potassium chloride, divalent ions, sugars, etc. can be added to the PCR buffer solution in advance, as in the examples of this specification.
本発明は、第二に、ｐＨ４．２～６.９の酸性反応液中でのＤＮＡポリメラーゼ反応で対象遺伝子を増幅させる工程（Ｉ）の後に、該工程（Ｉ）で得られた増幅産物又は反応副産物に基づき前記対象遺伝子を検出する工程（ＩＩ）を含む、遺伝子検出法に係る。
増幅産物又は反応副産物に基づく対象遺伝子の検出は、当業者に公知の任意の方法・手段を用いて、定性的、半定量的、又は、定量的に実施することが出来る。
例えば、増幅産物である増幅遺伝子を電気泳動した後、これを染色することによって、定性的又は半定量的に検出することが出来る。又は、反応副産物であるピロリン酸を蛍光試薬で検出する方法もある。
或いは、工程（Ｉ）で生じたピロリン酸及び水素イオン等の反応副産物の夫々の量を測定し、該反応副産物の測定量に基づき対象遺伝子の量を決定する（定量的に検出する)ことが出来る。この方法では、反応副産物の量と測定対象遺伝子の量との一定の相関関係に基づく検量線等を利用することができる。特に、本発明の工程（Ｉ）で生じたピロリン酸の量の測定には、当業者に公知の任意の方法・手段を使用することができる。特に、本発明の工程（Ｉ）では酸性条件下で測定対象遺伝子の増幅が可能であるので、酸性条件下で行われる、ピロリン酸を特異的に検出するピリジルボロン酸により電位変化を測定する方法を効果的に使用することが出来る。また、本発明の工程（Ｉ）で生じたピロリン酸をヒポキサンチン－グアニンホスホリボシルトランスフェラーゼ、キサンチンオキシダーゼ又はキサンチンデヒドロゲナーゼを組み合わせた方法や、ピロリン酸を無機ピロホスファターゼなどで２分子のリン酸とし、そのリン酸を測定することで、より高感度の測定、例えば、ルミノールと無機ピロホスファターゼ、ピルビン酸オキシダーゼ及びペルオキシダーゼを組み合わせた方法などにより、ピロリン酸を吸光度法で測定できる。さらに遺伝子増幅反応に於いてピロリン酸から生じるプロトン（水素イオン）の測定には、水素イオンを検出するガラス電極やイオン感応性電界効果トランジスタにより電位変化を測定する測定方法などを使用することができる。本発明の工程（Ｉ）で生じたピロリン酸、水素イオンなどは、適宜、遺伝子増幅反応溶液から分離し、測定することができる。該反応溶液からのピロリン酸、水素イオンなどの分離方法としては、測定に影響の無い方法であれば特に限定されないが、例えば、ペーパークロマトグラフィー分離、マイクロ流体デバイスでの分離などが挙げられる。
Secondly, the present invention relates to a gene detection method comprising step (I) of amplifying a gene of interest by a DNA polymerase reaction in an acidic reaction solution having a pH of 4.2 to 6.9, and then step (II) of detecting the gene of interest based on the amplification product or reaction by-product obtained in step (I).
Detection of the target gene based on the amplification product or reaction by-product can be carried out qualitatively, semi-quantitatively, or quantitatively using any method or means known to those skilled in the art.
For example, the amplified gene, which is the amplification product, can be subjected to electrophoresis and then stained to allow qualitative or semi-quantitative detection. Alternatively, pyrophosphate, which is a reaction by-product, can be detected with a fluorescent reagent.
Alternatively, the amounts of the reaction by-products such as pyrophosphate and hydrogen ions generated in step (I) can be measured, and the amount of the target gene can be determined (quantitatively detected) based on the measured amount of the reaction by-product. In this method, a calibration curve based on a certain correlation between the amount of the reaction by-product and the amount of the gene to be measured can be used. In particular, any method or means known to those skilled in the art can be used to measure the amount of pyrophosphate generated in step (I) of the present invention. In particular, since the gene to be measured can be amplified under acidic conditions in step (I) of the present invention, a method of measuring a change in potential using pyridylboronic acid, which specifically detects pyrophosphate, performed under acidic conditions can be effectively used. In addition, pyrophosphate generated in step (I) of the present invention can be measured by absorptiometry using a method in which hypoxanthine-guanine phosphoribosyltransferase, xanthine oxidase, or xanthine dehydrogenase is combined with pyrophosphate to convert it into two molecules of phosphate and then the resulting phosphate is measured, for example, a method in which luminol is combined with inorganic pyrophosphatase, pyruvate oxidase, and peroxidase to obtain a more sensitive measurement. Furthermore, for measuring protons (hydrogen ions) generated from pyrophosphate in a gene amplification reaction, a measurement method in which a potential change is measured using a glass electrode that detects hydrogen ions or an ion-sensitive field effect transistor can be used. Pyrophosphate, hydrogen ions, and the like generated in step (I) of the present invention can be appropriately separated from the gene amplification reaction solution and measured. The method for separating pyrophosphate, hydrogen ions, and the like from the reaction solution is not particularly limited as long as it does not affect the measurement, and examples thereof include paper chromatography separation and separation using a microfluidic device.
更に、本発明は上記の本発明方法を実施するための、対象遺伝子を増幅するに必要な前述の各成分、例えば、ＤＮＡポリメラーゼ、緩衝剤、反応試薬（プライマー及びｄＮＴＰ等）並びに、糖類等の各種添加剤等を含む、遺伝子増幅用キットを提供する。当該キットは、安定化剤又は緩衝剤等の当業者に公知の他の任意成分を適宜含有させ、前記酵素等試薬成分の安定性を高めても良い。測定に影響の無い成分であれば特に限定されないが、例えば、卵白アルブミン、糖アルコール類、カルボキシル基含有化合物、酸化防止剤、界面活性剤等を例示できる。Furthermore, the present invention provides a gene amplification kit for carrying out the above-mentioned method of the present invention, which includes the above-mentioned components necessary for amplifying a target gene, such as DNA polymerase, a buffer, reaction reagents (primers, dNTPs, etc.), and various additives such as sugars. The kit may contain other optional components known to those skilled in the art, such as stabilizers or buffers, as appropriate, to enhance the stability of the enzyme and other reagent components. There are no particular limitations on the components as long as they do not affect the measurement, and examples of such components include ovalbumin, sugar alcohols, carboxyl group-containing compounds, antioxidants, surfactants, etc.
又、本発明は、当該遺伝子増幅用キットを含み、上記遺伝子検出法（測定法）を実施するための遺伝子検出装置または遺伝子検出システムを提供する。係る遺伝子検出装置または遺伝子検出システムには、当該遺伝子増幅用キットに加えて、本発明方法に於ける対象遺伝子の増幅工程（Ｉ）で得られた増幅産物又は反応副産物に基づき該遺伝子を検出する工程（ＩＩ）を実施する様々な手段・方法に応じて、それらの各手段・方法に必要とされる当該技術分野に於いて公知である任意の試薬、装置、器具及びキット等を適宜含むことができる。The present invention also provides a gene detection device or gene detection system that includes the gene amplification kit and is used to carry out the gene detection method (measurement method). In addition to the gene amplification kit, the gene detection device or gene detection system may appropriately include any reagents, devices, instruments, kits, etc. that are known in the technical field and are required for each of the various means and methods for carrying out step (II) of detecting a gene based on an amplification product or reaction by-product obtained in step (I) of amplifying a target gene in the method of the present invention.
以下、実施例によって本発明を具体的に説明するが、本発明の技術的範囲は以下の実施例によって限定されるものではない。The present invention will be described in detail below with reference to examples, but the technical scope of the present invention is not limited to the following examples.
（超好熱菌由来のポリメラーゼの調製）
Ｔｈｅｒｍｏｃｏｃｃｕｓ ｗａｉｏｔａｐｕｅｎｓｉｓ由来のＤＮＡポリメラーゼ遺伝子（非特許文献３）を組み込んだプラスミドｐＥＴ２８ｂ（＋）で大腸菌Ｒｏｓｅｔｔａ ２(ＤＥ３)ｐＬｙｓＳを形質転換し、発現株として用いた。発現株について、カナマイシンを終濃度５０μg／ｍＬ及びクロラムフェニコールを終濃度３４μｇ/ｍＬ含むＬＢ培地により３７℃で４時間培養後、終濃度０．２ｍＭとなるようにＩＰＴＧを添加した。さらに、培養液を２５℃で終夜培養後、集菌を行い、得られた菌体を超音波破砕し、無細胞抽出液を調製した。調製した無細胞抽出液について遠心分離を行い、得られた上清の一部を用いて電気泳動法により目的酵素の発現を確認した。次いで残りの上清をアフィニティカラム（商品名：ＨｉＴｒａｐ Ｈｅｐａｒｉｎ ＨＰ、ＧＥヘルスケア製）により夾雑タンパクを除去し、０.１５ｍｇ／ｍＬのＴｈｅｒｍｏｃｏｃｃｕｓ属由来のＤＮＡポリメラーゼを得た。なお、Ｔｈｅｒｍｕｓ属由来のＤＮＡポリメラーゼはタカラバイオ製（商品名：Ｅｘ Ｔａｑ ＨＳ）、Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓバクテリオファージ由来のＤＮＡポリメラーゼは関東化学製（商品名：ｐｈｉ２９ ＤＮＡポリメラーゼ）を用いた。
(Preparation of polymerase derived from hyperthermophilic bacteria)
Escherichia coli Rosetta 2 (DE3) pLysS was transformed with plasmid pET28b (+) incorporating a DNA polymerase gene (Non-Patent Document 3) derived from Thermococcus waiotapuensis, and used as an expression strain. The expression strain was cultured at 37 ° C. for 4 hours in LB medium containing kanamycin at a final concentration of 50 μg / mL and chloramphenicol at a final concentration of 34 μg / mL, and then IPTG was added to a final concentration of 0.2 mM. Furthermore, after overnight culture at 25 ° C., the cells were collected, and the obtained cells were ultrasonically disrupted to prepare a cell-free extract. The prepared cell-free extract was centrifuged, and the expression of the target enzyme was confirmed by electrophoresis using a portion of the obtained supernatant. The remaining supernatant was then passed through an affinity column (product name: HiTrap Heparin HP, manufactured by GE Healthcare) to remove contaminating proteins, thereby obtaining 0.15 mg/mL of Thermococcus-derived DNA polymerase. Thermus-derived DNA polymerase was manufactured by Takara Bio (product name: Ex Taq HS), and Bacillus subtilis bacteriophage-derived DNA polymerase was manufactured by Kanto Chemical (product name: phi29 DNA polymerase).
尚、上記Ｔｈｅｒｍｏｃｏｃｃｕｓ属由来のポリメラーゼＤＮＡの塩基配列は以下のとおりである。
［Ｔｈｅｒｍｏｃｏｃｃｕｓ属由来のＤＮＡポリメラーゼ ］
ATGGGGATCCTGGACGCAGACTACATTACGGAAGATGGCAAGCCGGTCATTCGTGTGTTCAAGAAAGAAAAGGGCGAATTCAAAATCAATTATGATCGTGACTTTGAACCGTATATTTACGCTCTGCTGAAAGATGACAGCGCGATCGAAGATATTAAAAAGATCACCGCTGAACGTCACGGTACCACGGTCCGTGTGACGCGCGCCGAACGTGTTAAAAAGAAATTTCTGGGTCGCCCGGTTGAAGTCTGGAAACTGTATTTCACCCATCCGCAGGATGTGCCGGCTATTCGTGACAAAATCCGCGAACACCCGGCGGTGGTTGATATTTATGAATACGACATCCCGTTTGCAAAGCGTTATCTGATTGATAAAGGCCTGATCCCGATGGAGGGTAACGAAGAACTGCGCATGCTGGCGTTTGACATTGAAACCCTGTACCATGAAGGCGAAGAATTCGGCGAAGGTCCGATTCTGATGATCAGCTATGCGGATGAAGAAGGTGCCCGTGTGATTACCTGGAAAAATATCGACCTGCCGTATGTTGAAAGTGTCTCCACGGAAAAAGAAATGATTAAGCGCTTTCTGAAAGTGATCCAGGAAAAAGATCCGGACGTTCTGATTACCTATAACGGCGATAATTTTGACTTCGCGTACCTGAAGAAACGTTCAGAAACGCTGGGTGTTAAGTTCATTCTGGGCCGCGATGGTTCGGAACCGAAAATCCAACGTATGGGCGACCGCTTTGCCGTGGAAGTTAAAGGTCGCATCCACTTCGATCTGTACCCGGTGATTCGTCGCACCATCAACCTGCCGACCTATACGCTGGAAACGGTGTACGAAGCCATTTTTGGCCAGCCGAAAGAAAAGGTTTATGCAGAAGAAATCGCACAAGCTTGGGAAAGTGGCGAAGGTCTGGAACGCGTTGCCCGTTATTCCATGGAAGATGCGAAGGCCACCTACGAACTGGGTAAAGAATTTTTCCCGATGGAAGCACAGCTGAGCCGTCTGGTCGGCCAAAGCCTGTGGGATGTGTCTCGCAGCTCTACCGGTAACCTGGTGGAATGGTTCCTGCTGCGTAAAGCCTATGAACGCAACGAACTGGCACCGAATAAACCGGATGAACGTGAACTGGCACGTCGCGCAGAATCTTATGCGGGCGGTTACGTCAAGGAACCGGAAAAAGGCCTGTGGGAAAACATCGTGTACCTGGATTACAAGTCACTGTACCCGTCGATTATCATTACCCATAACGTTAGTCCGGATACGCTGAATCGTGAAGGCTGCCGCGAATATGACGTGGCACCGCAGGTTGGTCACCGCTTTTGTAAAGATTTTCCGGGCTTCATTCCGTCCCTGCTGGGTGACCTGCTGGAAGAACGTCAGAAGGTCAAGAAAAAGATGAAAGCGACCGTGGATCCGATCGAACGCAAGCTGCTGGACTATCGTCAACGCGCAATCAAAATTCTGGCTAACAGCTATTACGGCTATTACGGTTATGCAAATGCTCGTTGGTACTGCCGCGAATGTGCGGAATCTGTGACCGCCTGGGGCCGTCAGTACATTGAAACCACGATGCGCGAAATCGAAGAAAAGTTTGGTTTCAAAGTTCTGTATGCTGATACCGACGGCTTTTTCGCGACGATTCCGGGTGCGGATGCCGAAACCGTCAAAAAGAAAACGAAGGAATTCCTGAACTACATCAACCCGCGTCTGCCGGGCCTGCTGGAACTGGAATATGAAGGCTTCTACCGTCGCGGCTTTTTCGTTACCAAGAAAAAGTATGCCGTCATTGATGAAGAAGACAAAATCACCACGCGTGGCCTGGAAATTGTGCGTCGCGATTGGTCAGAAATCGCAAAAGAAACCCAGGCTCGCGTTCTGGAAGCGATTCTGAAACATGGTGATGTCGAAGAAGCCGTGCGTATCGTTAAGGAAGTCACGGAAAAACTGTCGCGCTATGAAGTGCCGCCGGAAAAACTGGTTATTTACGAACAAATCACCCGCAACCTGCGTGATTATCGTGCAACGGGTCCGCACGTCGCAGTGGCTAAGCGTCTGGCAGCGCGTGGCATCAAAATTCGTCCGGGTACCGTTATTAGTTACATCGTCCTGAAAGGCCCGGGTCGTGTGGGTGATCGCGCGATTCCGTTTGATGAATTCGACCCGGCCAAACATCGCTATGACGCAGAATATTACATTGAAAATCAGGTCCTGCCGGCAGTGGAACGTATCCTGCGTGCATTTGGTTATCGTAAAGAAGATCTGCGCTACCAGAAAACCAAGCAAGCAGGCCTGGGTGCTTGGCTGAAACCGAAGACGTAA
The base sequence of the polymerase DNA derived from the genus Thermococcus is as follows:
[Thermococcus-derived DNA polymerase]
（Ｔｈｅｒｍｏｃｏｃｃｕｓ属由来のＤＮＡポリメラーゼにおける酸性条件下での遺伝子増幅反応液中の緩衝液の影響）
ポリメラーゼ連鎖反応（ＰＣＲ）時の終濃度として、表１に示す組成のものをＰＣＲ緩衝液とした。各ＰＣＲ緩衝液に、２．５ｍＭ ｄＮＴＰを４μＬ、０．１％ ＢＳＡを２．５μＬ、０．０１％ Ｔｒｉｔｏｎ Ｘ－１００を５μＬ、１０μＭ フォワードプライマー（１）：CAGTCGTCATGCATTGCCTGCTC、及びリバースプライマー（１）：GTAGGCGCAATCACTTTCGTCTACTCCGを各２μＬ、６．４ｎｇ／μＬのλ―ＤＮＡを４μＬ、実施例１で取得したＴｈｅｒｍｏｃｏｃｃｕｓ属由来のＤＮＡポリメラーゼを１μＬ添加し、５０μＬのＰＣＲ増幅反応液を調製した。ＰＣＲの反応温度と時間は、表２に示す条件で実施した。
(Effect of buffer in gene amplification reaction solution under acidic conditions on Thermococcus-derived DNA polymerase)
The PCR buffer solution had the composition shown in Table 1 as the final concentration during polymerase chain reaction (PCR). 4 μL of 2.5 mM dNTP, 2.5 μL of 0.1% BSA, 5 μL of 0.01% Triton X-100, 2 μL each of 10 μM forward primer (1): CAGTCGTCATGCATTGCCTGCTC and reverse primer (1): GTAGGCGCAATCACTTTCGTCTACTCCG, 4 μL of 6.4 ng/μL λ-DNA, and 1 μL of DNA polymerase derived from the genus Thermococcus obtained in Example 1 were added to each PCR buffer solution to prepare 50 μL of PCR amplification reaction solution. The PCR reaction temperature and time were performed under the conditions shown in Table 2.
ＰＣＲ増幅後、実施例２で調した実施品１～７について、アガロース電気泳動にて目的遺伝子の増幅を確認したところ、図１のとおり、５ｍＭ ＮａＨ２ＰＯ４－ＮａＯＨ ｐＨ５．５及び６．０、並びに、５ｍＭ ＮａＨ２ＰＯ４－Ｎａ２ＨＰＯ４ ｐＨ６．０を用いたサンプル（実施品５～７）で、目的遺伝子の増幅が確認された。 After PCR amplification, amplification of the target gene was confirmed by agarose electrophoresis for Samples 1 to 7 prepared in Example 2. As shown in Figure 1, amplification of the target gene was confirmed in the samples (samples 5 to 7) using 5 mM NaH2PO4- NaOH pH 5.5 and 6.0, and 5 mM NaH2PO4 - Na2HPO4 pH 6.0.
（Ｔｈｅｒｍｕｓ属由来ＤＮＡポリメラーゼにおける酸性条件下での遺伝子増幅反応液中の緩衝液の影響）
ＰＣＲ反応時の終濃度として、表３及び表４に示す組成のものをＰＣＲ緩衝液とした。各ＰＣＲ緩衝液に、２．５ｍＭ ｄＮＴＰを４μＬ、１０μＭ フォワードプライマー（１）及びリバースプライマー（１）を各０．４μＬ、６．４ｎｇ／μＬのλ―ＤＮＡを１μＬ、Ｅｘ Ｔａｑ ＨＳポリメラーゼを０．２５μＬ、終濃度１０％となるようにトレハロースを添加し（実施品８、９、１０のみ添加）、５０μＬのＰＣＲ増幅反応液を調製した。ＰＣＲの反応温度と時間は、表５に示す条件で実施した。
(Effect of buffer in gene amplification reaction solution under acidic conditions using Thermus-derived DNA polymerase)
The PCR buffer solutions were prepared with the compositions shown in Tables 3 and 4 as the final concentrations during the PCR reaction. 4 μL of 2.5 mM dNTP, 0.4 μL each of 10 μM forward primer (1) and reverse primer (1), 1 μL of 6.4 ng/μL λ-DNA, 0.25 μL of Ex Taq HS polymerase, and trehalose to a final concentration of 10% (only samples 8, 9, and 10 were added) were added to each PCR buffer solution to prepare 50 μL of PCR amplification reaction solution. The PCR reaction temperature and time were as shown in Table 5.
ＰＣＲ増幅後、実施例４で調製した実施品８～１６について、アガロース電気泳動にて目的遺伝子の増幅を確認したところ、図２のとおり、２ｍＭ ＮａＨ２ＰＯ４－Ｎａ２ＨＰＯ４（ｐＨ６．０）を用いたサンプル（実施品８）では、目的遺伝子の増幅が確認されたが、クエン酸緩衝液を用いたサンプルは、いずれも増幅が見られなかった。また、２ｍＭ ＮａＨ２ＰＯ４－ＫＯＨ（ｐＨ６．０）を用いたサンプル（実施品１１）では、目的遺伝子の増幅が確認されたが、ＮａＨ２ＰＯ４－ＫＯＨ（ｐＨ５．０、又は、５．５）を用いたサンプルは増幅が見られなかった。さらにマッキルベイン緩衝液（ｐＨ５．０、又は、５．５、又は、６．０）を用いたサンプルでは、増幅が見られなかった。 After PCR amplification, the amplification of the target gene was confirmed by agarose electrophoresis for the samples 8 to 16 prepared in Example 4. As shown in Figure 2, the amplification of the target gene was confirmed in the sample (sample 8 ) using 2 mM NaH2PO4 - Na2HPO4 (pH 6.0), but no amplification was observed in any of the samples using citrate buffer. Furthermore, the amplification of the target gene was confirmed in the sample (sample 11) using 2 mM NaH2PO4 - KOH (pH 6.0), but no amplification was observed in the samples using NaH2PO4- KOH (pH 5.0 or 5.5). Furthermore, no amplification was observed in the samples using McIlvaine buffer (pH 5.0, 5.5, or 6.0).
（Ｔｈｅｒｍｏｃｏｃｃｕｓ属由来ＤＮＡポリメラーゼにおける酸性条件下での遺伝子増幅 反応液中の緩衝液及び塩化カリウムの濃度の影響）
ＰＣＲ反応時の終濃度として、表６に示す組成のものをＰＣＲ緩衝液とした。各ＰＣＲ緩衝液に、２．５ｍＭ ｄＮＴＰを４μＬ、０．１％ ＢＳＡを２．５μＬ、０．０１％ Ｔｒｉｔｏｎ Ｘ－１００を５μＬ、１０μＭ フォワードプライマー（１）及びリバースプライマー（２）：GCATTGCCCGTCAGGCTAATTCTGAAを各２μＬ、６．４ｎｇ／μＬのλ―ＤＮＡを２．５μＬ、実施例１で取得したＴｈｅｒｍｏｃｏｃｃｕｓ属由来のＤＮＡポリメラーゼを１μＬ添加し、５０μＬのＰＣＲ増幅反応液を調製した。また、ＰＣＲの反応温度と時間は、表７に示す条件で実施した。
(Gene amplification under acidic conditions using Thermococcus DNA polymerase: Effects of buffer and potassium chloride concentrations in the reaction solution)
The PCR buffer solution had the composition shown in Table 6 as the final concentration during the PCR reaction. 4 μL of 2.5 mM dNTP, 2.5 μL of 0.1% BSA, 5 μL of 0.01% Triton X-100, 2 μL each of 10 μM forward primer (1) and reverse primer (2): GCATTGCCCGTCAGGCTAATTCTGAA, 2.5 μL of 6.4 ng/μL λ-DNA, and 1 μL of the Thermococcus DNA polymerase obtained in Example 1 were added to each PCR buffer solution to prepare 50 μL of PCR amplification reaction solution. The PCR reaction temperature and time were performed under the conditions shown in Table 7.
ＰＣＲ増幅後、実施例６で調製した実施品１７～２２について、アガロース電気泳動にて目的遺伝子の増幅を確認したところ、図３のとおり、５０ｍＭ ＮａＨ２ＰＯ４－ＮａＯＨ（ｐＨ５．５）を用いたサンプルでは、５ｍＭ ＫＣｌ及び３０ｍＭ ＫＣｌを添加したサンプル（実施品１７及び１８）において目的遺伝子の増幅が確認されたが、５０ｍＭ ＫＣｌを添加したサンプルにおいては増幅が見られなかった。また、１００ｍＭ ＮａＨ２ＰＯ４－ＮａＯＨ（ｐＨ５．５）を用いたサンプルでは、５、３０、５０ｍＭ ＫＣｌを添加したいずれのサンプルでも増幅が見られなかった。 After PCR amplification, amplification of the target gene was confirmed by agarose electrophoresis for Samples 17 to 22 prepared in Example 6. As shown in Figure 3, in the sample prepared using 50 mM NaH2PO4 - NaOH (pH 5.5), amplification of the target gene was confirmed in the samples (samples 17 and 18) to which 5 mM KCl and 30 mM KCl were added, but amplification was not observed in the sample to which 50 mM KCl was added. Furthermore, in the sample prepared using 100 mM NaH2PO4 - NaOH (pH 5.5), amplification was not observed in any of the samples to which 5, 30, or 50 mM KCl was added.
（遺伝子増幅反応におけるマグネシウム濃度の影響）
ＰＣＲ反応時の終濃度として、表８に示す組成のものをＰＣＲ緩衝液とした。各ＰＣＲ緩衝液に、２．５ｍＭ ｄＮＴＰを４μＬ、０．１％ ＢＳＡを２．５μＬ、０．０１％ Ｔｒｉｔｏｎ Ｘ－１００を５μＬ、１０μＭ フォワードプライマー（１）及びリバースプライマー（２）を各２μＬ、６．４ｎｇ／μＬのλ―ＤＮＡを１μＬ、実施例１で取得したＴｈｅｒｍｏｃｏｃｃｕｓ属由来のＤＮＡポリメラーゼを１μＬ添加し、５０μＬのＰＣＲ増幅反応液を調製した。ＰＣＲの反応温度と時間は、表７に示す条件で実施した。
(Effect of magnesium concentration on gene amplification reaction)
The PCR buffer solution had the composition shown in Table 8 as the final concentration during the PCR reaction. 4 μL of 2.5 mM dNTP, 2.5 μL of 0.1% BSA, 5 μL of 0.01% Triton X-100, 2 μL each of 10 μM forward primer (1) and reverse primer (2), 1 μL of 6.4 ng/μL λ-DNA, and 1 μL of the Thermococcus DNA polymerase obtained in Example 1 were added to each PCR buffer solution to prepare 50 μL of PCR amplification reaction solution. The PCR reaction temperature and time were performed under the conditions shown in Table 7.
ＰＣＲ増幅後、実施例８で調製した実施品２３～２７について、アガロース電気泳動にて目的遺伝子の増幅を確認したところ、図４のとおり、ＰＣＲ反応時の終濃度で２、４、６ｍＭ ＭｇＣｌ２を含むＰＣＲ緩衝液を用いたサンプル（実施品２４～２６）において、目的遺伝子の増幅が確認された。また、ＰＣＲ増幅反応後の溶液中のピロリン酸をピロリン酸測定キット（商品名：ＰＰiＬiｇｈｔ Ｉｎｏｒｇａｎｉｃ Ｐｙｒｏｐｈｏｓｐｈａｔｅ Ａｓｓａｙ、ＬＯＮＺＡ製）を用いて測定したところ、表９のとおりとなり、ＰＣＲ反応時の終濃度で２ｍＭ ＭｇＣｌ２を含むＰＣＲ緩衝液を用いた時に、最もピロリン酸量が高かった。なお、ピロリン酸量は最も高い値を１００％として相対値で示した。 After PCR amplification, the amplification of the target gene was confirmed by agarose electrophoresis for the working samples 23 to 27 prepared in Example 8. As shown in FIG. 4, the amplification of the target gene was confirmed in the samples (working samples 24 to 26) using a PCR buffer containing 2, 4, and 6 mM MgCl2 as the final concentration during the PCR reaction. In addition, the pyrophosphate in the solution after the PCR amplification reaction was measured using a pyrophosphate measurement kit (trade name: PPiLight Inorganic Pyrophosphate Assay, manufactured by LONZA), and the results were as shown in Table 9. The amount of pyrophosphate was the highest when a PCR buffer containing 2 mM MgCl2 as the final concentration during the PCR reaction was used. The amount of pyrophosphate was shown as a relative value with the highest value set to 100%.
（遺伝子増幅反応における塩化カリウム濃度の影響）
ＰＣＲ反応時の終濃度として、表１０示す組成のものをＰＣＲ緩衝液とした。各ＰＣＲ緩衝液に、２．５ｍＭ ｄＮＴＰを４μＬ、０．１％ ＢＳＡを２．５μＬ、０．０１％ Ｔｒｉｔｏｎ Ｘ－１００を５μＬ、１０μＭ フォワードプライマー（１）及びリバースプライマー（２）を各２μＬ、６．４ｎｇ／μＬのλ―ＤＮＡを１μＬ、実施例１で取得したＴｈｅｒｍｏｃｏｃｃｕｓ属由来のＤＮＡポリメラーゼを１μＬ添加し、５０μＬのＰＣＲ増幅反応液を調製した。ＰＣＲの反応温度と時間は、表７に示す条件で実施した。
(Effect of potassium chloride concentration on gene amplification reaction)
The PCR buffer solution had the composition shown in Table 10 as the final concentration during the PCR reaction. 4 μL of 2.5 mM dNTP, 2.5 μL of 0.1% BSA, 5 μL of 0.01% Triton X-100, 2 μL each of 10 μM forward primer (1) and reverse primer (2), 1 μL of 6.4 ng/μL λ-DNA, and 1 μL of the Thermococcus-derived DNA polymerase obtained in Example 1 were added to each PCR buffer solution to prepare 50 μL of PCR amplification reaction solution. The PCR reaction temperature and time were performed under the conditions shown in Table 7.
ＰＣＲ増幅後、実施例１０で調製した実施品２８～３３について、アガロース電気泳動にて目的遺伝子の増幅を確認したところ、図５のとおり、ＰＣＲ反応時の終濃度で３０、５０、７５、又は１００ｍＭ ＫＣｌを含むＰＣＲ緩衝液を用いたサンプル（実施品３０～３３）において、目的遺伝子の増幅が確認された。また、ＰＣＲ増幅反応後の溶液中のピロリン酸をピロリン酸測定キット（商品名：ＰＰiＬiｇｈｔ Ｉｎｏｒｇａｎｉｃ Ｐｙｒｏｐｈｏｓｐｈａｔｅ Ａｓｓａｙ、ＬＯＮＺＡ製）を用いて測定したところ、表１１のとおり、ＰＣＲ反応時の終濃度で７５、又は１００ｍＭ ＫＣｌを含むＰＣＲ緩衝液を用いた時に、最もピロリン酸量が高かった。なお、ピロリン酸量は、最も高かった値を１００％として相対値で示した。
After PCR amplification, the amplification of the target gene was confirmed by agarose electrophoresis for the working samples 28 to 33 prepared in Example 10. As shown in Figure 5, the amplification of the target gene was confirmed in the samples (working samples 30 to 33) in which the PCR buffer solution contained 30, 50, 75, or 100 mM KCl as the final concentration during the PCR reaction. In addition, the pyrophosphate in the solution after the PCR amplification reaction was measured using a pyrophosphate measurement kit (trade name: PPiLight Inorganic Pyrophosphate Assay, manufactured by LONZA). As shown in Table 11, the amount of pyrophosphate was the highest when the PCR buffer solution contained 75 or 100 mM KCl as the final concentration during the PCR reaction. The amount of pyrophosphate was shown as a relative value with the highest value taken as 100%.
（酸性条件下での遺伝子増幅反応におけるトレハロース添加効果）
ＰＣＲ反応時の終濃度として、表１２に示す組成のものをＰＣＲ緩衝液とした。各ＰＣＲ緩衝液に、２．５ｍＭ ｄＮＴＰを４μＬ、１０μＭ フォワードプライマー(１)及びリバースプライマー(１)を各０.４μＬ、６．４ｎｇ／μＬのλ―ＤＮＡを１μＬ、Ｅｘ Ｔａｑ ＨＳポリメラーゼを０．２５μＬ添加し、５０μＬの遺伝子増幅反応液を調製した。ＰＣＲの反応温度と時間は、表５に示す条件で実施した。
(Effect of adding trehalose on gene amplification reaction under acidic conditions)
The PCR buffer solution had the composition shown in Table 12 as the final concentration during the PCR reaction. 4 μL of 2.5 mM dNTP, 0.4 μL each of 10 μM forward primer (1) and reverse primer (1), 1 μL of 6.4 ng/μL λ-DNA, and 0.25 μL of Ex Taq HS polymerase were added to each PCR buffer solution to prepare 50 μL of gene amplification reaction solution. The PCR reaction temperature and time were as shown in Table 5.
ＰＣＲ増幅後、実施例１２で調製した実施品３４～３７について、アガロース電気泳動にて目的遺伝子の増幅を確認したところ、図６のとおり、全てのサンプルについて、目的遺伝子の増幅が確認された。また、反応後の溶液中のピロリン酸をピロリン酸測定キット（商品名：ＰＰiＬiｇｈｔ Ｉｎｏｒｇａｎｉｃ Ｐｙｒｏｐｈｏｓｐｈａｔｅ Ａｓｓａｙ、ＬＯＮＺＡ製）を用いて測定したところ、表１３のとおり、トレハロースを添加した方が遺伝子増幅反応で生じるピロリン酸量が多くなった。なお、ピロリン酸量は最も高かった値を１００％として相対値で示した。After PCR amplification, amplification of the target gene was confirmed for Samples 34 to 37 prepared in Example 12 by agarose electrophoresis, and as shown in Figure 6, amplification of the target gene was confirmed for all samples. In addition, pyrophosphate in the solution after the reaction was measured using a pyrophosphate measurement kit (product name: PPiLight Inorganic Pyrophosphate Assay, manufactured by LONZA). As shown in Table 13, the amount of pyrophosphate generated in the gene amplification reaction was greater when trehalose was added. The amount of pyrophosphate is shown as a relative value, with the highest value being 100%.
［比較例１］
（本発明と公知文献記載の遺伝子増幅反応組成における酸性条件下での遺伝子増幅反応の比較）
市販のＤＮＡポリメラーゼ（商品名：Ｅｘ Ｔａｑ ＨＳ、タカラバイオ製）の添付資料に記載の組成をもとに緩衝液のみ２ｍＭ ＮａＨ２ＰＯ４－Ｎａ２ＨＰＯ４（ｐＨ６．０）に変更し、ＰＣＲ反応時の終濃度として、表１４に示す比較品１の組成のものをＰＣＲ緩衝液とした。ＰＣＲ緩衝液を５μＬ、２．５ｍＭ ｄＮＴＰを４μＬ、１０μＭ フォワードプライマー(１)及びリバースプライマー(１)を各０.４μＬ、６．４ｎｇ／μＬのλ―ＤＮＡを１μＬ、Ｅｘ Ｔａｑ ＨＳポリメラーゼを０．２５μＬ添加し、５０μＬの遺伝子増幅反応液を調製した。ＰＣＲの反応温度と時間は、表５に示す条件で実施した。
[Comparative Example 1]
(Comparison of gene amplification reactions under acidic conditions between the gene amplification reaction compositions of the present invention and those described in publicly known publications)
Based on the composition described in the attached document of a commercially available DNA polymerase (trade name: Ex Taq HS, manufactured by Takara Bio), only the buffer solution was changed to 2 mM NaH 2 PO 4 -Na 2 HPO 4 (pH 6.0), and the final concentration during PCR reaction was the composition of Comparative Product 1 shown in Table 14, which was used as the PCR buffer solution. 5 μL of PCR buffer solution, 4 μL of 2.5 mM dNTP, 0.4 μL each of 10 μM forward primer (1) and reverse primer (1), 1 μL of 6.4 ng/μL λ-DNA, and 0.25 μL of Ex Taq HS polymerase were added to prepare 50 μL of gene amplification reaction solution. The PCR reaction temperature and time were as shown in Table 5.
ＰＣＲ反応時の終濃度として、表１４の実施品３８の組成のものをＰＣＲ緩衝液とした。ＰＣＲ緩衝液に、２．５ｍＭ ｄＮＴＰを４μＬ、１０μＭ フォワードプライマー(１)及びリバースプライマー(１)を各０.４μＬ、６．４ｎｇ／μＬのλ―ＤＮＡを１μＬ、Ｅｘ Ｔａｑ ＨＳポリメラーゼを０．２５μＬ添加し、５０μＬの遺伝子増幅反応液を調製した。ＰＣＲの反応温度と時間は、表５に示す条件で実施した。また、表１４の実施品３９の組成のものをＰＣＲ緩衝液とし、ＰＣＲ緩衝液に、２．５ｍＭ ｄＮＴＰを４μＬ、０．１％ ＢＳＡを２．５μＬ、０．０１％、Ｔｒｉｔｏｎ Ｘ－１００を５μＬ、１０μＭ フォワードプライマー(１)及びリバースプライマー(２)を各２μＬ、６．４ｎｇ／μＬのλ―ＤＮＡを１μＬ、Ｔｈｅｒｍｏｃｏｃｃｕｓ属由来のＤＮＡポリメラーゼを１μＬ添加し、５０μＬの遺伝子増幅反応液を調製した。また、ＰＣＲの反応温度と時間は、表７に示す条件で実施した。
The PCR buffer solution had the composition of Example 38 in Table 14 as the final concentration during the PCR reaction. 4 μL of 2.5 mM dNTP, 0.4 μL each of 10 μM forward primer (1) and reverse primer (1), 1 μL of 6.4 ng/μL λ-DNA, and 0.25 μL of Ex Taq HS polymerase were added to the PCR buffer solution to prepare 50 μL of gene amplification reaction solution. The PCR reaction temperature and time were as shown in Table 5. The composition of Example 39 in Table 14 was used as the PCR buffer solution, and 4 μL of 2.5 mM dNTP, 2.5 μL of 0.1% BSA, 5 μL of 0.01% Triton X-100, 2 μL each of 10 μM forward primer (1) and reverse primer (2), 1 μL of 6.4 ng/μL λ-DNA, and 1 μL of Thermococcus-derived DNA polymerase were added to the PCR buffer solution to prepare 50 μL of gene amplification reaction solution. The PCR reaction temperature and time were set under the conditions shown in Table 7.
ＰＣＲ増幅後、比較例１及び実施例１６で調製した比較品１及び実施品３８、３９について、アガロース電気泳動にて目的遺伝子の増幅を確認したところ、図７のとおり、従来の遺伝子増幅組成では、遺伝子増幅が認められなかったが、緩衝液の種類や塩化カリウム濃度を変えることにより、酸性条件下で遺伝子増幅が認められた。After PCR amplification, amplification of the target gene was confirmed by agarose electrophoresis for Comparative Example 1 and Implementation Examples 38 and 39 prepared in Example 1 and Example 16. As shown in Figure 7, no gene amplification was observed with the conventional gene amplification composition, but by changing the type of buffer solution and potassium chloride concentration, gene amplification was observed under acidic conditions.
（Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓバクテリオファージ由来のＤＮＡポリメラーゼにおける酸性条件下での遺伝子増幅反応液中の緩衝液の影響）
ＰＣＲ反応時の終濃度として、表１５に示す組成のものをＰＣＲ緩衝液とした。ＰＣＲ緩衝液２μＬに、０.５μｇ／μＬ ｐＵＣ１９を１μＬ、１００μＭ Ｍ －１３Ｆプライマー：CAGTCGTCATGCATTGCCTGCTCを２μＬ、滅菌水を５μＬ添加した、１０μＬのテンプレート混合液を調製した。テンプレート混合液を９５℃、1分加熱後、３０℃まで０.１℃／秒で冷却した。冷却後、ＰＣＲ緩衝液２μＬに、２５ｍＭ ｄＮＴＰを０.８μＬ、１００ｍＭ ＤＴＴを１μＬ、１００Ｕ／ｍＬ ピロホスファターゼを０.２μＬ、５０μｇ／ｍＬ Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓバクテリオファージ由来のＤＮＡポリメラーゼを２μＬ、滅菌水を４μＬ添加した、１０μＬの反応混合液をテンプレート混合液に添加し、３０℃、１６時間のＰＣＲ反応を行った。
(Effect of buffer in gene amplification reaction solution under acidic conditions using DNA polymerase derived from Bacillus subtilis bacteriophage)
The PCR buffer had the composition shown in Table 15 as the final concentration during the PCR reaction. 10 μL of template mixture was prepared by adding 1 μL of 0.5 μg/μL pUC19, 2 μL of 100 μM M-13F primer: CAGTCGTCATGCATTGCCTGCTC, and 5 μL of sterile water to 2 μL of PCR buffer. The template mixture was heated to 95°C for 1 minute and then cooled to 30°C at 0.1°C/sec. After cooling, 10 μL of a reaction mixture prepared by adding 0.8 μL of 25 mM dNTP, 1 μL of 100 mM DTT, 0.2 μL of 100 U/mL pyrophosphatase, 2 μL of 50 μg/mL DNA polymerase derived from Bacillus subtilis bacteriophage, and 4 μL of sterile water to 2 μL of PCR buffer was added to the template mixture, and a PCR reaction was carried out at 30° C. for 16 hours.
ＰＣＲ増幅後、実施例１６で調製した実施品４０～４４について、アガロース電気泳動にて目的遺伝子の増幅を確認したところ、図８のとおり、３５ｍＭ クエン酸－クエン酸ナトリウム ｐＨ４.０、３５ｍＭ クエン酸－クエン酸ナトリウム ｐＨ４.５、３５ｍＭ ＮａＨ２ＰＯ４－ＮａＯＨ ｐＨ５．０及び、３５ｍＭ ＮａＨ２ＰＯ４－Ｎａ２ＨＰＯ４ ｐＨ６．０を用いたサンプル（実施品４１～４４）で、目的遺伝子の増幅が確認され、酸性条件下において室温付近で遺伝子増幅を行うことで、ｐＨ４台において酸性条件下でも遺伝子増幅させることができることが認められた。 After PCR amplification, amplification of the target gene was confirmed by agarose electrophoresis for Products 40 to 44 prepared in Example 16. As shown in Figure 8, amplification of the target gene was confirmed in samples (Products 41 to 44) using 35 mM citric acid-sodium citrate pH 4.0, 35 mM citric acid-sodium citrate pH 4.5, 35 mM NaH2PO4 - NaOH pH 5.0, and 35 mM NaH2PO4- Na2HPO4 pH 6.0. This indicates that gene amplification can be achieved even under acidic conditions at a pH of around 4 by performing gene amplification under acidic conditions at around room temperature.
（Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓバクテリオファージ由来のＤＮＡポリメラーゼにおける酸性条件下での遺伝子増幅反応液中の塩化カリウム濃度の影響）
ＰＣＲ反応時の終濃度として、表１６に示す組成のものをＰＣＲ緩衝液とした。ＰＣＲ緩衝液２μＬに、０.５μｇ／μＬ ｐＵＣ１９を１μＬ、１００μＭ Ｍ－１３Ｆプライマー：CAGTCGTCATGCATTGCCTGCTCを２μＬ、滅菌水を５μＬ添加した、１０μＬのテンプレート混合液を調製した。テンプレート混合液を９５℃、1分加熱後、３０℃まで０.１℃／秒で冷却した。冷却後、ＰＣＲ緩衝液２μＬに、２５ｍＭ ｄＮＴＰを０.８μＬ、１００ｍＭ ＤＴＴを１μＬ、１００Ｕ／ｍＬ ピロホスファターゼを０.２μＬ、５０μｇ／ｍＬ Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓバクテリオファージ由来のＤＮＡポリメラーゼを２μＬ、滅菌水を４μＬ添加した、１０μＬの反応混合液をテンプレート混合液に添加し、３０℃、１６時間のＰＣＲ反応を行った。
(Effect of potassium chloride concentration in gene amplification reaction solution under acidic conditions using DNA polymerase derived from Bacillus subtilis bacteriophage)
The PCR buffer had the composition shown in Table 16 as the final concentration during the PCR reaction. 10 μL of template mixture was prepared by adding 1 μL of 0.5 μg/μL pUC19, 2 μL of 100 μM M-13F primer: CAGTCGTCATGCATTGCCTGCTC, and 5 μL of sterile water to 2 μL of PCR buffer. The template mixture was heated to 95°C for 1 minute and then cooled to 30°C at 0.1°C/sec. After cooling, 10 μL of a reaction mixture prepared by adding 0.8 μL of 25 mM dNTP, 1 μL of 100 mM DTT, 0.2 μL of 100 U/mL pyrophosphatase, 2 μL of 50 μg/mL DNA polymerase derived from Bacillus subtilis bacteriophage, and 4 μL of sterile water to 2 μL of PCR buffer was added to the template mixture, and a PCR reaction was carried out at 30° C. for 16 hours.
ＰＣＲ増幅後、実施例１８で調製した実施品４５～４７について、アガロース電気泳動にて目的遺伝子の増幅を確認したところ、図９のとおり、ＰＣＲ反応時の終濃度で７５、１１５、又は、１２５ｍＭ ＫＣｌを含むＰＣＲ緩衝液を用いたサンプル（実施品４５～４７）において、目的遺伝子の増幅が確認された。After PCR amplification, amplification of the target gene was confirmed by agarose electrophoresis for Examples 45 to 47 prepared in Example 18. As shown in Figure 9, amplification of the target gene was confirmed in samples (Examples 45 to 47) using a PCR buffer containing 75, 115, or 125 mM KCl at a final concentration during the PCR reaction.
以上の結果から、本発明方法における遺伝子増幅反応では、ＤＮＡポリメラーゼを用いて、酸性条件下で測定対象の遺伝子を選択的且つ簡便に増幅させることができた。実施例２～７に示されるように、各種ＤＮＡポリメラーゼにおいて、緩衝液の種類や該緩衝液と塩化カリウムの濃度を適切な範囲に設定することよって酸性条件下での遺伝子増幅が変化することが分かった。また、実施例８～１１及び、実施例１８～１９に示されるように、反応液中のマグネシウムや塩化カリウム濃度を適切な範囲に設定し、実施例１２～１３に示されるように、トレハロースを反応液に添加することによっても酸性条件下での遺伝子増幅が変化することが分かった。また、比較例１及び実施例１４～１５に示されるように、従来の遺伝子増幅条件では、酸性条件下で遺伝子増幅が起こらなかったが、本発明方法に於いて、緩衝液の種類や塩化カリウム濃度を適切な範囲に設定することで、酸性条件下でも遺伝子増幅させることができることが分かった。さらに実施例１６～１７に示されるように、室温付近で遺伝子増幅を行う場合には、ｐＨ４台の酸性条件下でも遺伝子増幅させることができることが分かった。 From the above results, in the gene amplification reaction in the method of the present invention, the gene to be measured could be selectively and easily amplified under acidic conditions using DNA polymerase. As shown in Examples 2 to 7, it was found that gene amplification under acidic conditions changes for various DNA polymerases by setting the type of buffer and the concentration of the buffer and potassium chloride to an appropriate range. In addition, as shown in Examples 8 to 11 and Examples 18 to 19, it was found that gene amplification under acidic conditions also changes by setting the magnesium and potassium chloride concentrations in the reaction solution to an appropriate range, and as shown in Examples 12 to 13, it was found that gene amplification under acidic conditions changes. In addition, as shown in Comparative Example 1 and Examples 14 to 15, gene amplification did not occur under acidic conditions under conventional gene amplification conditions, but it was found that gene amplification can be achieved even under acidic conditions by setting the type of buffer and the potassium chloride concentration to an appropriate range in the method of the present invention. Furthermore, as shown in Examples 16 to 17, it was found that gene amplification can be achieved even under acidic conditions at a pH of 4 when gene amplification is performed near room temperature.
従来の遺伝子増幅条件では、酸性条件下でＤＮＡポリメラーゼを反応させる具体的な組成や条件が示されていなかった。これに対して、本発明に係る遺伝子増幅法に於いては、酸性条件下で測定対象の遺伝子を増幅させるための適切な緩衝液、各種塩類、添加物等の反応組成及び反応条件を見出し、酸性条件下で測定対象の遺伝子を増幅させることができた。その結果、本発明に係る遺伝子増幅法は、遺伝子増幅反応で産生したプロトンが蓄積しても問題なく反応が進む。その結果、優れた遺伝子検出技術として有用である、ピロリン酸を特異的に認識するピリジルボロン酸を用いた遺伝子検出法等に本発明の遺伝子増幅法で用いた酸性反応液を直ちに利用することが可能である。更に、本発明方法は、蛍光試薬を使用する必要がなくて安価である。In the conventional gene amplification conditions, specific compositions and conditions for reacting DNA polymerase under acidic conditions were not disclosed. In contrast, in the gene amplification method of the present invention, appropriate reaction compositions and reaction conditions, such as buffers, various salts, and additives, for amplifying the gene to be measured under acidic conditions were found, and the gene to be measured was amplified under acidic conditions. As a result, in the gene amplification method of the present invention, the reaction proceeds without any problems even if protons produced in the gene amplification reaction accumulate. As a result, it is possible to immediately use the acidic reaction solution used in the gene amplification method of the present invention in gene detection methods using pyridylboronic acid that specifically recognizes pyrophosphate, which are useful as excellent gene detection techniques. Furthermore, the method of the present invention is inexpensive because it does not require the use of fluorescent reagents.
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| WO2015145702A1 (en) | 2014-03-27 | 2015-10-01 | 国立大学法人東京医科歯科大学 | Diphosphate compound detector and diphosphate compound detection method |
| JP2015532593A (en) | 2012-08-23 | 2015-11-12 | ニユー・イングランド・バイオレイブス・インコーポレイテツド | Detection of amplification reaction products using pH sensitive dyes |
| JP2018538007A (en) | 2015-11-05 | 2018-12-27 | ユニヴァーシティ・オヴ・ユタ・リサーチ・ファウンデイション | Extreme reverse transcription PCR |
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| JP2007267738A (en) | 2006-03-31 | 2007-10-18 | Cem Corp | Microwave-assisted pcr amplification of dna |
| JP2015532593A (en) | 2012-08-23 | 2015-11-12 | ニユー・イングランド・バイオレイブス・インコーポレイテツド | Detection of amplification reaction products using pH sensitive dyes |
| WO2015145702A1 (en) | 2014-03-27 | 2015-10-01 | 国立大学法人東京医科歯科大学 | Diphosphate compound detector and diphosphate compound detection method |
| JP2018538007A (en) | 2015-11-05 | 2018-12-27 | ユニヴァーシティ・オヴ・ユタ・リサーチ・ファウンデイション | Extreme reverse transcription PCR |
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