JP3602550B2 - DNA sequence amplification method - Google Patents

Description

【００７９】
（解析結果）
大サイクル１回のみのもの（レーン１）では、主要な増幅産物は３種類（３本のバンド）あるが、特異的プライマーを代えてさらに増幅サイクルを行うと、１本のバンドになる。このバンドは、レーン２、３で大きさが異なる位置に泳動されるが、これは特異的プライマーがハイブリダイズする位置が、レーン３のものではより３’側であるためである。これらのバンドはレーン１ではほとんど認められないが、２回目の大サイクルを行うことによって、目的産物の特異性が向上した結果、１本のバンドのみが増幅されたものと考えられる。
【００８０】
尚、Ｔ−ＤＮＡ挿入配列の場合では、大サイクル１回目では、非特異的プライマーが結合する部位が異なる結果生じる増幅産物の種類は、平均２．２個であり、通常は最大５種類程度である。
【００８１】
レーン１で認められるバンドは、２回目の大サイクルでは消失し、両端が特異的プライマーである増幅産物であると推定される。これらの増幅産物は、レーン２、３で得られるバンドとは大きさは異なるが、単に低温サイクルで特異的プライマーがハイブリダイズした結果得られたものであり、既知配列に隣接する未知配列が増幅されたという意味においては目的産物であるということができる。
【００８２】
また、染色体ＤＮＡにＴＲ１の配列に類似する配列が存在し、その近傍にＴＲ１がハイブリダイズした結果、両端が特異的プライマーである増幅産物として生成した可能性もある。これらは、既知配列とは近傍の配列が異なるために、ＴＲ２やＴＲ３を使用した場合には増幅されず、バンドが消失したと考えられる。
【００８３】
いずれにしても、シロイヌナズナ染色体ＤＮＡのように長いＤＮＡ配列（約１０^１８塩基）を材料に用いても、本発明の方法により、目的とするＤＮＡ配列の特異的増幅が可能であることがわかった
また、出発材料にさらに多様性に富んだ小麦クロモゾームＤＮＡ（約１．５×１０^１０）を大量に加えても、特異的な増幅が認められ（レーン２〜４）、本発明の方法が極めて特異性の高いものであることがわかる。
【００８４】
【実施例２】
増幅産物を直接鋳型とする塩基配列の決定
次に、本発明の応用として、増幅産物を直接鋳型として用いた塩基配列決定の例を説明する。
【００８５】
実施例１で得られた大サイクル２回目反応液１０μｌ（１００〜２００ｎｇのＤＮＡを含む）に対し、０．５μｌの（α−^３２Ｐ）ｄＣＴＰ（〜１１０ＴＢｑ／ｍｍｏｌ）、１単位のＡｍｐｌｉＴａｑ ＤＮＡ ｐｏｌｙｍｅｒａｓｅ、４μｌのジメチルスルホキシド（ＤＭＳＯ）と１０ ｐｍｏｌｅの特異的プライマー（ＴＲ２）を含む緩衝液３０μｌを加えた。この反応液を各９μｌずつに４分割し、各分割液に対し１μｌの０．５ｍＭ ｄｄＧＴＰ， ５ｍＭ ｄｄＡＴＰ， ５ｍＭ ｄｄＴＴＰ， ５ｍＭ ｄｄＣＴＰのいずれかを添加し、９４℃２０秒、６０℃１分、７２℃２分のサイクルを４０回繰り返す反応を行った。この反応液を、通常のジデオキシ法に用いる塩基配列決定と同様に電気泳動を行って塩基配列を決定した。
【００８６】
得られた塩基配列を配列番号７に示す。塩基番号６９までは形質転換に用いたＴｉプラスミドの配列で、塩基番号７０〜２５１まではゲノムＤＮＡ中に挿入されたＴ−ＤＮＡに隣接する未知のシロイヌナズナゲノムＤＮＡ配列であった。尚、得られた未知配列は、シロイヌナズナゲノムＤＮＡのサザン分析の結果から、約２００塩基対を基本単位とする繰り返し配列であることが解明された。
【００８７】
図７に、他の塩基配列決定例における電気泳動の結果を示す。本発明では、ジデオキシヌクレオチドの取り込みとＤＮＡ合成産物の増幅を同時に行っているが、通常のジデオキシ法と同様にシークエンスラダーが形成されていることがわかる。
【００８８】
【実施例３】
Ｐ１ベクター挿入断片の増幅
次に、本発明の実施例として、Ｐ１ベクターに挿入されたＤＮＡの配列の増幅の例を説明する。
【００８９】
（１）Ｐ１ベクターが挿入された大腸菌ゲノムＤＮＡの調製
シロイヌナズナ染色体ＤＮＡ断片を含むＰ１ベクターを保有する大腸菌のコロニーを爪楊枝等で取り、直接反応液中に懸濁し、増幅反応に用いる鋳型とした。また、液体培地で培養した大腸菌からアルカリリシス法などによりＰ１プラスミドＤＮＡを調製してもよい。
【００９０】
（２）増幅反応の反応液の組成
Ｐ１ベクターのクローニングサイトの近傍に存在するＳｐ６プロモーターの配列の１部を有するオリゴヌクレオチドを３種用意し、特異的プライマーとした。各々のプライマーは、ハイブリダイズする部分がクローニングサイトから挿入配列方向である順に、Ｓｐ６−１（配列番号８：２２塩基、Ｔｍ６２．１℃）、Ｓｐ６−２（配列番号９：１７塩基、Ｔｍ５２．８℃）、及びＳｐ６−３（配列番号１０：１５塩基、Ｔｍ４２．４℃）である。
【００９１】
一方、非特異的プライマーとしては、実施例１に用いたＷ３を使用した。
ＤＮＡ増幅反応は、特異的プライマーを変えて２回以上行った。各反応における反応液組成は、特異的プライマー以外は実施例１と同様であり、特異的プライマーには、１回目の大サイクルにはＳｐ６−１を、２回目の大サイクルにはＳｐ６−２又はＳｐ６−３を、各々０．２μＭ使用した。
【００９２】
（３）増幅反応の反応温度サイクル
１回目のＤＮＡ増幅反応は、実施例１と同様にして行った。また、２回目の増幅反応は、９４℃１分、４５℃１分、７２℃３．５分の中温サイクルを２２回繰り返すことにより行った。
【００９３】
各々の反応後の反応液のうち、５μｌをとり、１．２〜１．５％のアガロースゲル電気泳動を行い、増幅産物のＤＮＡを分析を行った。結果を図８に示す。
レーン１、２は、各々異なるＰ１クローンを鋳型として、Ｓｐ６−１を特異的プライマーとした１回の大サイクルにより増幅を行った結果である。レーン３、４は、レーン１の試料を特異的プライマーにＳｐ６−２、Ｓｐ６−３を各々用いて２回目の大サイクルを行ったもの、レーン５、６はレーン２の試料をＳｐ６−２、Ｓｐ６−３を各々用いて２回目の大サイクルを行ったものについての結果である。
【００９４】
（解析結果）
レーン１の試料では、多数のバンドが認められるが、プライマーを代えて大サイクルを繰り返すと１本のバンドになる（レーン３、４）。これに対し、レーン２の試料では、大サイクル１回ですでに一本のバンドに集約されている。この結果は、増幅に用いる材料によっては、１回の大サイクルのみで充分であることを示している。特に、Ｐ１クローンのように鋳型配列が１０^５塩基程度の長さのものについては、１回で充分な場合が多いと考えられる。したがって、大サイクル１回目終了後の反応液について増幅産物の解析を行い、不十分であればさらにプライマーを代えて大サイクルを２回、あるいは３回行えばよい。
【００９５】
【実施例４】
次に、本発明の実施例としてＰ１ベクターに挿入されたＤＮＡの配列を、実施例３とは異なる反応温度サイクルで増幅した例を説明する。
【００９６】
（１）Ｐ１ベクターが挿入された大腸菌ゲノムＤＮＡの調製
実施例３と同様にして行なった。
【００９７】
（２）増幅反応の反応液の組成
特異的プライマーは実施例３に用いたＳｐ６−１を使用した。一方、非特異的プライマーとしては、実施例１に用いたＷ２を使用した。ＤＮＡ増幅反応における反応液組成は、実施例３と同様の組成を使用した。
【００９８】
（３）増幅反応の反応温度サイクル
１回目のＤＮＡ増幅反応は、実施例１の（第３工程）をＤＮＡ変性（９４℃３０秒）、アニーリング（５８℃１分）、ポリメラーゼ反応（７２℃３分）からなる中温サイクルを３０回繰り返した。反応後の反応液のうち、５μｌをとり、１．２〜１．５％のアガロースゲル電気泳動を行い、増幅産物のＤＮＡの分析を行なった。結果を図９に示す。
【００９９】
（解析結果）
この結果は、第３工程を中温サイクルのみで行なった場合も、目的とする増幅産物が得られることを示している。実施上特に、単一のバンドとすることを必要としない場合には、本実施例の方法で目的を達することが可能である。
【０１００】
【実施例５】
続いて、実施例１で得られたＴ−ＤＮＡを挿入したシロイヌナズナの染色体ライブラリーのうちの１株について、第３工程を中温サイクルのみで行った増幅例を説明する。
【０１０１】
（１）Ｔ−ＤＮＡ挿入配列を持つ植物の作製
実施例１と同様にして行った。
【０１０２】
（２）増幅反応の反応液の組成
特異的プライマーは実施例１に用いたＴＲ２あるいはＴＲ３を使用した。一方、非特異的プライマーとしては、実施例１に用いたＷ３を使用した。ＤＮＡ増幅反応における反応液組成は、実施例１と同様の組成を使用した。
【０１０３】
（３）増幅反応の反応温度サイクル
増幅反応は、実施例１の（第３工程）をＤＮＡ変性（９４℃３０秒）、アニーリング（５８℃１分）、ポリメラーゼ反応（７２℃３分）からなる中温サイクルを３０回繰り返した。特異的プライマーとしてＴＲ２あるいはＴＲ３を用いた増幅反応液から各々５μｌをとり、１．２〜１．５％のアガロースゲル電気泳動を行い、増幅産物のＤＮＡの分析を行なった。結果を図１０に示す。
【０１０４】
（解析結果）
いずれの特異的プライマーを用いた場合でも、増幅産物は、ほぼ単一のバンドとして得られた。ＴＲ３を用いた場合の増幅産物（レーン２）は、ＴＲ２を用いた場合の増幅産物（レーン１）よりも小さく、その差はＴ−ＤＮＡ中のＴＲ２及びＴＲ３と同一の配列を有する部位の距離６４塩基と一致していた。このことから、上記で得られた各々の増幅産物は、いずれも目的産物であることがわかった。
【０１０５】
この結果は、シロイヌナズナのように大きな染色体を材料にした場合でも、中温サイクルのみで目的とする増幅産物が得られることを示している。
【０１０６】
【発明の効果】
本発明の方法により、既知配列に隣接する未知配列を、ＤＮＡの環状化やオリゴヌクレオチドの連結といった複雑な操作を必要とせずに、しかも、効率よく増幅させることができる。
【０１０７】
【配列表】
【０１０８】

【０１０９】

【０１１０】

【０１１１】

【０１１２】

【０１１３】

【０１１４】

【０１１５】

【０１１６】

【０１１７】

【図面の簡単な説明】
【図１】従来のＰＣＲ法の原理を示す図。
【図２】公知の改変ＰＣＲ法の原理を示す図。
【図３】本発明に用いる増幅サイクルを示す図。
【図４】本発明の方法の概念を示す図。
【図５】Ｔ−ＤＮＡの配列の一部と、これに特異的プライマーがハイブリダイズする位置を示す図。
【図６】ＤＮＡ増幅産物の解析結果を示す電気泳動図。
【図７】塩基配列決定の電気泳動図。
【図８】ＤＮＡ増幅産物の解析結果を示す電気泳動図。
【図９】ＤＮＡ増幅産物の解析結果を示す電気泳動図。
【図１０】ＤＮＡ増幅産物の解析結果を示す電気泳動図。
【符号の説明】
図中、Ｍはマーカーを、図の左の数字は分子量を示す。[0001]
[Industrial applications]
The present invention relates to a method for amplifying a DNA sequence, and more particularly, to a method for amplifying a DNA sequence adjacent to a DNA sequence whose sequence is known in a simple and rapid manner.
[0002]
[Prior art]
Conventionally, a so-called PCR (Polymerase chain reaction) method has been widely used as a method of replicating a large amount of DNA having the same sequence to increase the number of molecules, that is, a method of amplifying DNA. This method, as shown in FIG.
(1) heat denaturation of double-stranded DNA into single-stranded DNA,
(2) annealing oligonucleotides (primers 1 and 2) corresponding to the sequences at both ends of the site to be amplified with the heat-denatured DNA,
(3) a polymerase reaction using the oligonucleotide as a primer,
Is a method of exponentially amplifying the DNA sequence by repeating the amplification cycle consisting of
[0003]
By this method, a very small amount of DNA can be amplified, but primers cannot be synthesized unless the sequences at both ends of the region to be amplified are known, so that the method can be applied only to known sequences. Was.
[0004]
Therefore, as a method for amplifying a DNA region of unknown sequence adjacent to a DNA sequence of known sequence, several improved methods based on the PCR method have been reported.
One is to cut an unknown sequence adjacent to either side or one side of the known sequence with a restriction enzyme and then circularize, using two specific primers specific to the known sequence portion and directed outward from each other, using ordinary PCR. This is a method in which the same amplification cycle as in the above method is repeated, and is called an inverse PCR (inverse PCR) method.
[0005]
In addition, a method called “bubble” PCR method cleaves an unknown sequence adjacent to one side of a known sequence with a restriction enzyme, ligates it to a double-stranded oligonucleotide, and prepares a primer and oligonucleotide specific to the known sequence. In this method, amplification cycles are repeated using specific primers. In this method, the oligonucleotide to be linked to the unknown sequence contains a non-complementary sequence at the center thereof, so that the primer binds only to a sequence synthesized by extension from a primer specific to the known sequence. , The array is designed
However, these methods require labor and time for selection of an appropriate restriction enzyme, circularization of DNA, or ligation of oligonucleotides. Further, in order to determine the base sequence using the product of the DNA amplification reaction, an operation for removing primers and nucleotides contained in the amplification reaction solution is required.
[0006]
In order to solve such a problem, there is disclosed a method of amplifying an unknown sequence linked to a known sequence using a single primer without cyclizing DNA or linking oligonucleotides (Parks). , CL, Shenk, T. et al., Nucleic Acids Res., 19, No. 25 (1991)). This method uses a primer having a sequence corresponding to a known sequence, an amplification cycle under mild conditions such that the primer hybridizes nonspecifically to an unknown sequence, and the primer specifically hybridizes. The known sequence and the unknown sequence are amplified by repeating each amplification cycle under such stringent conditions.
[0007]
In addition, a method of amplifying an unknown sequence by using nonspecific primers having an arbitrary sequence in addition to specific primers and annealing at different temperatures and performing an amplification cycle has been proposed (Parker, JD. et al., Nucleic Acids Res., 19, No. 11 (1991))). That is, as shown in FIG. 2, annealing is performed at a temperature at which the specific primer hybridizes to the DNA sequence to be amplified and at a temperature at which the non-specific primer hybridizes while containing a mismatch, and the amplification cycle is repeated. DNA near the known sequence can be amplified.
[0008]
[Problems to be solved by the invention]
The present invention has been made in view of the above, and a method capable of efficiently amplifying a sequence adjacent to a known sequence without requiring complicated operations such as circularization of DNA and ligation of oligonucleotides. The task is to provide
[0009]
[Means for Solving the Problems]
The present inventors have conducted intensive studies in order to solve the above-mentioned problems. As a result, the amplification primer was non-specifically hybridized to the target DNA sequence only once, and then the primer was specifically hybridized. The present inventors have found that the DNA sequence can be efficiently amplified by repeating the amplification cycle in which annealing is performed at a soybean temperature, and the present invention has been achieved.
[0010]
That is, the present invention provides a method for mixing a DNA sequence and an oligonucleotide primer having a partial sequence of the DNA sequence in a DNA polymerase reaction solution containing a DNA polymerase and deoxyribonucleotides, denaturing the DNA sequence, And a method of amplifying the DNA sequence by repeating an annealing cycle consisting of annealing with a DNA primer and a DNA polymerase reaction,
(A) one amplification cycle in which the annealing is performed at a temperature at which the oligonucleotide primer non-specifically hybridizes to an arbitrary position in the DNA sequence;
(B) a plurality of amplification cycles, wherein the annealing is performed at a temperature at which the oligonucleotide primer specifically hybridizes to a DNA sequence complementary to the sequence,
And a method for amplifying a DNA sequence.
[0011]
The present invention also provides a DNA sequence, an oligonucleotide primer having a partial sequence of the DNA sequence (specific primer), and an oligonucleotide primer having a lower Tm than the specific primer and having an arbitrary sequence (non-specific primer). ) Is mixed in a DNA polymerase reaction solution containing DNA polymerase and deoxyribonucleotide, and the above-mentioned DNA sequence is denatured, annealing of this DNA sequence with a DNA primer, and an amplification cycle consisting of a DNA polymerase reaction are repeated. In a method for amplifying a DNA sequence,
(A) one amplification cycle in which annealing is performed at a temperature at which the non-specific primer hybridizes non-specifically to the DNA sequence;
(B) multiple amplification cycles of annealing at a temperature at which the specific primer specifically hybridizes to the DNA sequence;
(C) multiple amplification cycles of annealing at a temperature at which the non-specific primer specifically hybridizes to a DNA sequence complementary to that sequence;
A method for amplifying a DNA sequence, comprising:
[0012]
In the present invention, "hybridize" means that a single-stranded DNA sequence and an oligonucleotide primer form a partial double strand by hydrogen bonding, and "annealing" or "annealing" This means that the conditions are set so that the single-stranded DNA sequence and the oligonucleotide primer hybridize.
[0013]
Unless otherwise specified, "specific" means that the primer and the DNA sequence hybridize without mismatch, and "non-specific" means that they hybridize with mismatch. . Hereinafter, the present invention will be described in detail.
[0014]
<1> Primers used for DNA sequence amplification method of the present invention
Specific primers are oligonucleotide primers that are specific for a portion of a DNA sequence, ie, have a sequence identical to a portion of a DNA sequence, the sequence of which is selected from known sequences adjacent to an unknown sequence.
[0015]
On the other hand, a non-specific primer is an oligonucleotide having an arbitrary sequence, and a site complementary to a non-strand having a sequence identical to that of a specific primer on a non-strand having a sequence identical to that of a specific primer (hereinafter referred to as an oligonucleotide). , "Target site") and serves as a primer for a DNA polymerase (hereinafter, simply referred to as "polymerase") reaction.
[0016]
The non-specific primer is preferably designed to have a lower Tm (melting temperature: temperature at which duplex formation and dissociation with a complementary sequence are at equilibrium) than the specific primer. 5 ° C. or higher, preferably 10 to 15 ° C. Incidentally, the Tm value can be predicted by the following formula.
[0017]
(Equation 1)
Tm = 69.3 ° C. + 0.41 (% GC ratio) ° C.-650 / L ° C.
(However, L represents the number of bases of the primer)
[0018]
The Tm value obtained by the above equation is a value in an aqueous solution containing no salt, and actually depends on the salt concentration, pH, and the like. In a buffer used for a normal polymerase reaction, the value is often higher than the value obtained above.
[0019]
By the way, the present invention is based on the fact that a specific primer is hybridized to a strand synthesized by a polymerase reaction starting from a non-specific primer, and the other strand is synthesized. Therefore, the non-specific primer must bind to a position where it can reach the site where the specific primer hybridizes by the polymerase reaction.
[0020]
In general, the DNA chain length synthesized by the polymerase reaction is about 10 kb under limited conditions, and under general conditions, the limit is about 5 kb. Is preferably shorter. For example, when the unknown sequence is a uniform sequence, the non-specific primer needs to be able to hybridize to the unknown sequence within 5000 to 10000 bases from the known sequence. That is, since there are four types of nucleotides, in order to allow a non-specific sequence to hybridize at a position within 5 to 10 kb, 6 (= log) ₄ 4094) or 7 (= log ₄ 16) Non-specific primers for bases must be used.
[0021]
However, when a non-specific primer having a chain length of about 6 to 7 bases is used, it hybridizes to a large number of unintended sites on the DNA sequence, and the polymerase reaction which occurs when the non-specific primer binds to the opposite strand. May be generated, and these reactions may antagonize the reaction of amplifying the target sequence, thereby inhibiting amplification of the target product.
[0022]
In order to satisfy the above conditions, a mixture of oligonucleotides having different sequences or an oligonucleotide containing an inosine residue capable of forming a hydrogen bond with a plurality of nucleotides is used as a non-specific primer to determine the number of bases capable of forming a hydrogen bond. With the use of a long-chain primer, a number of mismatches are caused at a considerably lower temperature than the Tm value to cause hybridization near the known sequence, and in subsequent reactions, hybridization is performed near the Tm of the primer. Preferably, the sequence specificity is improved. In this case, the chain length of the specific primer is preferably 20 to 25. On the other hand, the chain length of the non-specific primer is preferably 14 to 17. It is preferable that any of the primers has a sequence that does not form a secondary structure in the primer and that does not hybridize with each other.
[0023]
<2> DNA sequence amplification method of the present invention
The present invention provides a single amplification cycle in which a non-specific primer (or a specific primer when only a specific primer is used) is annealed at a temperature at which it hybridizes non-specifically to an arbitrary position in a DNA sequence. And can be applied to various conventionally known methods of performing an amplification cycle at different temperatures.
[0024]
In the following description, the amplification cycle in which non-specific primers (specific primers when only specific primers are used) are annealed at a temperature at which the DNA sequence non-specifically hybridizes (“low temperature”) is referred to as “a low temperature”. A low-temperature cycle, an amplification cycle that anneals at a temperature at which specific primers only (including the case where only specific primers are used) specifically hybridize to a DNA sequence ("high temperature"), a "high-temperature cycle" An amplification cycle in which annealing occurs at a temperature at which the target primer specifically hybridizes to a DNA sequence complementary to that sequence ("medium temperature") is referred to as a "medium temperature cycle."
[0025]
Hereinafter, each cycle will be described with reference to FIG. In FIG. 3, among the DNA sequences, the strand having the same sequence as the specific primer (the thin line in the figure) is called “+ strand”, and the other strand (the “thick line” in the figure) is called “− strand”.
[0026]
(1) Amplification cycle
(1) High temperature cycle
The high-temperature cycle is a reaction for selectively hybridizing only a specific primer to a corresponding site on a minus strand and amplifying an unknown sequence by a polymerase reaction. This cycle is the reaction with the highest specificity, and produces a small amount of by-products. Since specific primers can hybridize at lower temperatures, high temperature cycling is not necessarily required to amplify the DNA sequence, but is preferably performed to increase the specificity of the amplification product. Further, by using a plurality of specific primers, the yield of the amplification product can be increased.
[0027]
(2) Low temperature cycle
The low-temperature cycle is a reaction for hybridizing a non-specific primer to a non-specific position on the + strand and synthesizing a − strand. Non-specific primers hybridize to multiple sites and also to the minus strand, thus producing more by-products in this cycle. When the high-temperature cycle is performed after the low-temperature cycle, and then the low-temperature cycle is performed again, non-specific amplification occurs (*, ** marks in FIG. 3). As a result, non-specific primers are used for both the positive and negative strands. (A ** mark in the figure: hereinafter, "bidirectional non-specific amplification product"). Since such an amplification product is produced at a number of unintended sites in the DNA sequence, the yield of the target product is deteriorated.
[0028]
(3) Medium temperature cycle
When the high-temperature cycle is performed after the low-temperature cycle, the site of the + strand corresponding to the non-specific primer is replaced with a sequence completely complementary to the non-specific primer. Non-specific primers can hybridize nonspecifically to DNA sequences at low temperatures, but not at medium temperatures. However, even at a medium temperature, the above-mentioned substituted sequence is complementary and can hybridize, so that the minus strand can be synthesized. On the other hand, since * and ** products produced in the low-temperature cycle are not theoretically produced in the medium-temperature cycle, it is possible to suppress the production of bidirectional non-specific amplification products.
[0029]
In addition, since the specific primer can hybridize to the DNA sequence even in the low-temperature cycle and the medium-temperature cycle, a polymerase reaction starting from the specific primer also occurs, but non-specific amplification does not occur in this reaction. Cycles and intermediate temperature cycles are omitted. However, when a sequence similar to the sequence of the specific primer exists at a site other than the known sequence on the chromosomal DNA, non-specific amplification may occur. This amplification product is also a target product in the sense of amplifying a DNA sequence close to the specific primer.
[0030]
(2) Aspects of the present invention
The method of the present invention is carried out by combining the above-mentioned cycles, and basically consists of one low-temperature cycle, followed by repetition of a high-temperature cycle and a medium-temperature cycle. As described above, high-temperature cycling is not always required, but even at moderate temperatures, non-specific primers, which contain mismatches to some extent, hybridize to the DNA sequence, thus suppressing the generation of bidirectional non-specific amplification products. To do so, it is preferable to include a high-temperature cycle. If the high-temperature cycle is performed a plurality of times before the low-temperature cycle, the specificity of the amplification product can be further increased.
[0031]
The optimum combination and the number of times for each cycle vary depending on the type of primer used, the GC content of the DNA sequence to be amplified, and the like, but some embodiments are exemplified below.
[0032]
(I) When using only specific primers
Since the specific primer itself can non-specifically hybridize to the DNA sequence at a low temperature, amplification may be possible without using a non-specific primer.
[0033]
When only specific primers are used, it is preferable to perform the low-temperature cycle once and then repeat the high-temperature cycle, and more preferably to perform the high-temperature cycle several times before the low-temperature cycle.
[0034]
(Ii) When using a specific primer and a non-specific primer
Also in this case, similarly to the above, it is preferable to perform the low-temperature cycle once, and thereafter repeat the high-temperature cycle and the medium-temperature cycle, and it is more preferable to perform the high-temperature cycle a plurality of times before the low-temperature cycle.
[0035]
After each amplification cycle, amplification is performed again using another specific primer corresponding to a site downstream (3 'side) of the site where the specific primer used hybridizes to the DNA sequence. Thus, the specificity of the amplification product can be further improved. Instead of repeating the high-temperature cycle and the medium-temperature cycle, the medium-temperature cycle can be performed a plurality of times at an intermediate temperature between Tm of both primers.
[0036]
(3) Reaction conditions used in the present invention
Each reaction used in the method of the present invention is not particularly different from the conventional PCR method or its improved method, and it is sufficient to adopt generally used conditions.
[0037]
For example, the DNA sequence may be denatured at a temperature at which double-stranded DNA can be denatured into single-stranded DNA, for example, at 90 to 94 ° C. for 30 seconds to 2 minutes. It is preferable to use a heat-resistant polymerase such as Taq DNA polymerase so that the DNA polymerase is not inactivated at this stage or the annealing stage.
[0038]
Annealing, after denaturation, is in the range of the expected Tm of the specific primer in the high temperature cycle to + 10 ° C., about the expected Tm of the nonspecific primer in the medium temperature cycle, and less than the expected Tm of the nonspecific primer in the low temperature cycle. Usually, it is carried out at a temperature lower by about 20 ° C. for 1 to 5 minutes. In addition, it is preferable to lower the temperature over time after denaturation, especially in a low-temperature cycle, so that annealing can easily occur. Although theoretically annealing is performed at a temperature lower than Tm, as described above, since the expected Tm is a value in an aqueous solution containing no salt, the specific primer in the polymerase reaction solution has the above-mentioned temperature. The annealing can be performed as described above.
[0039]
The polymerase reaction is carried out at the optimal temperature for the DNA synthase used in the reaction. For example, when Taq DNA polymerase is used, the reaction is carried out at 70 to 72 ° C. for about 2 to 4 minutes.
[0040]
The setting and adjustment of the reaction temperature can be performed using a commercially available PCR device. The holding time of each step is appropriately set depending on the reaction rate of the enzyme, heat resistance, heating and cooling rates of the equipment used, and the like. For example, when the reaction rate of the enzyme is high, the reaction time can be short, and when the heat resistance of the enzyme is high, the denaturation time can be long.
[0041]
Furthermore, after amplifying the DNA sequence by the above method, another specific primer that hybridizes to a site downstream of the target site of the specific primer used is prepared, and the high-temperature cycle and the medium-temperature cycle are repeated to obtain the desired sequence. The product can be more selectively amplified.
[0042]
<4> Application of the DNA sequence amplification method of the present invention
(1) Application to base sequence determination
By applying the above-described DNA sequence amplification method to base sequence determination by the dideoxy method, the base sequence can be determined using an unlabeled primer.
[0043]
In the dideoxy method, DNA whose base sequence is to be determined, an oligonucleotide primer having a sequence complementary to a part of the base sequence, deoxynucleotide, dideoxynucleotide, and DNA polymerase coexist in a buffer solution. This is a method for determining the base sequence by conducting a polymerase reaction and analyzing a DNA synthesis product by electrophoresis. When the dideoxynucleotide added to the deoxynucleotide in a small amount is incorporated in the polymerase reaction, the DNA synthesis stops. Therefore, the base sequence can be determined by analyzing the DNA synthesis product by electrophoresis. In this method, the synthesis product is usually detected by autoradiography using an RI-labeled primer or an optical method using a fluorescent-labeled primer.
[0044]
In order to determine a nucleotide sequence by applying the present invention, for example, after amplifying a DNA sequence whose nucleotide sequence is to be determined using a specific primer and a non-specific primer, an appropriate amount of dideoxynucleotide is added to the amplification reaction solution. In addition, the high temperature cycle may be repeated. This allows the incorporation of dideoxynucleotides while amplifying the product of the polymerase reaction in proportion to the number of cycles.
[0045]
When the base sequence of an amplification product obtained by the conventional PCR method is determined by the dideoxy method, it is necessary to remove the primer used in the PCR method from the reaction solution before adding a primer for base sequence determination. Since the specific primer and the non-specific primer have different Tm, the specific primer can be used as it is as a primer for nucleotide sequence determination without removing these primers. In this respect, the same is true for the various modifications, but they cannot be used directly for sequencing due to their high by-product content. On the other hand, in the method of the present invention, since the purity of the target product is high, the amplification reaction solution can be used as it is for base sequence determination.
[0046]
(2) Other
The present invention can also be applied to chromosome walking using an amplification product as a probe, identification of transformed organisms, determination of a retrovirus insertion site, and the like.
[0047]
Since the method of the present invention can amplify an unknown sequence adjacent to a known sequence, a sequence such as a promoter or an intron can be obtained based on the sequence of cDNA. In addition, a gene encoding the protein can be amplified using a specific primer having a sequence deduced from the amino acid sequence of the protein.
[0048]
[Action]
Hereinafter, using a specific primer and a non-specific primer as embodiments of the present invention,
(1) a first step including a plurality of (x times) high-temperature cycles;
(2) a second step consisting of one low-temperature cycle;
(3) a third step which is a repetition (n times) of a combined cycle including a plurality of (y times) high temperature cycles and a plurality of (z times) medium temperature cycles;
The operation will be specifically described with reference to FIG. In addition, in order to facilitate understanding, the description will be made assuming that the DNA sequence as a starting material is one copy. Here, one copy means that when the starting material is an extremely long DNA fragment, such as genomic DNA, each is one molecule. Therefore, usually, a large number of DNA sequences are present in addition to the unknown sequence adjacent to the known sequence to be amplified.
[0049]
<1> First step
The first step is performed by performing a high-temperature cycle x times. In the first reaction cycle, two single-stranded DNAs are obtained by denaturation, one of which serves as a template, hybridizes in a state where the specific primer is completely matched to the known sequence portion, and is hybridized with the template sequence. Complementary DNA is synthesized. The other single-stranded DNA does not serve as a template here.
[0050]
In the second reaction cycle, the partially double-stranded DNA obtained in the first reaction cycle becomes single-stranded DNA again due to denaturation, and the specific primer becomes a primer in the same manner as in the first reaction cycle. Synthesized. When this is repeated x times, x single-stranded DNAs having the specific primer at the end are generated.
Apart from the above, the specific primer is unable to bind to other than the target site in the high-temperature cycle, and no non-specific reaction occurs, so that no by-products are generated.
[0051]
At this stage, the number of target products is (1 + x).
[0052]
<2> Second step
The second step consists of one low-temperature cycle, in which a non-specific primer is hybridized to an arbitrary portion of the DNA sequence including a mismatch, and DNA synthesis is performed. Here, the end of the newly generated DNA is the non-specific primer itself, which means that the sequence of the corresponding portion of the template has been replaced by the sequence of the non-specific primer. When DNA is synthesized from the specific primer in the subsequent medium / high temperature cycle, a sequence complementary to the non-specific primer is generated, and the non-specific primer can specifically hybridize to this site. And the generation of by-products can be suppressed.
[0053]
On the other hand, in the chromosomal DNA, a sequence that completely matches or approximates the sequence of the non-specific primer exists stochastically. Therefore, when the non-specific primer hybridizes near this sequence, the bidirectional non-specific amplification product Is produced and the product is amplified. In addition, amplification of by-products occurs as a result of nonspecific primer hybridizing to sites other than the target site, but specific primers cannot hybridize to the strand opposite to the generated strand, so further amplification is theoretically necessary. Does not happen. However, on the other hand, amplification of by-products can occur even at the medium temperature cycle because the non-specific primers hybridize to some extent including the mismatch even at the medium temperature cycle.
[0054]
In the conventional method, since the low-temperature cycle is repeatedly performed, a considerable amount of by-product is generated. However, in the present invention, the generation of the by-product can be suppressed by using only one low-temperature cycle.
[0055]
At this stage, the number of target products is (1 + x + 1), but since the template molecule itself has not been replaced with the sequence of the non-specific primer, the template to be used in the subsequent reaction is (x + 1).
[0056]
<3> Third step
The third step is performed by repeating a combined cycle consisting of y high-temperature cycles and z medium-temperature cycles n times. In this step, only the target product is amplified in the high temperature cycle, and both the target product and the bidirectional nonspecific product generated in the low temperature cycle are amplified in the medium temperature cycle. As a result, after performing the composite cycle n times, the target product is (x + 1) ｛(y + 2) 2 ^(Z-1) ｝ ⁿ Individual, 2 by-products ^zn Individual.
[0057]
In addition to the reactions described above, there are also amplification products formed by nonspecific hybridization of specific primers in a low-temperature cycle. For one molecule of the product thus generated, in the third step 2 ^{(Y + z) n} It is amplified twice. In addition, in a low-temperature cycle, nonspecific primers may hybridize to a site near or far from the target site of the specific primer, resulting in amplification products having different lengths. Such a product is also a product obtained by amplifying a target unknown sequence, and is therefore a target product in that sense, and does not pose a problem except for a case where the amplification product is to be unified.
[0058]
In the third step, it is preferable to determine x, y, z, and n so that the amount of bidirectional nonspecific amplification product with respect to the target product is minimized. The total number of cycles a = x + 1 + n (y + z) in all the steps is limited by the heat resistance of the enzyme. When using a polymerase derived from Thermus aquaticus (Perkin-Elmer; AmpliTaq DNA polymerase), a = about 50 Is a limit, and if a is considered constant, then (x + 1) ｛(y + 2) 2 ^(Z-1) ｝ ⁿ / 2 ^zn Is maximum when x = 4, y = 2, and z = 1. Also, n is determined by how many times a is set.
[0059]
After completion of the cycle consisting of the first to third steps (hereinafter, referred to as “large cycle”), if a large cycle is performed using another non-specific primer, the specificity of the amplification product can be improved. it can.
[0060]
【Example】
Hereinafter, examples of the method for amplifying a DNA sequence of the present invention and its application will be described.
[0061]
Embodiment 1
Amplification of plant genomic sequence adjacent to T-DNA insertion sequence
As an example of the method for amplifying a DNA sequence of the present invention, an example will be described in which T-DNA is inserted into a plant genome, and this T-DNA sequence is used as a known sequence to amplify a plant genome adjacent thereto.
[0062]
(1) Preparation of plant having T-DNA insertion sequence
Arabidopsis thaliana was infected with Agrobacterium tumefacience having pGDW32, which is a binary vector of Ti plasmid, by a method such as a leaf desk method to obtain a transformed plant having a T-DNA insertion sequence. Genomic DNA was extracted from these transformed plants by the urea-phenol method or the like.
[0063]
(2) Composition of reaction solution for amplification reaction
Three types of oligonucleotides TR1, TR2, and TR3 were prepared as specific primers that hybridize near the end of the T-DNA insertion sequence. These sequences are as described in the sequence listing as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and hybridize in order from the 5 'side to the 3' side of the T-DNA insertion sequence. These primers consisted of 23 bases, each having a Tm value of 60.6 ° C for TR1 and 57.1 ° C for TR2 and TR3. The positions where these primers hybridize to the known sequence are shown in FIG. This sequence shows a strand having the same sequence as the primer.
[0064]
As non-specific primers, three kinds of oligonucleotide mixtures W1, W2, and W3 having the sequences shown in SEQ ID NOs: 4, 5, and 6 were prepared. In each sequence, W represents A or T, and S represents G or C.
[0065]
W1 is a mixture of 64 kinds of oligonucleotides consisting of 15 bases, Tm value is 43.7 ° C., W2 is a mixture of 128 kinds of oligonucleotides consisting of 16 bases, Tm value is 46.6 ° C., and W3 is It is a mixture of 256 types of oligonucleotides consisting of 16 bases, and has a Tm value of 45.3 ° C.
[0066]
It is preferable that the method of the present invention is carried out twice or more by changing the primers to be used for DNA amplification. The reaction solution used in each reaction had a common composition except for the template and the primer. The basic composition is 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2 mM MgCl ₂ , 0.001% gelatin, and 100 μM dNTP.
[0067]
The reaction solution in the first large cycle contains 10 ng of template genomic DNA, 0.7 units of DNA polymerase (AmpliTaq DNA polymerase manufactured by Perkin-Elmer Cetus), 0.2 μM of TR1, and non-specific primers in the above basic composition. (One of 2 μM W1, 3 μM W2, and 4 μM W3) was added to a final volume of 20 μl. The reaction was carried out by overlaying 20 μl of mineral oil on this.
[0068]
In the DNA amplification reaction in the second and subsequent large cycles, the same non-specific primer was used as in the first reaction, but the specific primer can be hybridized to the 3 'side from the specific primer used in the first reaction. Specific primers (TR2, TR3) were used. To this reaction solution, 0.1 μl of the first reaction solution, 3.5 units of AmpliTaq DNA polymerase, 0.2 μM of a specific primer (TR2 or TR3), and a non-specific primer were added to make a total of 100 μl. The reaction was carried out by overlaying 20 μl of mineral oil on the surface.
[0069]
In selecting a non-specific primer to be used throughout each reaction, first, the reaction is performed using W1, and if a preferable amplification product is not obtained, the first reaction is performed sequentially using W2 and W3. It is good to carry out again from.
[0070]
(3) Reaction temperature cycle of amplification reaction
The first large cycle DNA amplification reaction was performed in the following steps using a commercially available device (thermal cycler manufactured by Perkin-Elmer Cetus).
[0071]
(First step)
A high-temperature cycle consisting of denaturation of DNA (94 ° C for 1 minute), annealing (65 ° C for 1 minute), and polymerase reaction (72 ° C for 3 minutes) was repeated 5 times.
[0072]
(2nd process)
After denaturation of the DNA at 94 ° C. for 1 minute, it was cooled to 25 ° C. for 5 minutes, kept at 25 ° C. for 5 minutes to perform annealing, heated to 72 ° C. for 5 minutes, and then reacted at 72 ° C. for 3 minutes. And a low-temperature cycle was performed.
[0073]
(3rd step)
Two high-temperature cycles consisting of DNA denaturation (94 ° C for 30 seconds), annealing (65 ° C for 1 minute) and polymerase reaction (72 ° C for 3 minutes), DNA denaturation (94 ° C for 30 seconds), annealing (45 ° C for 1 minute) A composite cycle consisting of a polymerase reaction (72 ° C., 3.5 minutes) and one intermediate temperature cycle was repeated 15 times.
[0074]
The amplification reaction in the second and subsequent large cycles includes two high-temperature cycles of 94 ° C for 1 minute, 62 ° C for 1 minute, and 72 ° C for 3 minutes, 94 ° C for 30 seconds, 45 ° C for 1 minute, and 72 ° C for 3.5 minutes. This was performed by repeating a combined cycle consisting of one intermediate temperature cycle 13 times.
[0075]
From the reaction solution after each reaction, 5 μl was taken and subjected to 1.2-1.5% agarose gel electrophoresis to analyze the DNA of the amplification product. The results are shown in FIG. Lane 1 shows the results of the amplification product obtained in the first large cycle, and lanes 2 and 3 show the results obtained by amplifying the product in the second large cycle.
[0076]
Further, 150 ng of wheat chromosome DNA was added to 1 ng of the starting DNA sequence, and the product was similarly amplified and analyzed. The results are shown in FIG. Lane 1 is for one large cycle, lanes 2 and 3 are for the second large cycle, and lane 4 is for the sample for lane 3 for the third large cycle.
[0077]
In the figure, "M" indicates a molecular weight marker, and the molecular weight of each band is shown on the left of the figure. Table 1 shows the number of large cycles and the types of primers used in the final cycle for each sample.
[0078]
[Table 1]

[0079]
(Analysis result)
In the case of only one large cycle (lane 1), there are three main amplification products (three bands), but when the amplification cycle is further performed by changing the specific primer, one band is obtained. This band migrates at different positions in lanes 2 and 3 because the position where the specific primer hybridizes is more 3 'in lane 3. Although these bands are scarcely observed in lane 1, it is considered that by performing the second large cycle, the specificity of the target product was improved, and as a result, only one band was amplified.
[0080]
In the case of the T-DNA insertion sequence, in the first large cycle, the number of types of amplification products resulting from the difference in the site to which the non-specific primer binds is 2.2 on average, and usually about 5 types at maximum. is there.
[0081]
The band observed in lane 1 disappeared in the second large cycle, and is presumed to be an amplification product having specific primers at both ends. Although these amplification products differ in size from the bands obtained in lanes 2 and 3, they were obtained simply as a result of hybridization of specific primers in a low-temperature cycle, and the unknown sequence adjacent to the known sequence was amplified. In the sense that it was done, it can be said that it is a target product.
[0082]
It is also possible that a sequence similar to the sequence of TR1 exists in the chromosomal DNA, and that TR1 hybridizes in the vicinity of the sequence, resulting in the generation of an amplification product having specific primers at both ends. It is considered that these were not amplified when TR2 or TR3 was used, and the bands disappeared, because their neighboring sequences were different from the known sequences.
[0083]
In any case, a long DNA sequence such as Arabidopsis chromosomal DNA (about 10 ¹⁸ Base) as a material, it was found that the method of the present invention allows specific amplification of a target DNA sequence.
In addition, wheat chromosome DNA (about 1.5 × 10 ¹⁰ ) Was added in a large amount (lanes 2 to 4), indicating that the method of the present invention has extremely high specificity.
[0084]
Embodiment 2
Determination of base sequence using amplification product directly as template
Next, as an application of the present invention, an example of base sequence determination using an amplification product directly as a template will be described.
[0085]
To 10 μl of the second-cycle reaction solution (containing 100 to 200 ng of DNA) obtained in Example 1, 0.5 μl of (α- ³² P) 30 μl of buffer containing dCTP (〜110 TBq / mmol), 1 unit of AmpliTaq DNA polymerase, 4 μl of dimethyl sulfoxide (DMSO) and 10 pmol of specific primer (TR2) were added. This reaction solution was divided into four portions of 9 μl each, and 1 μl of any of 0.5 mM ddGTP, 5 mM ddATP, 5 mM ddTTP, 5 mM ddCTP was added to each of the divided solutions, and the mixture was added at 94 ° C. for 20 seconds, 60 ° C. for 1 minute, 72 ° C. A reaction in which a cycle of 2 ° C. for 2 minutes was repeated 40 times was performed. The reaction solution was subjected to electrophoresis in the same manner as in the base sequence determination used in the usual dideoxy method, to determine the base sequence.
[0086]
The obtained base sequence is shown in SEQ ID NO: 7. Up to base No. 69 was the sequence of the Ti plasmid used for the transformation, and bases Nos. 70 to 251 were unknown Arabidopsis genomic DNA sequences adjacent to the T-DNA inserted into the genomic DNA. From the results of Southern analysis of Arabidopsis thaliana genomic DNA, it was revealed that the obtained unknown sequence was a repetitive sequence having a basic unit of about 200 base pairs.
[0087]
FIG. 7 shows the results of electrophoresis in another base sequence determination example. In the present invention, the incorporation of dideoxynucleotides and the amplification of the DNA synthesis product are performed at the same time, but it can be seen that a sequence ladder is formed as in the normal dideoxy method.
[0088]
Embodiment 3
Amplification of P1 vector insert
Next, as an example of the present invention, an example of amplification of a sequence of a DNA inserted into a P1 vector will be described.
[0089]
(1) Preparation of E. coli genomic DNA into which P1 vector has been inserted
A colony of Escherichia coli harboring the P1 vector containing the Arabidopsis chromosomal DNA fragment was picked with a toothpick or the like, and suspended directly in the reaction solution to use as a template for the amplification reaction. Alternatively, P1 plasmid DNA may be prepared from Escherichia coli cultured in a liquid medium by an alkaline lysis method or the like.
[0090]
(2) Composition of reaction solution for amplification reaction
Three kinds of oligonucleotides each having a part of the sequence of the Sp6 promoter existing near the cloning site of the P1 vector were prepared and used as specific primers. In each primer, Sp6-1 (SEQ ID NO: 8: 22 bases, Tm 62.1 ° C) and Sp6-2 (SEQ ID NO: 9: 17 bases, Tm52. 8 ° C.) and Sp6-3 (SEQ ID NO: 10: 15 bases, Tm 42.4 ° C.).
[0091]
On the other hand, W3 used in Example 1 was used as a non-specific primer.
The DNA amplification reaction was performed twice or more while changing the specific primer. The composition of the reaction solution in each reaction was the same as in Example 1 except for the specific primer. For the specific primer, Sp6-1 was used in the first large cycle, and Sp6-2 or Sp6-2 was used in the second large cycle. Sp6-3 was used at 0.2 μM each.
[0092]
(3) Reaction temperature cycle of amplification reaction
The first DNA amplification reaction was performed in the same manner as in Example 1. The second amplification reaction was performed by repeating a medium temperature cycle at 94 ° C. for 1 minute, 45 ° C. for 1 minute, and 72 ° C. for 3.5 minutes 22 times.
[0093]
From the reaction solution after each reaction, 5 μl was taken and subjected to 1.2-1.5% agarose gel electrophoresis to analyze the DNA of the amplification product. FIG. 8 shows the results.
Lanes 1 and 2 show the results obtained by performing amplification in one large cycle using Sp6-1 as a specific primer, using different P1 clones as templates. Lanes 3 and 4 show the results of the second large cycle using the sample of lane 1 as a specific primer and Sp6-2 and Sp6-3, respectively. Lanes 5 and 6 show the samples of lane 2 as Sp6-2 and Sp6-2, respectively. It is the result about what performed the 2nd large cycle using each Sp6-3.
[0094]
(Analysis result)
In the sample of lane 1, a large number of bands are observed, but when a large cycle is repeated with a different primer, one band is formed (lanes 3 and 4). On the other hand, in the sample of lane 2, one large cycle has already been collected into one band. This result indicates that only one large cycle is sufficient depending on the material used for amplification. In particular, when the template sequence is 10 ⁵ It is thought that one time is sufficient for those having a length of about a base. Therefore, the amplification product is analyzed for the reaction solution after the first large cycle, and if insufficient, the large cycle may be performed twice or three times by further changing the primers.
[0095]
Embodiment 4
Next, as an example of the present invention, an example will be described in which the sequence of the DNA inserted into the P1 vector is amplified by a different reaction temperature cycle from that in Example 3.
[0096]
(1) Preparation of E. coli genomic DNA into which P1 vector has been inserted
This was performed in the same manner as in Example 3.
[0097]
(2) Composition of reaction solution for amplification reaction
As a specific primer, Sp6-1 used in Example 3 was used. On the other hand, W2 used in Example 1 was used as a non-specific primer. The composition of the reaction solution in the DNA amplification reaction was the same as in Example 3.
[0098]
(3) Reaction temperature cycle of amplification reaction
In the first DNA amplification reaction, the (third step) of Example 1 was repeated 30 times at a medium temperature cycle consisting of DNA denaturation (94 ° C. for 30 seconds), annealing (58 ° C. for 1 minute), and polymerase reaction (72 ° C. for 3 minutes). Repeated. From the reaction solution after the reaction, 5 μl was taken and subjected to 1.2-1.5% agarose gel electrophoresis to analyze the DNA of the amplification product. FIG. 9 shows the results.
[0099]
(Analysis result)
This result indicates that the target amplification product can be obtained even when the third step is performed only by the intermediate temperature cycle. In particular, in the case where it is not necessary to form a single band in practice, the object can be achieved by the method of this embodiment.
[0100]
Embodiment 5
Subsequently, an amplification example in which the third step was performed only in the medium temperature cycle for one strain of the Arabidopsis chromosome library into which the T-DNA obtained in Example 1 was inserted will be described.
[0101]
(1) Preparation of plant having T-DNA insertion sequence
Performed in the same manner as in Example 1.
[0102]
(2) Composition of reaction solution for amplification reaction
As the specific primer, TR2 or TR3 used in Example 1 was used. On the other hand, W3 used in Example 1 was used as a non-specific primer. The same composition as in Example 1 was used for the composition of the reaction solution in the DNA amplification reaction.
[0103]
(3) Reaction temperature cycle of amplification reaction
In the amplification reaction, the (third step) of Example 1 was repeated 30 times at a medium temperature cycle consisting of DNA denaturation (94 ° C. for 30 seconds), annealing (58 ° C. for 1 minute), and polymerase reaction (72 ° C. for 3 minutes). 5 μl of each of the amplification reaction solutions using TR2 or TR3 as specific primers was subjected to 1.2-1.5% agarose gel electrophoresis, and the DNA of the amplification product was analyzed. The results are shown in FIG.
[0104]
(Analysis result)
When using any of the specific primers, the amplification product was obtained as a substantially single band. The amplification product when TR3 was used (lane 2) was smaller than the amplification product when TR2 was used (lane 1), and the difference was the distance between sites in T-DNA having the same sequence as TR2 and TR3. It was consistent with 64 bases. From this, it was found that each of the amplification products obtained above was a target product.
[0105]
This result indicates that even when a large chromosome such as Arabidopsis is used as a material, the target amplification product can be obtained only by the medium temperature cycle.
[0106]
【The invention's effect】
According to the method of the present invention, an unknown sequence adjacent to a known sequence can be efficiently amplified without requiring complicated operations such as circularization of DNA and ligation of oligonucleotides.
[0107]
[Sequence list]
[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[Brief description of the drawings]
FIG. 1 is a diagram showing the principle of a conventional PCR method.
FIG. 2 is a diagram showing the principle of a known modified PCR method.
FIG. 3 is a diagram showing an amplification cycle used in the present invention.
FIG. 4 is a diagram showing the concept of the method of the present invention.
FIG. 5 is a diagram showing a part of a T-DNA sequence and a position where a specific primer hybridizes thereto.
FIG. 6 is an electrophoretogram showing the results of analyzing a DNA amplification product.
FIG. 7 is an electrophoretogram of base sequence determination.
FIG. 8 is an electrophoretogram showing the results of analyzing a DNA amplification product.
FIG. 9 is an electrophoretogram showing the results of analyzing a DNA amplification product.
FIG. 10 is an electrophoretogram showing the results of analyzing a DNA amplification product.
[Explanation of symbols]
In the figure, M indicates a marker, and the number on the left of the figure indicates a molecular weight.

Claims (4)

The DNA sequence and an oligonucleotide primer having a partial sequence of the DNA sequence are mixed in a DNA polymerase reaction solution containing DNA polymerase and deoxyribonucleotide, and the DNA sequence is denatured, and the DNA sequence is mixed with the DNA primer. A method for amplifying the DNA sequence by repeating annealing and an amplification cycle comprising a DNA polymerase reaction,
(A) an oligonucleotide primer and any of the annealing line cormorants amplification cycle in a non-specific hybridising temperature to the position of the DNA sequence,
(B) a plurality of amplification cycles, wherein the annealing is performed at a temperature at which the oligonucleotide primer specifically hybridizes to a DNA sequence complementary to the sequence,
Look including the door in this order, amplification of a DNA sequence which is characterized in that only one amplification cycle (A). A DNA sequence, an oligonucleotide primer having a sequence identical to a part of the DNA sequence (specific primer), and an oligonucleotide primer having an arbitrary sequence having a lower Tm than the specific primer (non-specific primer) Are mixed in a DNA polymerase reaction solution containing DNA polymerase and deoxyribonucleotide, and the DNA is subjected to denaturation of the DNA sequence, annealing of the DNA sequence with a DNA primer, and an amplification cycle consisting of a DNA polymerase reaction, thereby repeating the DNA cycle. In a method of amplifying a sequence,
(A) amplification cycles annealing at a temperature nonspecific primers nonspecifically hybridized to DNA sequences,
(B) multiple amplification cycles of annealing at a temperature at which the specific primer specifically hybridizes to the DNA sequence;
(C) multiple amplification cycles of annealing at a temperature at which the non-specific primer specifically hybridizes to a DNA sequence complementary to that sequence;
Look including the door, amplification of DNA sequences of amplification cycles carried out only once, and performing after the amplification cycles (B) and (C) an amplification cycle (A) of (A). 2. The method for amplifying a DNA sequence according to claim 1, wherein an oligonucleotide primer having a sequence near the downstream side of the DNA sequence having the same sequence as the oligonucleotide primer is added to the polymerase reaction solution. 3. The method for amplifying a DNA sequence according to claim 2, wherein an oligonucleotide primer having a sequence near the downstream side of the DNA sequence having the same sequence as the specific primer is added to the polymerase reaction solution.

Images