JP2004313112A - Method for preparing dna design information and method for synthesizing dna - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method capable of obtaining a DNA without preparing a conventional gene library and readily synthesizing the DNA from data comprising DNA sequence information. <P>SOLUTION: The subject method for preparing DNA design information on DNA synthesis comprises (1) a step for comparing a partial sequence of a DNA desired to synthesize with an object DNA registered in object DNA database and extracting identical partial sequences (p1-2 and v3-4) and (2) a step for alternately arranging total even number of chains in the order of a sense chain and an antisense chain, starting from a sense chain on 5' end side by using base sequences of template single strands of the extracted partial sequences (p1, p2, v3 and v4) and non-extracted partial sequences (p3-p8), and arranging these chains so that adjacent sense chains and antisense chains and 5' ends and 3' ends have mutually complementary binding regions. The method for synthesizing DNA comprises using the resultant DNA design information. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

（式中、ａは糊代の塩基数、ｎは鎖間の塩基数、ＧＣ塩基数は鎖間のＧＣ含量、ＡＴ塩基数は鎖間のＡＴ含量、Ｔｍ’は鎖間のＡＴが最も続く場所のＴｍ値である）が成り立つ。あるいは、近似式（２）：
【数２】

（式中、ａは糊代の塩基数、ＧＣ塩基数は糊代のＧＣ含量、ＡＴ塩基数は糊代のＡＴ含量である）
が成り立つ。
【００２９】
次に、抽出した部分配列の隣接鎖間距離Ｘ（例えば、図５のｐ１−ｐ２間）測定する（Ｓ６０３）。ここで、Ｘ≧０ならば隣接鎖は重ならず、Ｘ＜０ならば隣接鎖が重なることを意味する。Ｘ≧０の場合はＳ６０５に進み、それ以外はＳ６０４にて鎖間調整する。短鎖が重なっている場合は、重なっているどちらかの隣接鎖を間引き、長鎖の場合は重なり部分Ｘを設定Ｔｍから予測される糊代長ａ、または０以下まで削る。
【００３０】
Ｓ６０５に進み、鎖間を埋めるための最適な補充鎖（オリゴオブジェクトＤＮＡ）を算出する。個々の予測糊代長ａのＴｍを上記式（１）または（２）で計算し、Ｔｍが設定値からずれていれば、糊代長を変更することで設定Ｔｍに近づける。鎖間が大きく空いていてオリゴＤＮＡ合成機の合成限界（設計時に設定した鎖長）を超えるときには、複数のオリゴオブジェクトＤＮＡを割り当てる。Ｓ６０６に進み、オリゴオブジェクトＤＮＡ、短鎖オブジェクトＤＮＡまたは長鎖オブジェクトＤＮＡの塩基配列の糊代部分の配列が異なるかをチェックする。同一の糊代を作らないことによりミスマッチ増幅を回避する。上記の条件に反する場合には、Ｓ６０４に戻って鎖間調整を繰り返す。Ｓ６０７で、ＤＮＡ合成のための設計情報を出力する。図５に、ＤＮＡ設計情報の処理結果の一例を示す。ここでは、抽出した部分配列（ｐ１、ｐ２、ｖ３およびｖ４）の鎖間を埋めるために、ｐ３〜ｐ８のオリゴオブジェクトＤＮＡを配置している。設計情報の出力方式は、紙媒体でも電子媒体でもよい。
【００３１】
（３）部分ＤＮＡおよびオリゴオブジェクトＤＮＡを準備する工程
上記で得られたＤＮＡ設計情報に基づいて、部品となる短鎖オブジェクトＤＮＡを短鎖オブジェクトＤＮＡ格納庫から出庫する。同じように、部品となる長鎖オブジェクトＤＮＡを長鎖ＤＮＡ格納庫から出庫する。長鎖オブジェクトＤＮＡの一部分が必要な場合には、必要な部分配列をプライマーで挟んでＰＣＲ法で切り出す。
【００３２】
データベースに登録されておらず、合成の必要なオリゴオブジェクトＤＮＡは、オリゴＤＮＡ自動合成機で合成する。オリゴＤＮＡ自動合成機は、入力された塩基配列を基に、チューブで結合され試薬、溶媒を入れたボトルのバルブを開閉させながら固相上に必要な試薬を順に送り出す装置である。ＤＮＡ合成機は、チューブから送り出される、固相上で反応させたい基以外を保護したヌクレオチド同士を、縮合剤を用いて結合させ、五炭糖の３’と５’の間でリン酸ジエステル結合を形成させるサイクルを次々と繰り返すことにより、３’→ ５’へＤＮＡ鎖を合成連結させる。オリゴオブジェクトＤＮＡ合成は、オリゴＤＮＡ受託合成メーカーに依頼して調達することもできる。
【００３３】
（４）モジュール化工程
モジュール化とは、工程（３）のＤＮＡ設計情報に基づいて、部分ＤＮＡおよびオリゴＤＮＡの連続鎖からなる２〜１０本、好ましくは２〜６本のモジュール単位を連結することをいう。本発明のＤＮＡ設計・合成方法では、部分ＤＮＡを一旦モジュール化し、そのモジュールをさらに連結することで、ＤＮＡの増幅に偏りが生じて全長ＤＮＡが得られないなどの事態を回避することができる。図５の設計情報例を用いたモジュール化の概念図を図７に示す。登録された短鎖オブジェクトＤＮＡ（ｐ１〜ｐ２）および合成オリゴオブジェクトＤＮＡ（ｐ３〜ｐ６）でモジュール１を形成し、登録された部分一致長鎖オブジェクトＤＮＡ（ｖ３〜４）でモジュール２を形成し、そして合成オリゴオブジェクトＤＮＡ（ｐ７〜ｐ８）でモジュール３を形成している。
【００３４】
以下に、ＰＣＲ法によるモジュール化を具体的に説明する。短鎖・長鎖オブジェクトＤＮＡおよび合成したオリゴオブジェクトＤＮＡ、ＰＣＲ法の原料であるｄＮＴＰｓ（デオキシヌクレオチド類）、塩、ならびに耐熱性ＤＮＡポリメラーゼで反応液を作る。耐熱性ポリメラーゼは、細菌由来のｐｏｌ Ｉ型酵素（例えばＴａｑ ＤＮＡポリメラーゼ）、始原菌由来のα型酵素（例えばｐｆｕ ＤＮＡポリメラーゼ、ＫＯＤ ＤＮＡポリメラーゼ）のいずれでもよく、さらにこれらを二種混合したものでもよい。これらは試薬メーカーから市販されているもの、例えばＴａｋａｒａ Ｅｘ Ｔａｑ、Ｐｙｒｏｂｅｓｔ ＤＮＡ Ｐｏｌｙｍｅｒａｓｅ（以上、ＴａＫａＲａ社製）、ＫＯＤ＋（ＴＯＹＯＢＯ社製）などを制限なく使用することができる。ＤＮＡポリメラーゼの濃度は、通常、０．２５単位／μｌである。バッファーは、前記市販の耐熱性ポリメラーゼに同梱された１０×バッファーを使用すればよい。例えばＴａｑポリメラーゼの１０×バッファーには、通常、Ｔｒｉｓ−ＨＣｌ１０〜２５ｍＭ、ＫＣｌ５０ｍＭ、およびＭｇＣｌ_２１〜５．０ｍＭが含まれている。
【００３５】
鋳型ＤＮＡの濃度は、通常、数十ｐｇ／μｌ〜数ｎｇ／μｌである。プライマーは、通常、０．２〜１０μＭ用意する。プライマーが少なすぎると反応が進まず、逆に多すぎると非特異的な増幅の原因となる場合がある。ｄＮＴＰｓは各０．２〜２ｍＭ用意する。
【００３６】
上記の反応液を加温し、鋳型ＤＮＡの二本鎖を乖離させる。次に、それぞれの一本鎖の相補的部分に重複するプライマー（隣の鋳型ＤＮＡ）が二本鎖を形成（アニール）する温度に冷却する。最後に、相補部分を起点として耐熱性ポリメラーゼ酵素によって５’→３’方向にＤＮＡ伸長を行なう。このサイクルを繰り返すことにより、連続した部分ＤＮＡおよびオリゴＤＮＡのモジュール化を達成する。
【００３７】
ＰＣＲ法の条件に関して、鋳型兼プライマーＤＮＡのアニール温度は、設計時に設定した糊代のＴｍに設定する。ＰＣＲ法の各ステップ（変性、アニール、伸長）にかける時間と温度は、例えば熱変性（９３〜９７℃）で３０秒、アニール（糊代のＴｍ）で３０秒、伸長反応（６８〜７２℃）で０．５〜１分×増幅する鎖長（ｋｂ）である。サイクル数は、合成したいＤＮＡ量に依存するが、一般に３０〜４０サイクルでよい。
【００３８】
（５）モジュール連結工程
上記で得られた連続するモジュールを、通常、２〜１０本、好ましくは２〜６本ＰＣＲ法で連結する。ＰＣＲ法の手順および条件は、（４）のモジュール化工程と同様である。図７の例では、プライマーとしてｐ１およびｐ８を用いて、モジュール１〜３を連結している。オブジェクトＤＮＡにベクターを使用した場合など、合成ＤＮＡを閉環する必要があるときには、セルフライゲーション操作を行う。Ｔ４ファージのＤＮＡカイネ−スおよびリガーゼが２本鎖ＤＮＡの平滑末端同士を直接結合するのに用いられる。市販のライゲーションキットを使用することもできる。
【００３９】
このように、本発明の設計・合成方法は、目的遺伝子の遺伝子ライブラリーを作製およびスクリーニングする必要がないので、１０ｋｂｐ以上の長鎖ＤＮＡでも３〜４日という短期間で作製可能である。
【００４０】
（６）サブクローニング工程
サブクローニングは、ＤＮＡ合成時にベクターＤＮＡをオブジェクトとして組み込んでおけば省略することができる。しかし、上記合成ＤＮＡにベクターＤＮＡが結合していない場合や、機能解析やタンパク質発現のためにベクターを乗せ換えるなどの必要がある場合には、以下の方法でサブクローニングする。
【００４１】
クローニングベクターの挿入部位に、目的特定遺伝子区分を挿入して使用する。クローニングベクターに挿入する方法は、従来公知の方法を使用することができる。挿入の準備として、クローニングベクターを特定部位で切断する制限酵素により、クローニングベクターの挿入目的部位を切断する。ＰＣＲ法により含成された合成ＤＮＡの末端を、クローニングベクターの挿入目的部位と連結可能なように制限酵素などで加工する。通常は、プライマー設計時にスムースに連結できるように細工する。最後に、ＰＣＲ法により含成されたＤＮＡと特定部位で切断したクローニングベクターとを、ＤＮＡライゲース酵素を用いて連結する。
【００４２】
（７）形質転換工程
次いで、（５）または（６）のキメラベクターを宿主細胞に導入する。宿主細胞は、クローニングベクターが安定かつ自律的に増殖可能なものであれば、制限なく使用することができる。宿主細胞の例には、大腸菌、ストレプトマイセス、サルモネラ・チフィムリウムなどの細菌；酵母などの真菌；ショウジョウバエＳ２、シロナヨトウＳｆ９などの昆虫細胞；ＣＨＯ、ＣＯＳ、Ｂｏｗｅｓ黒色腫細胞などの動物細胞；ならびに植物細胞が挙げられる。
【００４３】
ベクターの細胞への導入方法として、エレクトロポレーション、ＤＥＡＥ−デキストラン、リン酸カルシウム沈殿、リポソーム媒介融合（リポフェクション）、マイクロインジェクション、複製能のないウイルスによる形質導入などがある。宿主細胞に応じて導入法を使い分ける。
【００４４】
キメラベクターが挿入された宿主細胞は、適当な培地上にプレーティングを行なって培養し、単一のベクターを有する単一宿主細胞由来のコロニーを形成させる。培地中には、細胞の生育に必要な炭素源、窒素源、無機物などを含有させる。炭素源としては、グルコース、シュクロースなどの糖類；グリセロールなどのアルコール類；有機酸などが適宜使用される。窒素源としては、アンモニアまたはアンモニウム塩類、アミン類、その他の窒素化合物、あるいはペプトン、大豆加水分解物、発酵菌体分解物などの天然窒素源を用いることができる。無機塩としては、例えばリン酸一水素カリウム、リン酸二水素カリウム、硫酸マグネシウムがある。添加物として酵母、ビタミン類、成長促進因子などを添加してもよい。目的のキメラベクターをもった宿主細胞のみを培養するための選択マーカーとして、また雑菌の繁殖を防ぐために、ベクターの薬剤耐性遺伝子に対応する抗生物質を培地中に加える。例えば、アンピシリン耐性遺伝子を有するベクターを含む大腸菌を培養するには、大腸菌用ＬＢ培地に抗生物質アンピシリンを添加する。
【００４５】
クローニングベクターは宿主細胞内で多量に自己増幅する。ＤＮＡの合成が適確に行なわれたか否か、目的物の品質はどうかなどをチェックする方法には、宿主細胞コロニーのミニプレップ処理などでクローニングベクターを精製した後、挿入ＤＮＡの全長または部分長をベクターから切り出し、切り出されたＤＮＡを電気泳動でチェックする方法、ＰＣＲ方法によるチェック、コロニーハイブリダイゼーション法、カラーセレクションなどがある。
【００４６】
（８）遺伝子発現工程
宿主細胞に目的の合成ＤＮＡの産物（タンパク質）を作らせたい場合には、ベクターに発現ベクターを使用すればよい。発現ベクターは、プロモーターおよびターミネーターと連結したベクターである。プロモーターは、遺伝子の発現に用いる宿主に対応して適切なプロモーターであればいかなるものでもよい。形質転換する際の宿主がバチルス属菌である場合は、ＳＰＯ１プロモーター、ＳＰＯ２プロモーター、ｐｅｎＰなどが利用でき、動物細胞である場合には、ＳＶ４０由来のプロモーター、レトロウイルスのプロモーター、メタロチオネインプロモーター、ヒートショックプロモーター、サイトメガロウイルスプロモーター、ＳＲαプロモーターなどが利用できる。市販の発現ベクターには、例えばｐＵＣ系、ｐＢｌｕｅｓｃｒｉｐｔ ＩＩ、ｐＥＴおよびλｇｔ１１がある。合成ＤＮＡはリボソーム結合部位（ＳＤ配列）の後に組み込まれる。
【００４７】
遺伝子発現の形態には、宿主細胞内にタンパク質を生産させる方法、宿主細胞外にタンパク質を分泌させる方法、宿主細胞外膜上にタンパク質を生産させる方法などがある。タンパク質を宿主細胞で発現させた後、培養物から目的タンパク質を単離精製する。公知の分離操作、例えば、尿素などの変性剤や界面活性剤による処理、超音波処理、酵素消化、塩析、溶媒沈殿法、透析、遠心分離、限外濾過、ゲル濾過、ＳＤＳ−ＰＡＧＥ、等電点電気泳動、イオン交換クロマトグラフィー、疎水性クロマトグラフィー、アフィニティークロマトグラフィー、逆相クロマトグラフィーなどを適宜併用することができる。
【００４８】
前記クローニングベクターをマウスなどの動物の受精卵に導入し、または動物に直接注入することにより、トランスジェニック動物やノックアウト動物を作ることも可能である。
【００４９】
（９）ＤＮＡ保管・登録工程
上記工程で得られた新規なオリゴオブジェクトＤＮＡ、合成ＤＮＡ、合成ＤＮＡを組み込んだキメラベクターは、その一部を格納庫に保管する。例えば、共有を許される物は共有用の格納庫に保管し、一方、共有を許されない物は預かり用の格納庫に保管する。さらに、これらの保管されたオブジェクトＤＮＡの塩基配列情報を、格納情報と関連付けて、短鎖・長鎖オブジェクトデータベースに登録する。登録によって、次回のＤＮＡ合成に既存の資源を有効活用することが可能となる。
【００５０】
次に、本発明のＤＮＡ設計・合成方法の変形例を示す。図８は、ＤＮＡ合成の依頼元、ＤＮＡ設計情報の作製拠点およびＤＮＡの合成拠点が別個に存在する例である。本発明のＤＮＡ設計・合成方法では、短鎖・長鎖オブジェクトＤＮＡデータベースを充実させるほど効果が増すので、図８のようにＤＮＡ設計およびＤＮＡ合成の専門拠点を置くことは好ましい。
【００５１】
この使用態様では、ＤＮＡ合成を希望する依頼者は端末８０１からインターネットあるいは通信回線を介してＤＮＡ合成システムへアクセスする。ＤＮＡ合成システムは、サーバコンピュータ８０３、短鎖オブジェクトＤＮＡデータベース８０４、および長鎖オブジェクトＤＮＡデータベース８０５（適宜、共有用データベースと依頼者特定データベースに分かれている）からなり、さらにオブジェクトＤＮＡ格納庫８０７（適宜、共有または依頼者特定格納庫に分かれている）およびオリゴＤＮＡ自動合成機８０８へＤＮＡ設計情報８０６を送付する手段を有する。
【００５２】
図９は、上記ＤＮＡ合成システムにおける上記三者間の手続きの流れを示す図である。研究者、技術者などの依頼者は、依頼者端末８０１からインターネットを介してゲノムネットなどの遺伝子情報データベース８０２にアクセスし、また、学術情報誌を入手することによって、合成したいＤＮＡの配列情報を取得する（Ｓ９０１）。あるいは、用途や研究目的に沿って既知のＤＮＡ配列情報を基に一部改変したＤＮＡ配列情報を作製する。
【００５３】
依頼者は、合成を依頼したいＤＮＡ配列情報をインターネット、通信回線などを通じてＤＮＡ合成システム（サーバコンピュータ８０３）に送る（Ｓ９０２）。ＤＮＡ合成システムのサーバコンピュータ８０３が配列情報を受け取り、短鎖オブジェクトＤＮＡデータベース８０４および長鎖オブジェクトＤＮＡデータベース８０５を検索し、一致または部分一致する部分ＤＮＡを抽出する（Ｓ９０３）。得られた部分ＤＮＡの塩基配列情報とそれらの鎖間情報を用いて、ＤＮＡ合成のための設計情報８０６を作製する（Ｓ９０４）。作製されたＤＮＡ設計情報８０６は、依頼者へ送るか、あるいはＤＮＡ合成ために生産拠点へ送付する。ＤＮＡ設計情報の作製の情報拠点、オリゴＤＮＡ合成、ＰＣＲ、シーケンスなどの拠点が離れていてもかまわない。
【００５４】
ＤＮＡ生産拠点では、担当者が設計情報８０６に基づいて、短鎖オブジェクトＤＮＡ格納庫および長鎖ＤＮＡオブジェクトＤＮＡ格納庫から必要なオブジェクトＤＮＡを出庫し、足りないオリゴオブジェクトＤＮＡについてはオリゴＤＮＡ自動合成機で合成する（Ｓ９０５）。得られた部品とＰＣＲ法を用いてオブジェクトＤＮＡのモジュール化とモジュール連結を行なう（Ｓ９０６、Ｓ９０７）。連結された合成ＤＮＡ８１０は、検査後、その状態であるいは凍結乾燥などの処理を施した後に依頼者へ届ける。また、適宜のサブクローニング後（Ｓ９０８）、クローニングベクターを宿主細胞に接種して形質転換し（Ｓ９０９）、形質転換細胞内でベクターを自律増殖させ、あるいは遺伝子を発現させてタンパク質を得て（Ｓ９１０）から、ＤＮＡ、タンパク質などを依頼者に届けてもよい。形質転換したノックアウト動物またはやトランスジェニック動物８１１を供給することもできる。
【００５５】
本発明のＤＮＡ設計・合成方法は、上記の使用態様以外にも応用または変形が可能である。その一例は、オブジェクトＤＮＡデータベース；合成したいＤＮＡの部分配列と、オブジェクトＤＮＡデータベースに登録されたオブジェクトＤＮＡとを照合して、一致した部分配列を抽出するデータベース抽出手段；ならびに抽出された部分配列および非抽出の部分配列の鋳型一本鎖の塩基配列を用いて、５’末端側のセンス鎖から始めてセンス鎖、アンチセンス鎖の順に交互に合計偶数本配列し、しかも隣接するセンス鎖とアンチセンス鎖との５’末端同士および３’末端同士が互いに相補的な結合領域を有するように配列する設計情報作製手段を備えたＤＮＡ設計システムである。また別の一例は、（１）合成したいＤＮＡの部分配列と、オブジェクトＤＮＡデータベースに登録されたオブジェクトＤＮＡとを照合して、一致した部分配列を抽出する工程、ならびに（２）抽出された部分配列および非抽出の部分配列の鋳型一本鎖の塩基配列を用いて、５’末端側のセンス鎖から始めてセンス鎖、アンチセンス鎖の順に交互に合計偶数本配列し、しかも隣接するセンス鎖とアンチセンス鎖との５’末端同士および３’末端同士が互いに相補的な結合領域を有するように配列する工程をコンピュータに実行させるためのプログラムである。さらにまた別の一例は、該プログラムを記録したコンピュータ読取可能な記録媒体である。
【００５６】
【実施例】
以下に、実施例を用いて本発明のＤＮＡ設計・合成方法をさらに詳細に説明する。
〔実施例１〕
塩基配列（配列番号１）の概要を図１０に示すＤＮＡの合成を行なった。この塩基配列は、図１１の制限酵素地図に示されるように、クローニングベクターｐＢｌｕｅｓｃｒｉｐｔ ＩＩ ＳＫ（＋）（２９６１ｂｐ、以下「ｐＢＳＫ＋」と略す）のマルチクローニングサイト（ＭＣＳ）に、１１２ｂｐのＤＮＡ断片を挿入した全長３０７３ｂｐの環状のＤＮＡである。図１０のＴ７−Ｔ３間がＭＣＳであり、また、下線で示した領域が今回ｐＢＳＫ＋へ挿入する配列である。
【００５７】
（１）データベース抽出
目的の環状ＤＮＡを、図３のシステムのデータベース抽出処理制御部３０３に、図４に示す手順でデータベース抽出処理を実行させた。あらかじめ、短鎖オブジェクトデータベースには、既存の各種プライマーを登録しておき、一方、長鎖オブジェクトデータベースには、ｐＢＳＫ＋を登録しておいた。データベース抽出の結果を図１２に示す。短鎖オブジェクトＤＮＡデータベースからは、図１２のＤＮＡ１（斜線矢印部分）が見つかった。長鎖オブジェクトＤＮＡデータベースからは、図１２のＤＮＡ２（白抜き矢印部分）が見つかった。ＤＮＡ２がｐＢＳＫ＋であることから、本データベース抽出処理が順調に遂行されたことを確認した。
【００５８】
（２）ＤＮＡ設計情報作製
上記抽出された部分ＤＮＡとその鎖間情報とから、図３のシステムの設計情報作製処理制御部３０４に、図６に示す手順でＤＮＡ設計情報作製処理を実行させた。設計条件として、モジュール内およびモジュール間のＴｍを６０℃とし、オリゴオブジェクトＤＮＡの最大設計長を５９塩基とした。短鎖オブジェクトＤＮＡデータベースから抽出したＤＮＡ１を使用すると、糊代が足りなくなるため間引きし、新たにオリゴオブジェクトＤＮＡとしてＰ１〜Ｐ４を作るという演算結果を得た。ＤＮＡ合成のための設計情報の概観を図１３に示す。また、各オブジェクトＤＮＡの塩基配列情報を図１４および１５に示す。オブジェクトＤＮＡのモジュールの組み合わせとＴｍを表１にまとめた。
【００５９】
【表１】

【００６０】
（３）オブジェクトＤＮＡの準備
上記設計情報に従ってオブジェクトＤＮＡを準備した。まず、上記Ｐ１〜Ｐ４を、オリゴＤＮＡ合成機を用いて合成した。次いで、Ｐ５およびＰ６の切り出しに用いるプライマーＶ１（配列番号７）およびＶ２（配列番号８）を、オリゴＤＮＡ合成機を用いて合成した。長鎖オブジェクトＤＮＡの格納庫からｐＢＳＫ＋を出庫し、上記プライマー（Ｖ１およびＶ２）を用いてＰＣＲ法でＰ５およびＰ６を切り出した。ＰＣＲ法の反応液の組成および温度サイクルを、それぞれ、表２および図１６に示す。
【００６１】
【表２】

【００６２】
（４）モジュール化
Ｐ１〜Ｐ４を鋳型としたＰＣＲ法（プライマーとしてＰ１およびＰ４を使用）によって、モジュール２を作製した。ＰＣＲ法の反応液の組成を表３に示す。温度サイクル条件は図１６と同様に指定した。
【００６３】
【表３】

【００６４】
図１７にモジュール１および２の電気泳動結果を示す。図中、レーン▲１▼はｐＢＳＫ＋をプライマーＶ１、Ｖ２でＰＣＲを行った後の産物を電気泳動したものであり、２０００〜３０００塩基あたりにバンドが確認された。レーン▲２▼はＰ１〜Ｐ４をプライマーＰ１、Ｐ４でＰＣＲを行った産物を電気泳動したものであり、５００塩基未満のバンドが確認された。
【００６５】
（５）モジュール１とモジュール２との連結
（４）のモジュール１および２をエタノール沈殿後、サンプル濃度を調整し、次いで、モジュール１および２を鋳型としてＶ１およびＰ１プライマーでＰＣＲを行った。表４および図１８に反応液の組成および温度サイクル条件を示す。
【００６６】
【表４】

【００６７】
上記ＰＣＲ産物のサンプル濃度を変えて電気泳動した結果を図１９に示す。図１９において、モジュール１、モジュール２、およびモジュール１＋２（モジュール１と２との連結）と予想されるバンドを確認した。
【００６８】
（６）セルフライゲーション
電気泳動結果から、モジュール１＋２のバンドを切り出し、Ｇｅｌ Ｅｘｔｒａｃｔｉｏｎ Ｋｉｔ（Ｑｉａｇｅｎ社製）で精製後、末端部ブラントエンド（７１５、７１６）のセルフライゲーション（１６℃×２時間）を行って、直鎖状ＤＮＡを閉環した。ライゲーション反応液の組成を表５に示す。
【００６９】
【表５】

【００７０】
（７）形質転換
セルフライゲーションによって末端同士を再結合したキメラベクターを、コンピテントな状態の大腸菌（ＤＨ５α株）に形質転換した。形質転換された大腸菌はＬＢ−ＡＭＰ培地で順調に増殖した。培地から１０個の大腸菌コロニーを選択してミニプレップした後、得られた精製物を電気泳動にかけた。その結果を図２０に示す。電気泳動結果の中からレーン▲６▼、▲７▼および▲８▼を選択し、そのシーケンスを行った。その際、ＰＴ７プライマーでインサート部分を順方向にシーケンスし、ＰＴ３プライマーでインサート部分を逆方向にシーケンスした。シーケンスの結果から目的配列がマルチクローニングサイトに挿入されていることを確認した。
【００７１】
【発明の効果】
本発明のＤＮＡ設計・合成方法は、ＤＮＡの複製ではなく合成であるため、従来のような遺伝子ライブラリーを準備して、スクリーニングする必要がない。したがって、所望のＤＮＡを短期間で取得することが可能になる。本発明の方法はまた、合成するＤＮＡ配列情報を変更することによって自由な遺伝子設計が容易になる。さらに制限酵素処理を省略できるので、クローニングベクターの作製が従来方法よりも簡単になる。クローニングベクター自体の加工も可能となる。そして、一度作製したオリゴＤＮＡ、合成ＤＮＡ（ＰＣＲ産物）などの再利用が可能である。オブジェクトデータベースを充実させることによって、ＤＮＡ合成コストの一層の削減が図れる。
【００７２】
【配列表】

【図面の簡単な説明】
【図１】本発明のＤＮＡ設計・合成方法の全工程を示したフローチャートである。
【図２】ＤＮＡ塩基配列情報からデータベースを検索する概念図である。
【図３】本発明のＤＮＡ設計情報の作製方法を実行するコンピュータシステムの一例である。
【図４】図３のシステムにおいて、データベース抽出処理制御部が実行する作業のフローチャートである。
【図５】本発明のＤＮＡ設計・合成方法における設計情報作製の概念図である。
【図６】図３のシステムにおいて、設計情報作製処理制御部が実行する作業のフローチャートである。
【図７】本発明のＤＮＡ設計・合成方法におけるモジュール化の概念図である。
【図８】本発明のＤＮＡ設計・合成方法の応用例である。
【図９】図８のＤＮＡ合成システムにおける手続きの流れを示すフローチャートである。
【図１０】本発明に従う実施例１で合成したいＤＮＡの塩基配列の説明図である。
【図１１】図１０の合成したいＤＮＡの塩基配列を制限酵素地図で表したものである。
【図１２】図１０のＤＮＡ塩基配列情報をオブジェクトＤＮＡデータベース検索した結果を示す図である。
【図１３】図１０のＤＮＡ合成のための設計情報を示した図である。
【図１４】ＤＮＡ合成に用いるオブジェクトＤＮＡ（ｐ５）の塩基配列である。
【図１５】ＤＮＡ合成に用いるオブジェクトＤＮＡ（ｐ１〜ｐ４）の塩基配列である。
【図１６】本発明に従う実施例１において、オブジェクトＤＮＡ（ｐ５、ｐ６）を切り出すために行ったＰＣＲ法の温度サイクルを示す図である。
【図１７】実施例１のモジュール１および２の電気泳動結果を示す図面代用写真である。
【図１８】本発明に従う実施例１において、モジュール１と２とを連結するために行ったＰＣＲ法の温度サイクルを示す図である。
【図１９】図１８のＰＣＲ産物の電気泳動結果を示す図面代用写真である。
【図２０】形質転換後のベクター精製物の電気泳動結果を示す図面代用写真である。
【符号の説明】
２０１ 短鎖オブジェクトＤＮＡデータベース
２０２ 長鎖オブジェクトＤＮＡデータベース
３０１ 主制御部
３０３ データベース抽出処理制御部
３０４ 設計情報作製処理制御部
８０３ サーバコンピュータ
８０４ 短鎖オブジェクトＤＮＡデータベース
８０５ 長鎖オブジェクトＤＮＡデータベース
８０６ ＤＮＡ設計情報
８０７ オブジェクトＤＮＡ格納庫
８０８ オリゴＤＮＡ自動合成機
８１０ 合成ＤＮＡ
８１１ ノックアウト動物またはやトランスジェニック動物[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for producing DNA design information for DNA synthesis, and a method for synthesizing DNA using the obtained design information. More specifically, the present invention relates to DNA synthesis without using a gene library based on DNA base sequence information. Related to the method of synthesis.
[0002]
[Description of the Prior Art]
Genetic cloning technology is a basic technology used by researchers and engineers to analyze the structure and function of individual DNAs and proteins. This technique involves inserting a specific gene segment into a cloning vector that can autonomously replicate in a host cell (the inserted gene segment is referred to as a “chimeric vector”), and introducing the chimeric vector into a host cell. Is a technology that replicates a large number of gene segments. In the gene cloning technique, by arranging a promoter for RNA / protein synthesis around a gene section to be replicated, it is possible to cause host cells to synthesize a large amount of RNA / protein directed by the inserted DNA. Then, by observing the expression of the specific gene inserted into the vector, the function of the gene in the cell can be analyzed.
[0003]
To perform gene cloning, it is necessary to prepare a gene library containing the target DNA. A gene library is a group of cloning vectors formed by ligating genomic DNA or cDNA of a target organism to a vector. The target DNA is amplified using the gene library and the polymerase chain reaction (hereinafter, referred to as “PCR method”). In the PCR method, a DNA sandwiched between both primers is obtained by sandwiching both ends of a DNA sequence to be amplified with complementary primers and using a heat-resistant polymerase such as Taq DNA polymerase to repeat dissociation of complementary strand → annealing → extension. This is a technique for amplifying a large number of categories. In general, a primer having a maximum of about 200 bases is generally produced by an oligo DNA synthesizer by itself, or an order is custom-made to an oligo DNA synthesis maker to purchase a synthesized primer.
[0004]
[Problems to be solved by the invention]
The genomes of many organisms and the nucleotide sequences of genes have been decoded, and in the post-genome era, it has become extremely easy to obtain nucleotide sequence information of genes from various gene databases. There is also a situation where it is desired to actually obtain DNA based on the obtained base sequence information.
[0005]
However, since the conventional gene cloning technique is a combination of a gene library and DNA replication by the PCR method, it is necessary to first prepare a gene library to be a replication source. However, starting from the production of a gene library requires a considerable period of time (for example, one month or more). In order to insert two or more gene segments into a cloning vector, two or more recombination operations are required, resulting in a large burden in terms of work and time. In addition, although it is desired to insert a long-chain DNA into a cloning vector, the long-chain DNA is easily fragmented into a large number with a restriction enzyme, and the insertion is very difficult. It is necessary to design the restriction enzyme so that it does not work in unexpected places, and the longer the chain, the more difficult it is to design.
[0006] An object of the present invention is to provide a method that can obtain DNA without preparing a conventional gene library and that can easily synthesize DNA from data of DNA base sequence information. It is in.
[0007]
[Means for Solving the Problems]
The present inventor considers long-chain DNAs forming complementary base pairs and short-chain DNAs not forming complementary base pairs, for which the base sequence information of DNA is already known, as objects (parts) for synthesis. We found that we could solve the problem.
[0008]
That is, the present invention provides (1) a step of comparing a partial sequence of a DNA to be synthesized with DNA registered in an object DNA database to extract a matched partial sequence; Using the template single-strand of the extracted partial sequence, a total of an even number of template DNAs are arranged alternately in the order of the sense strand and the antisense strand, starting from the sense strand on the 5 ′ end side, and the adjacent sense strand and antisense strand (3) a method for preparing DNA design information for DNA synthesis, comprising the steps of preparing sequence information in which the 5 ′ end and the 3 ′ end have a binding region complementary to each other. Preparing a partial DNA according to the sequence information; (4) connecting two to ten continuous partial DNAs by a first polymerase chain reaction to obtain a module; And (5) connecting two to ten consecutive modules by a second polymerase chain reaction to obtain a synthetic DNA.
[0009]
In the method for producing DNA design information and the method for synthesizing DNA of the present invention (hereinafter abbreviated as “DNA design / synthesis method”), the object DNA database refers to genomic DNA, cDNA, EST, primer, PCR derived from various organisms. This is a database in which base sequence information of DNA such as products and vectors is stored in association with information / location actually obtained. Unlike the conventional gene library, the object DNA database does not need to store the entire chromosome gene population. In the method of the present invention, target DNAs are produced by organically combining partial DNAs of different origins.
[0010]
Since the DNA design / synthesis method of the present invention is a synthesis method based on DNA sequence information, the sequence information of the DNA to be synthesized needs to be known. The DNA sequence information can be obtained from a genetic database on the Internet, such as GenBank, DDBJ, or EMBL, or academic literature. If this DNA sequence information is used as it is for synthesis, natural DNA can be synthesized. Furthermore, a DNA variant can be synthesized by manipulating the DNA sequence information according to the purpose.
[0011]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the DNA design / synthesis method of the present invention will be described with reference to the accompanying drawings. FIG. 1 is a flowchart showing all steps of the DNA design / synthesis method of the present invention. The method for preparing DNA design information includes steps (1) and (2), and the method for synthesizing DNA includes steps (1) to (5). Hereinafter, the steps of FIG. 1 will be described step by step.
[0012]
(1) Step of searching sequence information in object database
In the DNA design / synthesis method of the present invention, first, sequence information of DNA to be synthesized is subjected to database search. FIG. 2 is a conceptual diagram for searching a database from DNA base sequence information. DNA may be linear or circular. The length of the base sequence to be synthesized is within a range that can be extended by the PCR method incorporated in the DNA design / synthesis method of the present invention. If the base length is increased by the progress of the PCR method, the base length that can be synthesized by the DNA design / synthesis method of the present invention also increases.
[0013]
First, as shown in FIG. 2, a target DNA base sequence is searched in the short-chain object DNA database 201. The short-chain object DNA is used as a primer or a template DNA. The base length of the short-chain object DNA is usually 10 to 200 bases, preferably 15 to 120 bases. The short-chain object DNAs extracted as primers have different base sequences from each other among the primers and do not form a complementary pair (the relationship between the sense strand and the antisense strand). In the example shown in FIG. 2, p1 and p3 are extracted as perfectly matching short-chain object DNA.
[0014]
Next, the target DNA base sequence is searched in the long-chain object DNA database 202. The long-chain object DNA is used as a template DNA in the DNA design / synthesis method of the present invention. The long-chain object DNA is not particularly limited in its form as long as the DNA is actually obtained, and includes cDNA, genomic DNA, synthetic DNA, and vector. The long-chain object DNA database 202 contains a part or all of the genomic / cDNA libraries of various organisms such as humans, Arabidopsis, Drosophila, bacteria, and the like, as well as commercially available cDNA / genomic libraries, which have been elucidated by public gene structure analysis projects. Some or all of can be used.
[0015]
It is preferable to register vector DNA in the long-chain object DNA database 202. Conventionally, when subcloning a PCR product from a gene library into a cloning vector, a restriction enzyme treatment was required prior to ligation of the vector with the PCR product. However, the treatment with the restriction enzyme may cause fragmentation of the produced PCR product, which makes it particularly difficult to incorporate long-chain DNA into a vector. In the DNA design / synthesis method of the present invention, the above-described restriction enzyme treatment is not required because the vector DNA can be incorporated as an object into the synthesis. Therefore, it becomes easy to incorporate the long chain DNA into the vector. Furthermore, in the DNA designing / synthesizing method of the present invention, it is very easy to prepare a vector partially different from an existing cloning vector (for example, base substitution, dilution, SNP), and to customize by partially omitting it. Furthermore, in the method of the present invention, it is easy to incorporate two or more gene segments into one cloning vector.
[0016]
The types of cloning vectors include plasmids, phages, viruses, cosmids, yeast artificial chromosomes, and the like. Appropriate vectors are used depending on the length of the inserted gene, application, and the like. Plasmid vectors are small extranuclear genes present in many bacteria, and can usually incorporate 5 to 10 kbp of exogenous DNA. At present, plasmid vectors are produced and sold by reagent companies and research institutions according to their functions. For example, plasmids derived from E. coli include pBR322, pBR325, pUC12, pUC13, pUC18, pUC19, pBluescript II and pET, plasmids derived from Bacillus subtilis include pUB110, pTP5 and pC194, and plasmids derived from yeast include pSH19 and pSH19. There is pSH15.
[0017]
Lambda phage is a virus that infests Escherichia coli and is a vector that can embed 15 to 20 kbp of exogenous DNA. Representative examples of the phage include λgt10, λgt11, λZAPII. Cosmids are plasmids with the phage lambda cos site and can incorporate up to about 40 kbp of foreign DNA. Viral vectors such as baculovirus, vaccinia virus and retrovirus can also be used. The detoxified virus vector is particularly useful as a vector for gene therapy.
[0018]
The above-mentioned vector incorporates a selection marker for discriminating the presence or absence of vector introduction and the success or failure of cloning. These include, but are not limited to, tetracycline or ampicillin resistance in E. coli and other bacteria; dihydrofolate reductase or neomycin resistance for eukaryotic cell culture.
[0019]
In the long-chain object DNA database 202, PCR products, modules, and DNAs of chimeric vectors obtained by the DNA design / synthesis method of the present invention are also appropriately registered. These reuses contribute to the reduction of cost and time for producing oligo object DNA.
[0020]
Even for genes having the same function between species, their nucleotide sequences partially differ depending on the evolution of the organism. In the collation operation between the partial sequence of the DNA to be synthesized and the long-chain object DNA database 202 in the present invention, it is preferable to also extract DNA that partially matches the target DNA. By actively using partially matching DNA, the use of a database can be promoted, and the need for oligo object DNA synthesis can be further reduced. For example, if the partial match sequence is 200 bases or more, it can be used as a template DNA. In the example of FIG. 2, although a DNA object that completely matches the long chain object DNA database 202 was not found, a partially matching long chain DNA object v3 and its antisense strand v4 are extracted.
[0021]
The collation operation between the partial sequence of the DNA to be synthesized and the object DNA database as described above is usually executed by a computer program stored in a computer. FIG. 3 is an example of a system that executes a database extraction process. The system includes an external storage device 302 connected to a main controller 301 that is programmed to control the entire system as a whole. An input device 306 such as a keyboard and a mouse, a display device 307 for monitoring input data, and an output device 308 for outputting a calculation result are connected to the main control unit 301 via an input / output control unit 305. The main control unit 301 has a control program such as an OS, a program for preparing DNA design information (including a program read from an external storage device), and an internal memory for temporarily storing data. 303 and a design information production processing control unit 304. The external storage device 302 is a storage means such as an HD, an MO, a CD, and a DVD, and stores a short-chain object DNA database 201 and a long-chain object DNA database 202.
[0022]
FIG. 4 shows an example of a procedure executed by the database extraction processing control unit 303. First, an initial process for extracting a database of sequence information of DNA to be synthesized is performed. As an example of the initial processing, the base sequence of the target DNA is divided into 16 bases while shifting one base at a time from the 5 ′ end to the 3 ′ end (S401), and the base sequence of each fragment is converted to hexadecimal numbers (for example, AA: 0, AT: 1... CG: e, CC: f), and further compressing the amount of information by 256-base conversion (S402). Next, the short-chain object database 201 is searched for each divided fragment (16 bases) (S403). When the candidate is searched, it is searched whether or not there is a completely matching portion in the target sequence (S404). The search is repeated over the entire length, taking into account the multiple matches. If they completely match, the matching object is extracted in association with the matching information (base sequence, direction, matching position) (S405). When all divided fragments have been searched, the process returns to S403, and the same processing is performed for the antisense (reverse) strand.
[0023]
When the search process using the short-chain object DNA database 201 is completed, the search of the long-chain object DNA database 202 is performed in the same manner as the short-chain object DNA database (S406 to 408). However, in this search, object DNA that partially matches the partial sequence of the base sequence of the target DNA is also extracted. Specifically, the end of the matching part is detected from the matching information, and the front part match (5 'direction) and the rear part match (3' direction) are detected by comparing with the contents registered in the database. The partial matching information (base sequence, direction, matching position) is sequentially created.
[0024]
The extraction processing of the short-chain and long-chain object DNA databases may employ, for example, a search method algorithm (BRAST, FASTA, etc.) known as a homology search, in addition to the above-described search method. However, at the time of the search, only DNA having a degree of coincidence of 100% is partially extracted.
[0025]
(2) Step of creating DNA design information
Design information for DNA synthesis is prepared using the template single strand of the partial sequence extracted in step (1) and its inter-strand information (that is, an unextracted partial sequence that fills the voids of the extracted partial sequence). . FIG. 5 shows a conceptual diagram of the design information production. As a design condition, starting from a sense strand having a 5 ′ end (p1 in FIG. 5), a total of even single strands are arranged alternately in the order of sense strand and antisense strand. The 5 'end of the sense strand is at least blunt with the antisense strand to be paired or protrudes like p1. If there is a partial sequence (p1, p2, v3 and v4 in FIG. 5) that matches the object DNA database, it is used, and if not, oligo object DNA (p3 to p8 in FIG. 5) is synthesized and Fill in. The oligo object DNA is usually designed in the range of 40 to 200 bases. When there is a large gap between the strands, they are allocated to a plurality of oligo objects, and they are alternately arranged with sense strands and antisense strands.
[0026]
The single strands are arranged such that adjacent 5 ′ ends and 3 ′ ends of the sense strand and the antisense strand have binding regions complementary to each other. The binding region in which the 5 ′ end and the 3 ′ end of the adjacent sense strand and antisense strand are complementary serves as a glue margin when a single strand is formed as a whole. In the PCR method, DNA extension is performed in the 5 ′ → 3 ′ direction starting from the glue margin portion, and one DNA is finally obtained as a whole while repeating the PCR cycle. The length of the glue margin is determined according to the annealing temperature of the PCR method. Usually, the Tm (melting temperature) of the paste margin is determined so as to be 50 to 70 ° C, preferably 55 to 65 ° C. It is also possible to make a temperature difference between Tm in one module and Tm between modules. For example, by setting the Tm within one module to be slightly lower than the Tm between modules, the length of the margin portion can be shortened, and as a result, the cost of producing oligo DNA is reduced.
[0027]
The above design processing work is executed by the control unit 304 of the design information creation processing of the computer system in FIG. FIG. 6 shows a flowchart of the contents executed by control unit 304. First, Tm (melting temperature) of the paste margin is set (S601). The Tm of a plurality of glue margins in each module is usually set to be substantially equal. The same applies to a plurality of glue allowances Tm between modules. Next, using the set Tm, the glue margin length a for replenishment chain calculation is predicted (S602). For the prediction, a relationship is generally used in which the higher the GC base content of the paste margin, the stronger the bonding strength of the paste margin and the higher the Tm. For example, the following approximate expression (1) is provided between Tm and the paste length.
[0028]
(Equation 1)

(Where a is the number of bases in the glue margin, n is the number of bases between the chains, GC base is the GC content between the chains, AT base is the AT content between the chains, and Tm ′ is the AT lasting between the chains. (Which is the Tm value of the place). Alternatively, approximate expression (2):
(Equation 2)

(Where a is the number of bases in the glue margin, the GC base number is the GC content in the glue margin, and the AT base number is the AT content in the glue margin)
Holds.
[0029]
Next, the distance X between adjacent chains of the extracted partial sequence (for example, between p1 and p2 in FIG. 5) is measured (S603). Here, when X ≧ 0, adjacent chains do not overlap, and when X <0, it means that adjacent chains overlap. If X ≧ 0, the process proceeds to S605. Otherwise, the inter-chain adjustment is performed in S604. If the short chains overlap, one of the overlapping adjacent chains is thinned out. If the short chains overlap, the overlapping portion X is trimmed to the glue length a predicted from the set Tm or to 0 or less.
[0030]
Proceeding to S605, an optimal replenisher strand (oligo object DNA) for filling in the inter-chain is calculated. The Tm of each predicted glue margin length a is calculated by the above equation (1) or (2), and if the Tm deviates from the set value, the glue margin length is changed to approach the set Tm. When the space between the strands is large and exceeds the synthesis limit of the oligo DNA synthesizer (chain length set at the time of design), a plurality of oligo object DNAs are allocated. Proceeding to S606, it is checked whether the base portion of the oligo object DNA, the short-chain object DNA or the long-chain object DNA has a different glue margin sequence. Avoiding mismatch amplification by not creating the same glue margin. If the above condition is not met, the process returns to S604 and the inter-chain adjustment is repeated. In S607, design information for DNA synthesis is output. FIG. 5 shows an example of the processing result of the DNA design information. Here, oligo object DNAs of p3 to p8 are arranged to fill in the spaces between the extracted partial sequences (p1, p2, v3 and v4). The output method of the design information may be a paper medium or an electronic medium.
[0031]
(3) Step of preparing partial DNA and oligo object DNA
Based on the DNA design information obtained as described above, the short-chain object DNA serving as a part is retrieved from the short-chain object DNA storage. Similarly, the long-chain object DNA as a part is delivered from the long-chain DNA storage. When a part of the long-chain object DNA is required, the necessary partial sequence is cut out by a PCR method with a primer interposed therebetween.
[0032]
Oligo object DNA that is not registered in the database and needs to be synthesized is synthesized by an oligo DNA automatic synthesizer. The automatic oligo DNA synthesizer is a device for sequentially sending necessary reagents onto a solid phase while opening and closing valves of bottles containing reagents and solvents, which are connected by tubes, based on an input base sequence. The DNA synthesizer uses a condensing agent to combine nucleotides, which are sent out of the tube and are protected on the solid phase except for the group to be reacted, with a phosphodiester bond between 3 ′ and 5 ′ of the pentose. Are repeated one after another, thereby synthesizing and linking a DNA strand from 3 ′ to 5 ′. Oligo object DNA synthesis can also be procured by requesting an oligo DNA contract synthesis manufacturer.
[0033]
(4) Modularization process
Modularization means connecting 2 to 10, preferably 2 to 6, module units consisting of continuous strands of partial DNA and oligo DNA based on the DNA design information in step (3). In the DNA design / synthesis method of the present invention, partial DNAs are once modularized and the modules are further connected to avoid a situation in which the amplification of DNA is biased and a full-length DNA cannot be obtained. FIG. 7 shows a conceptual diagram of modularization using the design information example of FIG. A module 1 is formed from the registered short-chain object DNA (p1 to p2) and a synthetic oligo object DNA (p3 to p6), and a module 2 is formed from the registered partially matched long-chain object DNA (v3 to 4). The module 3 is formed by the synthetic oligo object DNA (p7 to p8).
[0034]
Hereinafter, modularization by the PCR method will be specifically described. A reaction solution is prepared using short-chain / long-chain object DNA, synthesized oligo object DNA, dNTPs (deoxynucleotides) which are raw materials for PCR, salts, and a heat-resistant DNA polymerase. The thermostable polymerase may be either a pol I type enzyme derived from bacteria (eg, Taq DNA polymerase) or an α type enzyme derived from archaebacteria (eg, pfu DNA polymerase, KOD DNA polymerase), or a mixture of two types of these. Good. These may be commercially available from reagent manufacturers, such as Takara Ex Taq, Pyrobest DNA Polymerase (above, manufactured by TaKaRa), KOD + (manufactured by TOYOBO), and the like. The concentration of DNA polymerase is usually 0.25 units / μl. As the buffer, a 10 × buffer enclosed with the commercially available thermostable polymerase may be used. For example, a 10 × buffer of Taq polymerase typically contains 10-25 mM Tris-HCl, 50 mM KCl, and MgCl ₂ 1-5.0 mM.
[0035]
The concentration of the template DNA is usually several tens pg / μl to several ng / μl. Primers are usually prepared at 0.2 to 10 μM. If the number of primers is too small, the reaction does not proceed, while if it is too large, non-specific amplification may be caused. dNTPs are prepared at 0.2 to 2 mM each.
[0036]
The reaction solution is heated to dissociate the double strand of the template DNA. Next, the primers (adjacent template DNA) overlapping the complementary portions of each single strand are cooled to a temperature at which a double strand is formed (annealed). Finally, DNA extension is performed in the 5 ′ → 3 ′ direction using a thermostable polymerase enzyme starting from the complementary portion. By repeating this cycle, continuous modularization of partial DNA and oligo DNA is achieved.
[0037]
Regarding the conditions of the PCR method, the annealing temperature of the template / primer DNA is set to the bonding margin Tm set at the time of design. The time and temperature required for each step (denaturation, annealing, extension) of the PCR method are, for example, 30 seconds for thermal denaturation (93 to 97 ° C), 30 seconds for annealing (Tm of glue allowance), and an extension reaction (68 to 72 ° C). ) Is 0.5 to 1 minute × the chain length to be amplified (kb). The number of cycles depends on the amount of DNA to be synthesized, but generally may be 30 to 40 cycles.
[0038]
(5) Module connection process
The continuous modules obtained above are usually ligated by 2 to 10, preferably 2 to 6, PCR. The procedure and conditions of the PCR method are the same as in the modularization step (4). In the example of FIG. 7, modules 1 to 3 are connected using p1 and p8 as primers. When it is necessary to close the synthetic DNA such as when a vector is used as the object DNA, a self-ligation operation is performed. T4 phage DNA kinase and ligase are used to directly join blunt ends of double-stranded DNA. A commercially available ligation kit can also be used.
[0039]
As described above, according to the designing / synthesizing method of the present invention, it is not necessary to prepare and screen a gene library of a target gene, so that a long-chain DNA of 10 kbp or more can be prepared in a short period of 3 to 4 days.
[0040]
(6) Subcloning step
Subcloning can be omitted if vector DNA is incorporated as an object during DNA synthesis. However, when vector DNA is not bound to the above synthetic DNA, or when it is necessary to transfer the vector for functional analysis or protein expression, subcloning is performed by the following method.
[0041]
The target specific gene segment is inserted into the insertion site of the cloning vector and used. As a method for inserting into a cloning vector, a conventionally known method can be used. As a preparation for insertion, the insertion target site of the cloning vector is cut with a restriction enzyme that cuts the cloning vector at a specific site. The end of the synthetic DNA contained by the PCR method is processed with a restriction enzyme or the like so that it can be ligated to the insertion target site of the cloning vector. Usually, the primer is designed so that it can be connected smoothly when designing the primer. Finally, the DNA contained by the PCR method and the cloning vector cut at a specific site are ligated using a DNA ligase enzyme.
[0042]
(7) Transformation step
Next, the chimeric vector of (5) or (6) is introduced into a host cell. The host cell can be used without limitation as long as the cloning vector can be stably and autonomously propagated. Examples of host cells include bacteria such as Escherichia coli, Streptomyces, Salmonella typhimurium; fungi such as yeast; insect cells such as Drosophila S2 and Chinese armyworm Sf9; animal cells such as CHO, COS and Bowes melanoma cells; Cells.
[0043]
Methods for introducing vectors into cells include electroporation, DEAE-dextran, calcium phosphate precipitation, liposome-mediated fusion (lipofection), microinjection, transduction with a non-replicating virus, and the like. Different introduction methods are used depending on the host cell.
[0044]
The host cell into which the chimeric vector has been inserted is plated and cultured on an appropriate medium to form a colony derived from a single host cell having a single vector. The medium contains a carbon source, a nitrogen source, inorganic substances, and the like necessary for cell growth. As the carbon source, sugars such as glucose and sucrose; alcohols such as glycerol; and organic acids are appropriately used. As the nitrogen source, ammonia or ammonium salts, amines, other nitrogen compounds, or natural nitrogen sources such as peptone, hydrolyzate of soybean, and decomposition of fermentation cells can be used. Examples of the inorganic salt include potassium monohydrogen phosphate, potassium dihydrogen phosphate, and magnesium sulfate. Yeasts, vitamins, growth promoting factors and the like may be added as additives. An antibiotic corresponding to the drug resistance gene of the vector is added to the medium as a selectable marker for culturing only host cells having the target chimeric vector, and in order to prevent the propagation of various bacteria. For example, to culture Escherichia coli containing a vector having an ampicillin resistance gene, an antibiotic ampicillin is added to an LB medium for Escherichia coli.
[0045]
Cloning vectors self-amplify in host cells in large quantities. Methods for checking whether DNA synthesis has been performed properly and the quality of the target product include, for example, purifying a cloning vector by miniprep treatment of a host cell colony and then subjecting the inserted DNA to full length or partial length. From a vector and checking the cut-out DNA by electrophoresis, a check by a PCR method, a colony hybridization method, a color selection and the like.
[0046]
(8) Gene expression step
When a host cell is desired to produce a target synthetic DNA product (protein), an expression vector may be used as the vector. An expression vector is a vector linked to a promoter and a terminator. The promoter may be any promoter as long as it is appropriate for the host used for gene expression. When the host to be transformed is a bacterium belonging to the genus Bacillus, SPO1 promoter, SPO2 promoter, penP, and the like can be used. Promoters, cytomegalovirus promoters, SRα promoters and the like can be used. Commercially available expression vectors include, for example, the pUC system, pBluescript II, pET and λgt11. Synthetic DNA is incorporated after the ribosome binding site (SD sequence).
[0047]
Examples of the form of gene expression include a method of producing a protein in a host cell, a method of secreting the protein outside the host cell, and a method of producing the protein on the host cell outer membrane. After expressing the protein in host cells, the target protein is isolated and purified from the culture. Known separation operations, for example, treatment with a denaturant such as urea or a surfactant, ultrasonic treatment, enzyme digestion, salting out, solvent precipitation, dialysis, centrifugation, ultrafiltration, gel filtration, SDS-PAGE, etc. Electrofocusing, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, reverse phase chromatography, and the like can be appropriately used in combination.
[0048]
By introducing the cloning vector into a fertilized egg of an animal such as a mouse, or by directly injecting the animal, a transgenic animal or a knockout animal can be produced.
[0049]
(9) DNA storage and registration process
A part of the novel oligo object DNA, the synthetic DNA, and the chimeric vector into which the synthetic DNA is obtained in the above step are stored in a storage. For example, items that are allowed to be shared are stored in a storage hangar, while items that are not allowed to be shared are stored in a storage hangar. Further, the base sequence information of the stored object DNA is registered in the short-chain / long-chain object database in association with the stored information. By registering, existing resources can be effectively used for the next DNA synthesis.
[0050]
Next, modified examples of the DNA design / synthesis method of the present invention will be described. FIG. 8 shows an example in which a requester of DNA synthesis, a base for preparing DNA design information, and a base for synthesizing DNA are separately provided. In the DNA design / synthesis method of the present invention, since the effect increases as the short- and long-chain object DNA databases are enhanced, it is preferable to establish a specialized base for DNA design and DNA synthesis as shown in FIG.
[0051]
In this usage mode, a client who desires DNA synthesis accesses the DNA synthesis system from the terminal 801 via the Internet or a communication line. The DNA synthesis system includes a server computer 803, a short-chain object DNA database 804, and a long-chain object DNA database 805 (appropriately divided into a sharing database and a client identification database). And a means for sending the DNA design information 806 to the oligo DNA automatic synthesizer 808.
[0052]
FIG. 9 is a diagram showing a flow of a procedure between the three parties in the DNA synthesis system. A client such as a researcher or a technician accesses a gene information database 802 such as a genome network from the client terminal 801 via the Internet, and obtains an academic information magazine to obtain sequence information of DNA to be synthesized. It is acquired (S901). Alternatively, DNA sequence information partially modified based on known DNA sequence information according to the purpose of use or research purpose is created.
[0053]
The client sends the DNA sequence information to be requested for synthesis to the DNA synthesis system (server computer 803) via the Internet, a communication line, or the like (S902). The server computer 803 of the DNA synthesis system receives the sequence information, searches the short-chain object DNA database 804 and the long-chain object DNA database 805, and extracts a matching or partially matching partial DNA (S903). The design information 806 for DNA synthesis is created using the obtained base sequence information of the partial DNA and their interstrand information (S904). The prepared DNA design information 806 is sent to a client or to a production site for DNA synthesis. The information base for production of DNA design information and the bases for oligo DNA synthesis, PCR, sequence and the like may be far apart.
[0054]
At the DNA production base, a person in charge releases the required object DNA from the short-chain object DNA storage and the long-chain DNA object DNA storage based on the design information 806, and synthesizes the insufficient oligo object DNA by an automatic oligo DNA synthesizer. (S905). Using the obtained parts and the PCR method, modularization of the object DNA and module connection are performed (S906, S907). The linked synthetic DNA 810 is delivered to the client after inspection, in that state, or after processing such as freeze-drying. After appropriate subcloning (S908), the cloning vector is inoculated into a host cell for transformation (S909), and the vector is autonomously propagated in the transformed cell or a gene is expressed to obtain a protein (S910). , The DNA, protein, etc. may be delivered to the client. Transformed knockout or transgenic animals 811 can also be provided.
[0055]
The DNA design / synthesis method of the present invention can be applied or modified in addition to the above-mentioned usage modes. One example is an object DNA database; database extraction means for comparing a partial sequence of DNA to be synthesized with object DNA registered in the object DNA database to extract a matched partial sequence; Using the base sequence of the single strand of the template of the extracted partial sequence, the sense strand and the antisense strand are alternately arranged in order of the sense strand and the antisense strand starting from the sense strand on the 5 'end side, and the adjacent sense strand and antisense strand This is a DNA design system provided with a design information producing means for arranging the 5 ′ ends and the 3 ′ ends of the above to have a binding region complementary to each other. Another example is (1) a step of comparing a partial sequence of DNA to be synthesized with an object DNA registered in an object DNA database to extract a matched partial sequence, and (2) a step of extracting the matched partial sequence. Using the base sequence of the single-stranded template of the unextracted partial sequence, the sense strand and the antisense strand are alternately arranged in order of the sense strand and the antisense strand starting from the sense strand on the 5′-end side, and the adjacent sense strand and antisense strand are further sequenced. This is a program for causing a computer to execute a step of arranging the 5′-end and the 3′-end of the sense strand so that they have binding regions complementary to each other. Still another example is a computer-readable recording medium on which the program is recorded.
[0056]
【Example】
Hereinafter, the DNA design / synthesis method of the present invention will be described in more detail using examples.
[Example 1]
The base sequence (SEQ ID NO: 1) was synthesized as shown in FIG. As shown in the restriction enzyme map of FIG. 11, a 112 bp DNA fragment was inserted into the multiple cloning site (MCS) of the cloning vector pBluescript II SK (+) (2961 bp, hereinafter abbreviated as “pBSK +”). This is a circular DNA having a total length of 3073 bp. MCS is between T7 and T3 in FIG. 10, and the underlined region is the sequence to be inserted into pBSK + this time.
[0057]
(1) Database extraction
The database extraction processing control unit 303 of the system shown in FIG. 3 executed the database extraction processing for the target circular DNA according to the procedure shown in FIG. Various existing primers were registered in the short-chain object database in advance, while pBSK + was registered in the long-chain object database. FIG. 12 shows the result of the database extraction. DNA1 (shaded arrow part) in FIG. 12 was found from the short chain object DNA database. DNA2 (open arrow portion) in FIG. 12 was found from the long chain object DNA database. Since DNA2 was pBSK +, it was confirmed that the database extraction process was successfully performed.
[0058]
(2) Preparation of DNA design information
Based on the extracted partial DNA and the inter-chain information, the design information creation processing control unit 304 of the system in FIG. 3 was caused to execute the DNA design information creation processing in the procedure shown in FIG. As design conditions, Tm within the module and between modules was set to 60 ° C., and the maximum design length of the oligo object DNA was set to 59 bases. When DNA1 extracted from the short-chain object DNA database was used, the amount of glue became insufficient, so that thinning was performed, and a calculation result was obtained in which P1 to P4 were newly formed as oligo object DNA. FIG. 13 shows an overview of design information for DNA synthesis. 14 and 15 show the base sequence information of each object DNA. Table 1 shows the combinations and Tm of the module of the object DNA.
[0059]
[Table 1]

[0060]
(3) Preparation of object DNA
An object DNA was prepared according to the above design information. First, P1 to P4 were synthesized using an oligo DNA synthesizer. Next, primers V1 (SEQ ID NO: 7) and V2 (SEQ ID NO: 8) used for excision of P5 and P6 were synthesized using an oligo DNA synthesizer. PBSK + was discharged from the storage of the long-chain object DNA, and P5 and P6 were cut out by PCR using the above primers (V1 and V2). The composition of the reaction solution and the temperature cycle of the PCR method are shown in Table 2 and FIG. 16, respectively.
[0061]
[Table 2]

[0062]
(4) Modularization
Module 2 was prepared by a PCR method using P1 to P4 as templates (P1 and P4 were used as primers). Table 3 shows the composition of the reaction solution of the PCR method. The temperature cycle conditions were specified as in FIG.
[0063]
[Table 3]

[0064]
FIG. 17 shows the results of electrophoresis of modules 1 and 2. In the figure, lane (1) is the result of electrophoresis of the product after PCR of pBSK + with primers V1 and V2, and a band was observed around 2000 to 3000 bases. Lane (2) is a result of electrophoresis of a product obtained by PCR of P1 to P4 with primers P1 and P4, and a band of less than 500 bases was confirmed.
[0065]
(5) Connection between module 1 and module 2
After ethanol precipitation of the modules 1 and 2 of (4), the sample concentration was adjusted, and then PCR was performed with the V1 and P1 primers using the modules 1 and 2 as templates. Table 4 and FIG. 18 show the composition of the reaction solution and the temperature cycle conditions.
[0066]
[Table 4]

[0067]
FIG. 19 shows the results of electrophoresis performed by changing the sample concentration of the PCR product. In FIG. 19, bands expected to be module 1, module 2, and module 1 + 2 (connection between modules 1 and 2) were confirmed.
[0068]
(6) Self-ligation
From the results of the electrophoresis, the band of module 1 + 2 was cut out, purified by Gel Extraction Kit (manufactured by Qiagen), and subjected to self-ligation (16 ° C. × 2 hours) of terminal blunt ends (715, 716) to form linear chains. The DNA was closed. Table 5 shows the composition of the ligation reaction solution.
[0069]
[Table 5]

[0070]
(7) Transformation
The chimeric vector whose ends were recombined by self-ligation was transformed into competent Escherichia coli (DH5α strain). The transformed E. coli grew well in LB-AMP medium. After 10 E. coli colonies were selected from the medium and miniprepped, the resulting purified product was subjected to electrophoresis. FIG. 20 shows the result. Lanes (6), (7) and (8) were selected from the electrophoresis results, and the sequence was performed. At that time, the insert portion was sequenced in the forward direction with the PT7 primer, and the insert portion was sequenced in the reverse direction with the PT3 primer. From the sequence results, it was confirmed that the target sequence was inserted into the multiple cloning site.
[0071]
【The invention's effect】
Since the DNA design / synthesis method of the present invention is not DNA replication but synthesis, it is not necessary to prepare and screen a conventional gene library. Therefore, the desired DNA can be obtained in a short period of time. The method of the present invention also facilitates free gene design by changing DNA sequence information to be synthesized. Furthermore, since the restriction enzyme treatment can be omitted, the production of the cloning vector becomes simpler than the conventional method. Processing of the cloning vector itself is also possible. Then, the once-produced oligo DNA, synthetic DNA (PCR product), etc. can be reused. By enriching the object database, the cost of DNA synthesis can be further reduced.
[0072]
[Sequence list]

[Brief description of the drawings]
FIG. 1 is a flowchart showing all steps of the DNA design / synthesis method of the present invention.
FIG. 2 is a conceptual diagram for searching a database from DNA base sequence information.
FIG. 3 is an example of a computer system that executes the method for producing DNA design information of the present invention.
FIG. 4 is a flowchart of an operation performed by a database extraction processing control unit in the system of FIG. 3;
FIG. 5 is a conceptual diagram of design information preparation in the DNA design / synthesis method of the present invention.
FIG. 6 is a flowchart of an operation performed by a design information creation processing control unit in the system of FIG. 3;
FIG. 7 is a conceptual diagram of modularization in the DNA design / synthesis method of the present invention.
FIG. 8 is an application example of the DNA design / synthesis method of the present invention.
FIG. 9 is a flowchart showing a flow of a procedure in the DNA synthesis system of FIG.
FIG. 10 is an explanatory diagram of a base sequence of DNA to be synthesized in Example 1 according to the present invention.
FIG. 11 is a restriction map showing the nucleotide sequence of the DNA to be synthesized in FIG.
12 is a diagram showing a result of searching the DNA base sequence information of FIG. 10 by an object DNA database.
FIG. 13 is a diagram showing design information for DNA synthesis in FIG.
FIG. 14 is a base sequence of object DNA (p5) used for DNA synthesis.
FIG. 15 shows the base sequences of object DNAs (p1 to p4) used for DNA synthesis.
FIG. 16 is a diagram showing a temperature cycle of a PCR method performed to cut out object DNA (p5, p6) in Example 1 according to the present invention.
FIG. 17 is a photograph as a drawing showing electrophoresis results of modules 1 and 2 of Example 1.
FIG. 18 is a diagram showing a temperature cycle of a PCR method performed to connect modules 1 and 2 in Example 1 according to the present invention.
FIG. 19 is a drawing substitute photograph showing the results of electrophoresis of the PCR product of FIG. 18.
FIG. 20 is a photograph as a drawing showing the results of electrophoresis of the purified vector after transformation.
[Explanation of symbols]
201 Short chain object DNA database
202 Long Object DNA Database
301 Main control unit
303 Database extraction processing control unit
304 Design information production processing control unit
803 server computer
804 Short Object DNA Database
805 Long chain object DNA database
806 DNA design information
807 Object DNA Hangar
808 Oligo DNA automatic synthesizer
810 Synthetic DNA
811 knockout animals or transgenic animals

Claims (5)

(1) a step of comparing a partial sequence of DNA to be synthesized with an object DNA registered in an object DNA database to extract a matched partial sequence; and (2) an extracted partial sequence and a non-extracted partial sequence Starting from the sense strand on the 5 'end side, the sense strand and the antisense strand are alternately arranged in total in an order of even number using the base sequence of the single strand of the template, and the 5' of the adjacent sense strand and antisense strand A method for producing DNA design information for DNA synthesis, comprising a step of arranging such that the ends thereof and the 3 ′ ends thereof have complementary binding regions. The method according to claim 1, wherein in the step (1), a DNA partially matching the partial sequence of the DNA to be synthesized is also extracted. (1) collating the partial sequence of the DNA to be synthesized with the DNA registered in the object DNA database, and extracting a matched partial sequence;
(2) Using the template single-strand of the extracted partial sequence and the unextracted partial sequence, a total of even numbers are arranged alternately in the order of the sense strand and the antisense strand, starting from the sense strand on the 5 ′ end side, and are adjacent to each other. (3) arranging the sense strand and the antisense strand such that the 5′-ends and the 3′-ends of the antisense strand have binding regions complementary to each other; (3) preparing a partial DNA according to the sequence;
(4) a step of connecting 2 to 10 continuous partial DNAs by a first polymerase chain reaction to form a module, and (5) a step of connecting 2 to 10 continuous partial modules by a second polymerase chain reaction. A method for synthesizing DNA, comprising the step of obtaining a synthetic DNA by ligation. 4. The DNA synthesis method according to claim 3, wherein in step (1), DNA that partially matches DNA to be synthesized is also extracted. (6) The DNA synthesis method according to claim 3 or 4, further comprising a step of registering the newly prepared partial DNA and the synthesized DNA in an object DNA database.

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