JP2004531258A - Method of template-adjusted ligation direction localization for non-random shuffling of polynucleotides - Google Patents

Abstract

A method of gene shuffling-directed ligation wherein at least two fragments are hybridized adjacently onto an assembling template. The present invention aims, inter alia, to generate novel polynucleotides that differ from the reference sequence in some respects. The invention further includes sequences resulting from the method, hosts, vectors containing the same, and proteins translated therefrom.

Description

【００８４】
Ｃ．試薬
【００８５】
下記の表ＩＩに引用した制限及び改修酵素は、供給業者の推薦に従って用いた。
【００８６】
【表ＩＩ】

【００８７】
用いた緩衝液は、下記の表ＩＩＩに報告する。
【００８８】
【表ＩＩＩ】

【００８９】
ＩＩＩ．テンプレートの準備
【００９０】
オリゴヌクレオチドＭ１及びＭ２をプライマーとして用い、ＰＣＲ反応ステップによって野生型ｐｏｎＢ遺伝子を増幅した（図４）。１０μｌの重合緩衝液、１０μｌのｄＮＴＰｓ２ｍＭ、各２０ピコモルのオリゴヌクレオチドＭ１及びＭ２、そして５ＵのＴａｑＤＮＡ重合酵素を含む最終量が１００μｌの混合物へ、野生型遺伝子（７）を持つ５０ｎｇのｐＰＯＮＢＰＢＲプラスミドを加えることによって、五つのＰＣＲ反応を準備した。これらの混合物を、次のプログラムに従ってパーキン・エルマー（Perkin-Elmer）９６００サーモサイクラーで温置した。（９４℃−２分）−（９４℃１５秒−６０℃３０秒−７２℃１分）ｘ２９サイクル−（７２℃−３分）。
【００９１】
５つのＰＣＲ産物を混合して、１％のＴＢＥアガロース・ゲル上に装填した。泳動とエチジウム臭化物でのゲルの染色の後、２６ｂｐ及び９０ｂｐの二つの断片によって囲まれたｐｏｎＢ遺伝子増幅産物に対応する、２６５１ｂｐの縞模様を、紫外線による透視法で視覚化した。それから、それをメスで切り剥がし、クイアジェン社のクイアクイック・システム（QUIAquick system, QIAGEN）で精製した。精製したすべてのＤＮＡを、１２０μｌの緩衝液Ｔで溶離した。このＤＮＡの濃度は、２６０ｎｍにおけるその吸光度測定から、約１００ｎｇ／μｌであった。
【００９２】
ＩＶ．ライブラリの準備
【００９３】
Ａ．突然変異遺伝子の増幅
【００９４】
１０の突然変異体の遺伝子を、個別に、オリゴヌクレオチドＮ及びＥとのＰＣＲ反応によって増幅した。これらのオリゴヌクレオチドは、各々、制限部位ＮｃｏＩ及びＥｃｏＲＩを導入するため、これらの二つの部位で得られる産物のクローニングが可能となる。
【００９５】
１０μｌの重合緩衝液、１０μｌのｄＮＴＰｓ２mM、各々２０ピコモルのオリゴヌクレオチドＮ及びＥ、そして ５U のＴａｑＤＮＡ重合酵素を含む最終量で１００μｌの混合物へ、突然変異遺伝子を含む５０ｎｇのプラスミドを加えることによって、各ＰＣＲ反応を準備した。この混合物を、次のプログラムに従ってパーキン・エルマー９６００サーモサイクラーで温置した。（９４℃−２分）−（９４℃１５秒−６０℃３０秒−７２℃１分）ｘ２９サイクル−（７２℃−３分）。
【００９６】
最終量で３０μｌ内に３μｌの制限緩衝液Ａと５ＵのＰｖｕＩＩ酵素とを含む混合物内に、１時間３７℃で５μｌの各ＰＣＲ産物を温置することによって、遺伝子増幅の特殊性を、ＰｖｕＩＩエンドヌクレアーゼの制限プロフィールで実証した。その消化反応の１５μｌをＴＢＥ１％アガロース・ゲル上に装填した。泳動、そしてエチジウム臭化物での染色後、ゲルを紫外線にさらした。制限断片の視覚化は、各突然変異遺伝子の遺伝子増幅が特殊であることの確認を可能にした。
【００９７】
平行して、３μｌの各ＰＣＲ反応を、ＴＢＥ１％アガロース・ゲル上に装填した。泳動後、ゲルを上記のように処理した。各縞模様の輝度は、遺伝子増幅が同じ収量を持つことの査定を可能にした。
【００９８】
Ｂ．制限断片ライブラリの生成
【００９９】
５０μｌの各１０ＰＣＲを混合して、１％ＴＢＥアガロース・ゲル上に装填した。泳動とエチジウム臭化物での染色後、１０の突然変異遺伝子の増幅産物に対応する、２５７２ｂｐにおける縞模様をメスで採取して、クイアクイック・システム（QIAGEN）で精製した。精製したすべてのＤＮＡを、１２０μｌの緩衝液Ｔで溶離した。このＤＮＡの濃度は、２６０ｎｍにおけるその吸光度から、約１００ｎｇ／μｌであった。
【０１００】
制限断片のライブラリを生成するために、１２μｌの制限緩衝液Ｂ、１．２μｌのＢＳＡ（１０ｍｇ／ｍｌ）、２５Ｕの酵素ＢｓａＩ、そして４μｌの水を含む混合物内で、１時間５０℃、１００μｌのこのＤＮＡを温置した。それから、２μｌの制限緩衝液Ｂ、２μｌＢＳＡ（１ｍｇ／ｍｌ）、５０Ｕの酵素ＨｉｎｆＩ、そして１１．５μｌの水を混合物に加えて、それを３７℃で１時間温置した。その消化混合物をクイアクイック・コラム（QIAGEN）上で精製して、３０μｌの緩衝液Ｔで溶離した。１μｌの溶離物を１％ＴＢＥアガロース・ゲル上に装填し、その消化が完全であり、六つの制限断片が生成され、結果的に、５９０ｂｐ、５００ｂｐ、４７２ｂｐ、４３８ｂｐ、２９８ｂｐ及び２７４ｂｐの、六つの断片ライブラリが生成されたことを実証した。このＤＮＡの濃度は、２６０ｎｍにおけるその吸光度から、約２５０ｎｇ／μｌであった。
【０１０１】
Ｖ．組み換えライゲーション反応（ＲＬＲ）
【０１０２】
熱安定性ＤＮＡリガーゼが存在する状態で、完全なテンプレートを持つ１０の突然変異体の遺伝子（すなわち、野生型ｐｏｎＢ遺伝子）から、制限断片ＨｉｎｆＩ−ＢｓａＩの所定量を温置することによってＲＬＲ反応を行った。下記の表ＩＶに、ＲＬＲのための混合物の組成を報告する。
【０１０３】
【表ＩＶ】

【０１０４】
負の制御は、ＲＬＲ４の反応とまったく同じであるが、熱安定性ＤＮＡリガーゼを含まない。これらの異なる混合物を鉱油の滴で覆って、次のプログラムに従って２００μｌマイクロチューブ内でパーキン・エルマー９６００サーモサイクラーで温置した。（９４℃、５分）−（９４℃、１分−６５℃、４分）ｘ３５サイクル。
【０１０５】
それから、各ＲＬＲ反応の１０μｌを、１０μｌの重合緩衝液、１０μｌの２ｍＭｄＮＴＰｓ、各々４０ピコモルのオリゴヌクレオチドＡ１及びＡ２、そして５ＵのＴａｑＤＮＡ重合酵素を含む最終量が１００μｌのＰＣＲ反応混合物に加えた。この混合物を、次のプログラムに従ってパーキン・エルマー９６００サーモサイクラーで温置した。（９４℃、５分）−（９４℃、３０秒−４６℃、３０秒−７２℃、１分）ｘ２９サイクル−（７２℃、２分）。このＰＣＲ反応は、図４に示すように、オリゴヌクレオチドＡ１及びＡ２がテンプレートとハイブリダイズすることができないため、テンプレートを増幅しないで、ＲＬＲ反応中に形成されたライゲーション産物の特定な増幅を可能にした。
【０１０６】
各ＲＬＲ反応の５μｌと、先のＰＣＲ反応の各々の１０μｌとを、１％ＴＢＥアガロース・ゲル上に装填した。エチジウム臭化物で染色後、図５に示すように、ゲルを紫外光にさらした。
【０１０７】
このゲルの分析は、負の制御と同様、ＲＬＲ４反応のみが、制限断片を可視のまま含むことを明らかにする（トラック４及び５）。
【０１０８】
負の制御（トラック１０）に対するＰＣＲ産物の欠如は、ＰＣＲ反応が特定である（完全なテンプレートの増幅が全くない）ことだけではなく、選択条件の下で不純ＰＣＲ産物を生成するために、混合物内に存在する制限断片をプライマーで置換することはできないということをも明示している。平行して、トラック６、７及び８内の約２５００ｂｐにおける独特な縞模様の存在は、ＲＬＲ産物が、ＲＬＲ１、２及び３反応のためのＰＣＲによって増幅できることを表している。したがって、これら三つのＲＬＲ反応は、六つのライブラリの制限断片から開始して、一つ以上の完全な遺伝子の再生を可能にした。
【０１０９】
ＶＩ．増幅産物の分析
【０１１０】
Ａ．クローニング
【０１１１】
ＲＬＲ１、２及び３反応のＰＣＲ増幅産物を、ウィザードＰＣＲプレプス・システム（プロメガ）で精製して、４５μｌの緩衝液Ｔ内で溶離した。６μｌの各精製ＰＣＲを、３μｌの制限緩衝液Ｃ、３μｌのＢＳＡ（１ｍｇ／ｍｌ）、２０ＵのＥｃｏＲＩ酵素、１０ＵのＮｃｏＩ酵素、そして１５μｌの水を含む混合物内で、１時間３７℃で温置した。
【０１１２】
平行して、二つのベクター（ｐＡＲＡＰＯＮＢ及びｐＥＴ２６ｂ＋）を、クローニングのために準備した。これらのベクターを３７℃で２時間、３μｌの制限緩衝液Ｃ、３μｌのＢＳＡ（１ｍｇ／ｍｌ）、２０ＵのＥｃｏＲＩ酵素、１０ＵのＮｃｏＩ酵素、そして１９μｌの水を含む混合物内で、３μｇのプラスミドを温置することによって直線化した。
【０１１３】
直線化したベクターと、消化したＰＣＲとを、クイアクイック・システム（QUIAGEN）を用いて、ＴＢＥ１％アガロース・ゲル上で精製した。各ベクターあるいは各消化ＰＣＲを、３０μｌの緩衝液Ｔ中で溶離した。
【０１１４】
各ベクターで消化したＰＣＲの各々のライゲーションを、下記の表Ｖに説明する条件で行い、そして１６時間１６℃で温置した。
【０１１５】
【表Ｖ】

【０１１６】
２００μｌの化学反応が可能なＭＣ１０６１細胞（４）を、熱ショック（５）によって１０μｌの各ライゲーションで変換し、これらの変換細胞を選択媒体上に広げた。
【０１１７】
ライゲーション制御ＴＬｐＡＲ及びＴＬｐＥＴの変換後、クローンは全く得られなかった。このことは、ＮｃｏＩ−ＥｃｏＲＩベクターｐＡＲＡＰＯＮＢ及びｐＥＴ２６ｂ＋が、分子内ライゲーションを起こせないことを示している。
【０１１８】
Ｂ．ＰＣＲによるスクリーニング
【０１１９】
ベクターｐＡＲＡＰＯＮＢでのライゲーションの変換後に得られたクローンの第一のスクリーニングをＰＣＲによって行う。各ライゲーションＬｐＡＲ１、ＬｐＡＲ２及びＬｐＡＲ３から１４づつ４２のコロニーを個々に、５μｌの重合緩衝液、各々４０ピコモルのオリゴヌクレオチドＡ１及びＡ２、５μｌの２ｍＭｄＮＴＰｓ、そして５ＵのＴａｑＤＮＡ重合酵素を含む、最終量で５０μｌのＰＣＲ混合物内で再懸濁した。ＰＣＲ混合物へ、コロニーの代わりに５０ｎｇのプラスミドｐＢＲ３２２を加えることで、負の制御を得た。これら４３の試験管を、次のプログラムに従ってパーキン・エルマー９６００サーモサイクラーで温置した。（９４℃、５分）−（９４℃、３０秒−４６℃、３０秒−７２℃、１分）ｘ２９サイクル−（７２℃、２分）。それから、これらのＰＣＲ反応の各々５μｌを、２μｌの制限緩衝液Ａ、２μｌのＢＳＡ（１ｍｇ／ｍｌ）、そして５Ｕの制限酵素ＰｖｕＩＩを含む最終量で２０μｌの混合物内で、１時間３７℃で温置した。
【０１２０】
これらの消化の各々１０μｌを、非消化ＰＣＲの５μｌの各々と並列にＴＢＥ１％アガロース・ゲル上に装填した（これによって、制限消化によって得た断片でのＰＣＲの非特異的な縞模様に関する混乱の発生を避けた）。泳動、そしてこのゲルのエチジウム臭化物での染色後、初期突然変異体のどの断片が、遺伝子全体を再構築するのに他のものと関連するのかを判定するために、酵素ＰｖｕＩＩによる消化から生じる縞模様を分析した。このスクリーニングは、一つの突然変異を伴う２７の遺伝子、二つの突然変異を伴う七つの遺伝子、そして突然変異をもはや伴わない八つの遺伝子の存在を明示する。
【０１２１】
Ｃ．プラスミドＤＮＡミニプレパレーションによるスクリーニング
【０１２２】
ベクターｐＥＴ２６ｂ＋でのライゲーションの変換から生じた２１のクローン（各ライゲーションから７つのクローン）から、プラスミドＤＮＡ（５）を抽出することによって第二のスクリーニングを行った。各クローンに対して得たプラスミドＤＮＡの５μｌを、１μｌの制限緩衝液Ｃ、６Ｕの酵素ＰｓｔＩ、３Ｕの酵素ＮｃｏＩ、そして６Ｕの酵素ＥｃｏＲＩを含む最終量で１０μｌの混合物内で、１時間３７℃で温置した。これらの消化の各々５μｌをＴＢＥ１％アガロース・ゲル上に装填した。泳動と、このゲルのエチジウム臭化物での染色後、初期突然変異体のどの断片が、遺伝子全体を再構築するのに他のものと関連したのかを判定するために、ＰｓｔＩ酵素による消化から生じた縞模様を分析した。このスクリーニングは、一つ突然変異を伴う１３の遺伝子、二つの突然変異を伴う五つの遺伝子、そして突然変異をもはや伴わない三つの遺伝子の存在を明示する。
【０１２３】
Ｄ．組み換えの統計的分析
【０１２４】
酵素ＨｉｎｆＩ及びＢｓａＩの切断部位に対する各突然変異の位置から判断して（図６参照）、ＲＬＲを介して初期遺伝子の突然変異０、１、２、３あるいは４個を伴う遺伝子を得る確率を計算することが可能である。
【０１２５】
ＲＬＲ反応が全くランダムであると仮定して、確率Ｐは次の通りである。
【０１２６】
【数１】

【０１２７】
行った二つのスクリーニングは、下記の表ＶＩに報告するように、これらの統計上の予想に近い結果をもたらす。したがって、ＲＬＲ反応が準ランダムであることを示す。ゼロ突然変異を伴う遺伝子の損失によって、一つの突然変異を伴う遺伝子の割合が僅かに高いことが観察できる。この現象は、既に観察されているｐｏｎＢ遺伝子の弱い毒性に、また、非活性化突然変異を伴う遺伝子の選択を好むベクターｐＡＲＡＰＯＮＢ及びｐＥＴ２６ｂ＋の軽い発現漏れに帰することができるだろう。
【０１２８】
【表ＩＶ】

【０１２９】
例ＩＩＩ
【０１３０】
例ＩＩＩは、制御された消化を用いる本発明の実施例を表す。
【０１３１】
Ｉ．材料及び方法
【０１３２】
Ａ．菌種、ゲノム及びプラスミドＤＮＡ
【０１３３】
すべてのＤＮＡ操作に対して、標準技術及び処理を用いた。発現プラスミドｐＥＴ２６ｂ＋（Novagen）を繁殖するために、大腸菌ＭＣ１０６１ＤＥ３細胞を用いた。
【０１３４】
Ｂ．オリゴヌクレオチド
【０１３５】
ＰＣＲのためのすべての合成オリゴヌクレオチド・プライマーは、ＭＷＧバイオテク（MWG Biotech）が合成した。センス・プライマー５’ＡＧＧＡＡＴＴＣＣＡＴＡＴＧＣＧＡＡＡＧＡＡＡＡＧＡＣＧＧＧＧＡ３’、そしてアンチセンス・プライマー５’ＡＴＡＡＡＧＣＴＴＴＣＡＣＴＴＧＡＴＧＡＧＣＣＴＧＡＧＡＴＴＴＣ３’を、テルモトガ・ネアポリターナ・キシラナーゼＡ遺伝子を増幅してＮｄｅＩ及びＨｉｎｄＩＩＩ制限部位（下線部分）を導入するために用いた。ＮｄｅＩ部位は初期コドン（太字）を含んでいた。
【０１３６】
Ｃ．酵素
【０１３７】
制限酵素、ＤＮＡ重合酵素及び熱安定性リガーゼは、ネブ（NEB）及びエピセンター （EPICENTRE）から購入し、製造業者の勧めに従って用いた。
【０１３８】
Ｄ．ＤＮＡ増幅、クローニング及び発現
【０１３９】
ＰＣＲ増幅をＰＥ ９６００サーモサイクラーで行った。テルモトガ・ネアポリターナ・キシラナーゼＡアンプリコンを、プライマー特定制限エンドヌクレアーゼで消化し、ｐＥＴ２６ｂ＋上の適合性のある部位内へライゲートさせて、大腸菌ＭＣ１０６１ＤＥ３へ変換した。このｐＥＴ２６ｂ＋ＸｙｎＡ発現ベクターを含むＭＣ１０６１ＤＥ３クローンを、カナマイシン（６０μｇ／ｍｌ）を含むＬＢ内で、３７℃で繁殖させた。
【０１４０】
Ｅ．生化学的な特徴
【０１４１】
ＸｙｎＡを発現する大腸菌に対して、直接的に熱不活性化実験を行った。４℃で５分間６０００ｇで遠心分離後、２００ｍＭ酢酸塩緩衝液ｐＨ５．６中で細胞を再懸濁した。再懸濁は、試料毎の細胞量を標準化するために、適当な量で行った。それから、異なる時間、適当な温度で１５０μｌの細胞を温置した。これらの細胞１００μｌを、２００ｍＭ酢酸塩緩衝液ｐＨ５．６での０．５％（ｗ／ｖ）キシラン１００μｌへ加え、１０分間８０℃で温置した。それから、２００μｌの３，５−ジニトロサリチル酸を加え、５分間沸騰させ、氷上で５分間冷却し、そして５分間１２０００ｇで遠心分離した。１５０μｌをμタイタープレート内へ移し、そして５４０ｎｍで ＯＤ を測定した。
【０１４２】
最適な温度の実験のため、２００ｍＭ酢酸塩緩衝液ｐＨ５．６での０．５％（ｗ／ｖ）キシラン１００μｌを、１００μｌの再懸濁細胞に加え、そして異なる温度で１０分間温置した。それから、２００μｌの３，５−ジニトロサリチル酸を加え、５分間沸騰させ、氷上で５分間冷却し、そして１２０００ｇで５分間遠心分離した。１５０μｌをμタイタープレート内へ移し、５４０ｎｍでＯＤを測定した。
【０１４３】
ＩＩ．結果
【０１４４】
Ａ．ＸｙｎＡの、熱安定性が低い突然変異体の生成
【０１４５】
ＸｙｎＡタンパク質の熱安定性が低い突然変異体を生成するため、図７に示すように、１％アガロース・ゲルを用いてＷＴ・ＸｙｎＡ遺伝子に対してエラー・プローンＰＣＲを行った。その産物をプライマー特定制限エンドヌクレアーゼで消化し、ｐＥＴ２６ｂ＋上の適合性がある部位内へライゲートして、大腸菌ＭＣ１０６１ＤＥ３へ変換し、エラー・プローン・ライブラリを生成した。
【０１４６】
このエラー・プローン・ライブラリからの一つのクローン（突然変異体３３）は、ＷＴタンパク質に比べ、非常に低い熱安定性を持つようであった。最適な温度及び熱不活性化の判定を含む敏速な生化学的分析を行い、ＷＴのものと比較した。最適な温度に関して、突然変異体３３は、（９０℃を超える）ＷＴに比べ、７８℃前後が最適な温度であったが、８２℃で３０分間、あるいは９５℃で１分間定温放置した後の突然変異体３３からは、活性の残留が全く検出されなかった。そこで、図８から不活性化定数を計算した。８２℃における突然変異体３３の熱不活性化を、８２℃で０．１２０／分と推測した。これらの温度では、ＷＴタンパク質に対する熱不活性化の検出は、全くなかった、あるいは低かった。
【０１４７】
Ｂ．シャフリング実験
【０１４８】
それから、異なる熱安定性を持つ突然変異体を生成するために、Ｌシャフリング（商標）技術を用いて突然変異体３３及びＷＴ遺伝子を再結合した。異なる突然変異体が予期された。ＷＴ最適温度を持つ突然変異体、ＷＴよりも熱安定性が低い突然変異体、そして突然変異体３３の最適温度の場合よりも熱安定性が高い突然変異体。
【０１４９】
１）断片ライブラリ
【０１５０】
ＷＴ及び突然変異体３３のＰＣＲ増幅後、その産物を、ＨｉｎｃＩＩ、ＢａｍＨＩ、ＸｈｏＩ、ＳｐｈＩ、ＥｃｏＲＩ及びＥｃｏＲＶの、六つの制限酵素の混合物で消化し、八つの断片（１２０から７００ｐｂまで）を生成した。３％アガロース・ゲルを用いる、六つの制限エンドヌクレアーゼの混合物でのＰＣＲ産物の断片化、図９を参照。
【０１５１】
２）シャフリング実験
【０１５２】
ＲＬＲ（組み換えライゲーション反応）は、変性及びライゲーションあるいはハイブリダイゼーションのステップを数サイクル繰り返して、熱安定性リガーゼと共に、（図９に示す）標準化した断片、そしてテンプレートとしてのＮｄｅＩ／ＨｉｎｄＩＩＩで消化したｐＥＴ２６＋ＸｙｎＡを用いて行った。
【０１５３】
負の制御を、熱安定性リガーゼ（Ｂ）を含まずに同じ条件で行った。その結果を図１０、１％アガロース・ゲルを用いるＬシャフリング（商標）実験に示す。図１０は、熱安定性リガーゼがない場合、断片が組み換えに用いられないことを示す。それから、反応混合物にＤｐｎＩを加えることによって、テンプレートの選択的消化を行った。
【０１５４】
３）クローニング産物
【０１５５】
ＰＣＲ・Ｐｆｕ増幅（図１１、１％アガロース・ゲルを用いるＬシャフリング（商標）産物へのＰＣＲ・Ｐｆｕ）を、５’センス及び５’アンチセンス合成プライマーと上記プロトコルとを用いて、Ａ及びＢ（図９、負の制御）両方のＤｐｎＩ消化Ｌシャフリング（商標）産物に対して行った。クローニングのための多量の増幅Ｌシャフリング（商標）産物を得たにも拘わらず、テンプレート増幅は全く起こらなかった。したがって、Ｌシャフリング（商標）産物は、プライマー特定制限エンドヌクレアーゼで消化され、ｐＥＴ２６ｂ＋上の適合性がある部位内へライゲートされ、そして大腸菌ＭＣ１０６１ＤＥ３へ変換されてＬシャフリング（商標）・ライブラリを生成した。
【０１５６】
４）生化学的な特徴
【０１５７】
８２℃で３０分間定温放置後に残る活性に関して、Ｌシャフリング（商標）・ライブラリからいくつかのクローンを選択した。
【０１５８】
クローン２４、４１及び５６（図１２、９５℃における突然変異体の熱不活性化）は、突然変異体３３の最適温度を持ち、そしてクローン６はＷＴキシラナーゼの最適温度を持つ。これらの実験条件において、９５℃で１２０分間定温放置した後、ＷＴキシラナーゼは１００％の活性を維持した。対照的に突然変異体３３については、９５℃で１分間の後、活性の残留は全く検出されなかった。図１３は、二つの親核とは異なる特性を示したＬシャフリング（商標）・ライブラリからの四つの突然変異体を示す。
【０１５９】
例ＩＶ
【０１６０】
例ＩＶは、ランダムな消化を用いる本発明の実施例を表す。
【０１６１】
Ｉ．材料及び方法
【０１６２】
Ａ．菌種、ゲノム及びプラスミドＤＮＡ
【０１６３】
すべてのＤＮＡ操作に対して、標準技術及び処理を用いた。大腸菌ＭＣ１０６１ＤＥ３細胞を用いて、発現プラスミドｐＥＴ２６ｂ＋（Novagen）を繁殖した。
【０１６４】
Ｂ．オリゴヌクレオチド
【０１６５】
ＰＣＲのためのすべての合成オリゴヌクレオチド・プライマーは、ＭＷＧバイオテクが合成した。センス・プライマー５’ＡＧＧＡＡＴＴＣＣＡＴＡＴＧＣＧＡＡＡＧＡＡＡＡＧＡＣＧＧＧＧＡ３’、そしてアンチセンス・プライマー５’ＡＴＡＡＡＧＣＴＴＴＣＡＣＴＴＧＡＴＧＡＧＣＣＴＧＡＧＡＴＴＴＣ３’を用いて、テルモトガ・ネアポリターナ・キシラナーゼＡ遺伝子を増幅し、ＮｄｅＩ及びＨｉｎｄＩＩＩ制限部位（下線部分）を導入した。センス・プライマー５’ＧＧＡＡＴＴＣＣＡＴＡＴＧＧＣＧＧＣＧＧＣＡＧＣＣＧＧＣＡ３’、そしてアンチセンス・プライマー５’ＧＧＡＡＴＴＣＣＴＡＣＴＧＣＣＧＣＴＣＣＧＡＴＴＧＴＧＧ３’を用いて、アシドバクテリア・カプスラーツム・キシラナーゼ遺伝子を増幅し、ＮｄｅＩ及びＥｃｏＲＩ制限部位（下線部分）を導入した。ＮｄｅＩ部位は初期コドン（太字）を含んでいた。
【０１６６】
Ｃ．酵素
【０１６７】
制限酵素、ＤＮＡ重合酵素及び熱安定性リガーゼは、ネブ及びエピセンターから購入し、製造業者の勧めに従って用いた。
【０１６８】
ＩＩ．結果
【０１６９】
テルモトガ・ネアポリターナ遺伝子（３．２ｋｂ）及びアシドバクテリア・カプスラーツム遺伝子（１．２ｋｂ）は再結合した。
【０１７０】
Ａ．断片ライブラリ
【０１７１】
テルモトガ・ネアポリターナとアシドバクテリア・カプスラーツム遺伝子上でＰＣＲ増幅を行ってから、ＤＮアーゼＩでの消化を行った。
【０１７２】
図１３、１％アガロース・ゲルを用いるテルモトガ・ネアポリターナ（Ａ）及びアシドバクテリア・カプスラーツム（Ｂ）遺伝子のＤＮアーゼＩ断片化を参照。
【０１７３】
Ｂ．シャフリング実験
【０１７４】
（図１３に示す）熱安定性リガーゼ及び熱安定性フラップで標準化した断片で、変性及びハイブリダイゼーションあるいはライゲーションを数サイクル繰り返して、ＲＬＲを行った。
【０１７５】
同じ条件の下で、ただし熱安定性リガーゼそして／あるいは熱安定性フラップがない状態で、負の制御を行った（Ａ、Ｂ及びＣ）。その結果を図１４、１％アガロース・ゲルを用いるＬシャフリング（商標）実験に示す。図１４は、熱安定性リガーゼ及び熱安定性フラップがない場合、断片が再結合しないことを示す。図１４において、Ａは、リガーゼ及びフラップ活性がない断片を表し、Ｂは、リガーゼのみを持つ断片を表し、Ｃは、フラップのみを持つ断片を表し、そしてＤは、シャフリング状態を表す。
【０１７６】
例Ｖ
【０１７７】
例Ｖは、例ＩＩＩの材料及び方法を用いたが、ステップ（ｂ）及び（ｃ）の異なるサイクル数で実験した。図１５Ａ、ｎサイクルのステップ（ｂ）及び（ｃ）を用いるＬシャフリング（商標）、そして図１５Ｂ、対応するＬシャフリング（商標）産物のＰＣＲ増幅を参照。図１５Ａ及びＢに示すように、組み換えポリヌクレオチドを得るためには、少なくとも一つのサイクル（ｎ＝１）が必要である。
【０１７８】
例ＶＩ
【０１７９】
例ＶＩは、例ＩＩＩの材料及び方法を用いたが、
次に示す七つの量の断片で実験した。
１：１ｘ
２：２ｘ
３：３ｘ
４：４ｘ
５：１１ｘ
６：１４ｘ
７：１７ｘ
【０１８０】
図１６、断片の増量を用いるＬシャフリング（商標）実験は、これらの７つの量に対する結果を示す。
【０１８１】
前述のプレゼンテーションは、本発明の範囲を限定することを意図するものではない。本発明の実施例を、添付図面を参照しながら詳細に説明したが、ここで説明した方法への変更が可能であることは、本技術の熟練者には明白である。本技術の熟練者は、これらの、そして他の種々の変更及び実施例を、本発明の範囲から外れることなく行うことが可能である。本発明の範囲は、従来の技術を考慮に入れて、請求項及びそれと同等のものに参照して判定されるべきである。
【０１８２】
（参照文献）

【図面の簡単な説明】
【０１８３】
添付の図面を参照する。
【図１Ａ】従来のＤＮＡシャフリングを概略的に表す。
【図１Ｂ】従来のＳｔＥＰ（図１Ｂ）を概略的に表す。
【図２−１】本発明のプロセスの実施例、そしてその変形及び用途を概略的に表す。
【図２−２】本発明のプロセスの実施例、そしてその変形及び用途を概略的に表す。
【図２−３】本発明のプロセスの実施例、そしてその変形及び用途を概略的に表す。
【図３】ｐｏｎＢ遺伝子の各突然変異体による突然変異の１０領域（ＰｖｕＩＩ及びＰｓｔＩ）の位置を表す。
【図４】ｐｏｎＢ遺伝子配列に比較するために用いたプライマーの位置を表す。
【図５】アガロース・ゲル上における、ＲＬＲ反応のＲＬＲ及びＰＣＲ反応生成物の泳動を表す。
【図６】制限断片に比較した突然変異の位置を表す。
【図７】１％アガロース・ゲルを用いるＷＴ・ＸｙｎＡ遺伝子に対するＰＣＲの結果にエラーが起こり易いことを表す。
【図８】８２℃における、突然変異体３３の熱不活性化を表す。
【図９】３％アガロース・ゲルを用いる、六つの制限エンドヌクレアーゼによるＰＣＲ産物の断片化の結果を表す。
【図１０】１％アガロース・ゲルを用いる、Ｌシャフリング（商標）実験の結果を表す。
【図１１】１％アガロース・ゲルを用いて、Ｌシャフリング（商標）産物に対してＰＣＲ・Ｐｆｕを使用したときの結果を表す。
【図１２】９５℃における、突然変異体の熱不活性化を表す。
【図１３】１％アガロース・ゲルを用いる、 Thermotoga neapolitana （Ａ）及び Acidobacterium capsulatum （Ｂ）遺伝子のＤＮアーゼＩ断片化の結果を表す。
【図１４】１％アガロース・ゲルを用いる、Ｌシャフリング（商標）実験の結果を表す。
【図１５Ａ】ステップ（ｂ）及び（ｃ）をｎサイクル用いるＬシャフリング（商標）の結果を表す。
【図１５Ｂ】対応するＬシャフリング（商標）産物のＰＣＲ増幅を示す。
【図１６】断片の量を増加させた、Ｌシャフリング（商標）実験の結果を表す。【Technical field】
[0001]
The present invention relates generally to genetic modification, and more particularly to the field known as directed evolution, molecular propagation or DNA shuffling. An object of the present invention is to generate a novel sequence having improved properties compared to a reference sequence. When performed outside an organism, this process is a technique for in vitro evolution. The invention further relates to sequences generated by the method, libraries of such sequences, hosts and vectors containing such sequences, and proteins converted therefrom, to arrays simulating the methods of the invention, And an array capable of performing the following. The present invention further relates to intermediate products of the method and to reaction mixtures of specific types of polynucleotide fragments and organized templates.
[Background Art]
[0002]
Various techniques are known for promoting ex vivo recombination of polynucleotide sequences. The most well-known conventional techniques include polymerization-dependent sexual PCR (many cycles without the addition of primers) and DNA shuffling, including attachment extension (StEP).
[0003]
Typically, in DNA shuffling, including sexual PCR, DNase I randomly cleaves polynucleotide sequences to form oligonucleotide fragments, the fragments initiate polymerization or PCR extension, and the recombinant polynucleotide is amplified. You. At each hybridization step, crossovers occur at regions of homology between the sequences ("strand crossover"). FIG. 1A schematically illustrates this method.
[0004]
StEP consists of mixing different polynucleotide sequences containing different mutations in the presence of a pair of initiators. This mixture is subjected to a PCR reaction in which the hybridization and polymerization steps are integrated into a single, very short step. These conditions allow the initiator to hybridize, but also slow down the polymerization so that the initiator has time to synthesize only those fragments that after denaturation will randomly rehybridize to various polynucleotide sequences. . FIG. 1B schematically illustrates this method.
DISCLOSURE OF THE INVENTION
[Problems to be solved by the invention]
[0005]
Excessive reliance on polymerization has disadvantages. Such methods do not have control over the rate or location of recombination that occurs randomly during successive stages of the polymerization. The polymerization may also produce unwanted additional mutations or an insufficient number of mutations, depending on the conditions used and the polymerization enzyme. The latter occurs when long gaps are filled with residues that completely complement the opposing strand. Furthermore, after sufficient cycles, the fragments grow very long to become what are known as "mega-initiators" (6). Mega-initiators cause various problems, especially when the starting polynucleotide exceeds about 1.5 kb.
【The invention's effect】
[0006]
The present invention does not need to rely on the polymerization, size fractionation (separation of fragments by size) or amplification of the initial polynucleotide or fragment. In addition, Applicant believes that the present invention and embodiments have broad advantages except where expressly stated, but not desired, but not so.
BEST MODE FOR CARRYING OUT THE INVENTION
[0007]
First, the present invention provides control over the location of the recombination. Hybridization on the template allows precise control over where recombination occurs. For example, if the target protein contains an active site that one desires to maintain unchanged, the present invention can limit recombination to regions other than the active site. Furthermore, the present invention can achieve a high degree of recombination between adjacent sequence segments. The present invention is capable of moving nearby chunks, rather than treating them as "joining", in chunks to separate nearby laying arrays. Thus, in a sense, the present invention also achieves high resolution, fidelity and quality for genetic diversity. Indeed, embodiments of the invention that use non-random fragmentation can be used with fragments as short as 15 residues.
[0008]
The invention is also capable of producing more recombination and incorporation of fragments per reaction cycle, particularly in embodiments other than the ligation-only embodiment (defined below). In other words, a large variety of genes can be obtained. This is achieved directly by stimulating more total recombination events. It is also achieved indirectly by increasing overall efficiency. In particular, the use of directional ligation can improve the overall efficiency. Without directional ligation, sequences cut into "n" fragments would reassociate into a very wide variety of possible forms, even if some forms were useful. The present invention, on the other hand, facilitates the direct achievement of the desired form. Indeed, in some embodiments of the present invention, it is possible to obtain a recombinant polynucleotide after only a single reaction cycle.
[0009]
Typically, the present invention further increases efficiency because of the relatively low incidence of unshuffled nucleophilic clones and overlapping chimeras. Avoiding these unwanted by-products provides room for more novel chimeras. Conventional methods generate a selection library consisting of 30% to 70% nucleophilic DNA. All methods for directed evolution, molecular breeding or gene shuffling produce a sorted library of recombinant DNA molecules and express and screen these molecules. Screening is the most expensive and time consuming part of the process, as libraries contain hundreds of thousands to millions of recombinant molecules. This problem can be alleviated by eliminating the parent nuclear DNA from the selection library. Since the final population is made up of new and different polynucleotides, the removal of nucleophilic DNA is improved if the template is transient, as in the preferred embodiment of the present invention.
[0010]
Preferred embodiments of the method of the invention, particularly those using isolated strand templates or fragments, also facilitate low homology shuffling, for example, of distantly related members of a gene family. Because all are from the same strand, either the sense or antisense strand, of a single polynucleotide or multiple homologous polynucleotides, they do not complement one another, especially the population of single-stranded sequences. The term "isolated chain" is used to represent. For example, because isolated strand fragments are not complementary, or at least are not exactly complementary to other fragments in the reaction mixture, hybridization will occur with complementary fragments, such as "wild-type". Have no tendency towards an array. Thus, it is possible to adjust the hybridization temperature to the temperature of homology between sequences to maximize diversity and greatly increase the chance of finding the appropriate mutant at the minimum number of recombination cycles. (Note: the present invention may also include, for example, using a large amount of one parent nucleopolynucleotide to achieve a desired trend.)
[0011]
Furthermore, the present invention allows for the instant use of complexes that may contain introns, ie genomic DNA, with little preparation of a starting DNA library. Some other methods require time-consuming mRNA isolation and remodeling of the cDNA sequence to produce fragments that are shuffled or reassembled.
[0012]
Advantages and embodiments of the present invention are further described below.
[0013]
Summary of the Invention
[0014]
Although the present invention relates generally to genetic recombination, in the context of the present invention, `` recombination '' is intended to mean, as long as the term implies that the two strands are cut apart and rejoined to form a recombinant sequence. This is somewhat wrong. In other words, the invention does not rely on chain transfer or crossover. However, with this in mind, "recombination" and related terms are used in the text.
[0015]
In one embodiment, the method of the invention comprises a method of template-adjusted ligation orientation localization for non-random shuffling of polynucleotides.
The method includes the following.
a) Obtaining, directly or indirectly, single-stranded fragments of at least two homologous polynucleotides from a polynucleotide library.
b) forming at least one partially double-stranded polynucleotide by hybridizing said fragments to one or more devised organized templates until at least two fragments hybridize adjacently. . Note that at least one of the templates shares at least one homology region with the homologous polynucleotide.
c) treating said partially double-stranded polynucleotide to form at least one recombinant polynucleotide.
In this case, the process performs the following regardless of the order.
(I) Licens Nick. And
(Ii) Perform one or a combination of the following gap technologies as needed.
Filling gaps by further hybridizing the fragments to the template so as to increase the number of adjacent hybridizing fragments.
Filling short gaps by trimming overhanging flaps of partially hybridized fragments.
And filling short gaps through polymerization.
[0016]
In the above embodiment, any step, particularly steps (b) and (c), may be repeated as necessary. In another embodiment, the method of the invention produces a recombinant polynucleotide after only one, one cycle or a single operation of each step of the invention. In a preferred embodiment, the method further comprises the step (d) of selecting at least one of said recombinant polynucleotides having the desired properties. More preferably, the step occurs in vitro (outside the organism). In some preferred embodiments, the methods employ non-random fragmentation, transient templates and isolated strand templates or fragments, among others.
[0017]
In an alternative embodiment, the invention consists essentially of steps (b) and (c) above. In such a case, step (b) becomes “step (a)” and also includes hybridizing a single-stranded fragment of at least two homologous polynucleotides.
[0018]
In another alternative embodiment, the present invention comprises a method of template-adjusted ligation orientation localization for non-random low homology shuffling of gene families in vitro. Whether the homology is considered low depends on the situation, but homologies below 50% (eg, 40-70% or 20-45%) are typically considered low. In another alternative embodiment, the parent nucleopolynucleotides differ in length by more than two residues.
[0019]
In yet another alternative embodiment, the invention comprises a method of template-adjusted ligation orientation for in vitro non-random shuffling of a region containing a mutation in a polynucleotide allelic unit. This example further comprises locating restriction sites relative to regions containing mutations between allelic units and obtaining fragments corresponding to those restriction sites.
[0020]
The invention further includes sequences resulting from the method, libraries thereof, hosts and vectors containing the same, and proteins translated therefrom. It also includes arrays of logical names, such as computer algorithms that simulate the method of the present invention, or physical arrays, such as biochips, on which the method of the present invention is performed. The invention further relates to a reaction mixture of the polynucleotide fragment and the assembling template, which can be used for intermediates of the method and for performing some or all steps of the method.
[0021]
Definition
[0022]
As used herein, "ex vivo" refers to a location outside the body of an organism. "Homologous" polynucleotides differ from each other in at least one corresponding residue position. Thus, “homology” as used herein also includes those that are sometimes referred to as “partially heterologous”. For example, the homology between the parent nucleopolynucleotides ranges from 20% to 99.99%, preferably from 30 to 90%, more preferably from 40 to 80%. In some embodiments, the term "homologous" may indicate a sequence that is, for example, only about 20-45% identical at the corresponding residue position. Homologous sequences may or may not share a common ancestor or evolutionary origin with each other.
[0023]
"Polynucleotide" and "polynucleotide sequence" refer to single-stranded, isolated-strand, or partially or completely double-stranded, nucleic acid or ribonucleic acid sequences, including mRNA. When partially or completely double-stranded, each strand may be identical or different, unless otherwise indicated. The polynucleotide may be a gene or a part of a gene. "Gene" refers to a polynucleotide, or a portion thereof, that is associated with a known or unknown biological function or activity. Genes can be obtained from different methods, including extraction from nucleic acid sources, chemical synthesis, and synthesis by polymerization. "Nucleonucleotide" and "nucleophile" are interchangeable synonyms and refer to polynucleotides that have been fragmented to yield a donor. A parent nucleopolynucleotide is often obtained from a gene. "Recombinant polynucleotide", "mutant polynucleotide", "chimeric polynucleotide" and "chimera" generally refer to a polynucleotide produced by a method. However, these terms may refer to other chimeric polynucleotides, such as those in the initial library. “Reference sequence” refers to a polynucleotide, often derived from a gene, having desired or near characteristics, and is used as a target or reference for generating or evaluating other polynucleotides.
[0024]
"Polynucleotide library" and "DNA library" refer to a group, pool or bank of polynucleotides comprising at least two homologous polynucleotides or fragments thereof. The polynucleotide library consists of an initial library or a selection library. An "initial library", an "initial polynucleotide library", an "initial DNA library", a "nucleophilic library" and a "starting library" are at least two homologous nucleophilic polynucleotides, or polynucleotides comprising fragments thereof, Or it refers to a group, pool or bank of those fragments. The initial library consists of genomic or complex DNA and may include introns. It may also include an array generated by prior shuffling. Similarly, sorted libraries, or other limited libraries of recombinant polynucleotides or fragments, may be so referred to as may serve as initial libraries. "Screened library" refers to a polynucleotide library containing chimeras produced by an ingenious process or another recombination process.
[0025]
"Residue" refers to an individual nucleotide or ribonucleotide, rather than to multiple nucleotides or ribonucleotides. A residue may refer to a free residue that is not part of a polynucleotide or fragment, or may refer to a single residue that forms part of a polynucleotide or fragment.
[0026]
"Donor piece" and "fragment" generally refer to the fragmented portion of a parent nucleopolynucleotide. Fragments may also refer to supplemental or surrogate fragments that are added to the reaction mixture and / or originate from sources other than fragmentation of the parent nucleopolynucleotide. Many or all of the fragments should be shorter than the parent polynucleotide, and in preferred embodiments, most or all of the fragments are shorter than the assembled template.
[0027]
As used herein, "non-random" and "control" broadly refer to control or predictability, for example, with respect to the rate or location of recombination, achieved through the template and / or ligation orientation of the present invention. Non-random and control also refer narrowly to polynucleotide fragmentation techniques that allow for control over the size or sequence of the resulting fragment or that provide predictability. For example, by using a restriction enzyme to cleave a polynucleotide, the properties of the fragment can be controlled. It is noted that the invention may be considered non-random even when using random fragmentation (typically by DNase I digestion). In such cases, the organizational template of the present invention, and other features, still provide some control. However, in embodiments, fragmentation is non-random, ie, controlled.
[0028]
"Assembled template", "developed template" and "template" refer to polynucleotides used as scaffolds to anneal or hybridize fragments to form partial or complete double-stranded polynucleotides. Point. In a preferred embodiment, the template is longer than most or all of the donor strip. In such a case, the free donor pieces cannot be considered as templates for each other. The template may be obtained from a reference sequence, an initial library, a selection library, or elsewhere. The template consists of or is obtained from the parental nucleopolynucleotide of the initial library, but if the polynucleotide accidentally enters the shuffling process, for example, by falling into a hybridization step without fragmentation, the template Is not allowed. In other words, the template is neither completely random nor accidental. Rather, they are designed at least to a certain extent. The template may be a purpose-based design, creation, formulation, creation, derivation, and / or specific and desired polynucleotide sequence, or a desired polynucleotide sequence for use as a template by a human or by a computer operated by a human. Directly or indirectly through the selection of a sequence from a source likely to contain the sequence of Templates may be synthetic, resulting from shuffling or other artificial processing, or may be naturally occurring. "Transient template" refers to a template that is not itself incorporated into the final recombinant polynucleotide. This transientity results from the separation or disruption of the non-final recombinant polynucleotide template strands generated during the process.
[0029]
The term "isolated strand" refers to a population of single-stranded sequences that do not complement each other because they are from the same strand, sense or antisense, of a single polynucleotide or multiple homologous polynucleotides. . In other words, there are no sequences from opposing complementary strands and the population does not contain complementary sequences. For example, a population of isolated strand fragments may consist of fragments of the top strand of the parent nucleopolynucleotide and a population of isolated strand templates may consist of the bottom strand of one or more parent nucleopolynucleotides.
[0030]
"Ligation" refers to the formation of a phosphodiester bond between two residues.
[0031]
"Nick" refers to the lack of a phosphodiester bond between two residues hybridized to the same strand of a polynucleotide. Nicks include a lack of phosphodiester linkages by DNase or other enzymes, and also a lack of linkage between adjacently hybridized fragments that were simply not ligated. However, the nicks used herein do not include residue gaps.
[0032]
As used herein, "gap" and "residue gap" refer to the absence of one or more residues on one strand of a partially double-stranded polynucleotide. In some embodiments of the invention, short gaps (less than about 15 to 50 residues) are filled by a polymerase and / or flap trimming, and long gaps are customarily filled by a polymerase. In the present invention, long gaps may be filled only by hybridization or trimming.
[0033]
“Hybridization” has its general meaning, except that it involves the necessary cycles of denaturation and rehybridization.
[0034]
"Flanking fragments" refers to hybridizing fragments whose ends are smooth with respect to each other and are separated only by nicks, not by gaps.
[0035]
"Ligation only" refers to embodiments of the present invention that do not utilize, ie, do not require, polymerizing enzyme extension or flap trimming to fill the gap. In a ligation-only embodiment, all fragments hybridize contiguously. Note that ligation is also used in embodiments that are not ligation-only embodiments.
[0036]
“Ligation orientation” and “directed ligation” generally refer to or refer to a template adjustment process that allows ligation of fragments or residues in a relatively ordered or relatively predictable order. All embodiments of the invention rely on ligation orientation. For example, even in the ligation-only embodiment, the localization is in the ligation direction.
[0037]
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION
[0038]
One embodiment of the present invention is a template-tuned method of ligation orientation localization for non-randomly shuffling polynucleotides. The method comprises the following steps.
a) Obtaining, directly or indirectly, single-stranded fragments of at least two homologous polynucleotides from a polynucleotide library.
b) hybridizing these fragments to one or more devised organized templates until at least two fragments are hybridized adjacently. This forms at least one partially double-stranded polynucleotide. In this case, at least one of the templates shares at least one homology region with the homologous polynucleotide.
c) treating the partially double-stranded polynucleotide to form at least one recombinant polynucleotide.
This process includes the following steps, regardless of the order.
(I) Licens Nick. And
(Ii) If necessary, implement any of the following gap-filling techniques, or a combination thereof.
To increase the number of adjacent hybridized fragments, the gap is filled by further hybridizing the fragment to the template.
Fill short gaps by trimming the overhanging flaps of the partially hybridized fragments.
And fill short gaps through polymerization.
[0039]
Embodiments of the invention may use a polymerase, but in such embodiments, the polymerase may simply fill short gaps (eg, 15 to 50 residues or less) instead of long gaps. Used. In a preferred embodiment, the process does not use a polymerizing enzyme. In a ligation-only embodiment, the method does not use any gap-filling techniques, but instead relies on the ligation of completely adjacent fragments, often achieved after multiple hybridization events.
[0040]
Once the partially double-stranded polynucleotide has become sufficiently double-stranded, it is preferable to select (d) advantageous properties compared to those of one or more reference sequences. Advantageous properties may include, for example, the thermostability of the enzyme or activity under a particular pH or salinity. In many other possible applications, such enzymes may also be used to desizing textile fibers, bleach paper pulp, flavor dairy products, or biocatalyze the synthesis of new therapeutic molecules. Good.
[0041]
The process may also include disrupting the template strand, or separating it from the recombinant strand, before and after selection. It may further include amplifying the recombinant sequence prior to the selection in step (d), or cloning the recombinant polynucleotide sequence after separating the recombinant strand from the template. Any amplification technique may be used. PCR allows for selective amplification of the recombinant sequence, as there are initiators that can hybridize only to the ends of the recombinant sequence. However, unlike shuffling in sexual PCR, the present invention does not require amplification during the recombination reaction.
[0042]
Preferred screening techniques require in vitro transcription of the recombinant polynucleotide, followed by in vitro expression via in vitro translation of the mRNA. This technique eliminates the physiological problems of cells and the disadvantages associated with in vivo expression cloning. In addition, this technique is easily automated and allows for the screening of large numbers of recombinant sequences.
[0043]
Although embodiments of the present invention actually perform steps only once and do not need to go around ("non-iterative"), the present invention also provides for repetition of any of the steps, steps (a), (b) and And / or the repetition of (c). For example, the process may or may not require multiple hybridization events. The hybridization in step (b) and the additional hybridization in step (c) are intended to cover the required cycles of denaturation and rehybridization. If necessary, the ligated and / or unligated fragments generated by steps (b) and / or (c), rather than simply on the donor pieces generated by step (a) Alternatively, part or all of steps (b) and / or (c) may be repeated. Ligation-only embodiments typically require a large number of iterations. In addition to including repetition of steps, the present invention includes embodiments that allow for simultaneous processing of steps, known in the art as simultaneous processing capability.
[0044]
In a preferred embodiment, the initial library itself is generated by the present invention. In vivo or ex vivo screens can be used to form this library to repeat the process of the present invention. The recombinant sequences selected after the first stream of the process are optionally mixed with other sequences.
[0045]
Initial libraries can also be used in a manner known to those skilled in the art, for example, by starting with a wild-type gene, by managing successive steps of mutagenesis, by "error-prone" PCR (2). Can also be generated by random chemical mutagenesis, by random mutagenesis in vivo, or by combining genes from near or relatively distant families in the same or different species. . The initial library preferably results from a chain polymerization reaction under conditions that produce random local mutations. The invention may also consist of a synthetic sequence.
[0046]
Organizing template
[0047]
The organized template of step (b) or (c) is, for example, a polynucleotide from an initial library or a polynucleotide generated therefrom. Templates may be synthetic, may result from shuffling or other artificial processing, or may be naturally occurring. The template may be single-stranded or double-stranded. In the case of a double strand, it must be denatured in step (b) or the like before actual hybridization. If the template is directly incorporated in step (b), the template must be denatured or already in single-stranded form.
[0048]
The preferred embodiment uses an isolated strand template. In a more preferred embodiment, the isolated strand template uses the bottom strand from one parent nucleopolynucleotide and the top strand fragment from another nucleophile. This prevents the sequence from reannealing to its own complementary strand. In order to obtain an isolated strand DNA molecule, a vector of a family of filamentous phage such as Bluescript phagemid or M13mp18 can be used. Another method consists in generating double-stranded molecules by PCR using an initiator phosphorylated at 5 'and other non-phosphorylated initiators. Digestion of lambda phage by exonuclease breaks the 5 'phosphorylated DNA strand, leaving the unphosphorylated strand intact. Another method of generating isolated strand molecules consists of amplifying by asymmetric PCR starting from a methanol-denatured DNA template. Digestion with DpnI destroys the methanol-denatured strand, but leaves the amplification product intact, allowing purification after denaturation.
[0049]
The preferred embodiment also uses a transient template that is not incorporated into the final recombinant polynucleotide, eg, is not part of the polynucleotide that is transferred to the sorting library. One technique for conferring transients uses markers on the recombinant strand or template. For example, the template may be labeled with a hapten and separated, for example, by anchoring the carrier with an anti-hapten antibody or by initiating a biotin-streptavidin reaction. Another technique consists of synthesizing the transient template by PCR amplification with methanol-modified dATP, which allows the degradation of the template by the restriction endonuclease DpnI. In this case, the recombinant strand must not contain methanol-modified dATP. Transient templates can also be prepared by PCR amplification with dUTP, which allows for degradation with uracil DNA glycosylase. Conversely, it is possible to protect the recombinant strand by amplification in a selective PCR using an oligonucleotide with a 5 'phosphorothioate modified group. Thus, treatment with exonuclease allows for exclusive degradation of the template. In a most preferred embodiment, the transcript is conferred by using a template containing uracil, such as mRNA. mRNA has a high affinity for binding and can be removed by mRNA-specific enzymes. Such an mRNA template can be prepared in vivo or in vitro. In a more preferred embodiment, the use of an mRNA template requires that the process include at least three primers linked to a ligase.
[0050]
In yet another preferred embodiment, the template allows for directional orientation of blunt-ended multimolecular ligation. In this example, the template is a relatively complementary 3 'end of the first fragment and the 5' end of the second fragment adjacent to the first fragment in the parent polynucleotide. Consists of short single-stranded or double-stranded polynucleotides. This facilitates adjacent hybridization of these two ends on the template.
[0051]
Further, embodiments include any or all of the following. Templates and donor pieces are from different sources. The templates are individually added to the reaction mixture. And / or the template is modified in a particular way to increase chimerism.
[0052]
Donation piece
[0053]
Fragments can be derived from homologous polynucleotides, related genes, or from other genes. The nucleophilic DNA need not be characterized at all, but can be extracted from cells, clinical samples or the environment. Step (a) starts with a pre-fragmented single-stranded or double-stranded fragment from the library containing the initial fragment and / or fragments the single-stranded or double-stranded parent polynucleotide from the initial library. Starting with the sub-steps of Step (a) may include joining a separate library of fragments and / or fragmenting the parent nucleopolynucleotide from a separate starting library. It may also include fragmenting the parent nucleopolynucleotide from the same library in different ways, such as using different restriction enzymes. Further, step (a) may involve using more fragments from one parent nucleoside polynucleotide than another. For example, an experimenter using this process may bias the result by using more fragments or portions of polynucleotide X than fragments or portions of polynucleotide Y.
[0054]
In one embodiment, supplementary single- or double-stranded fragments of various lengths are added in step (b) or (c). These supplementary fragments are capable of replacing some of the fragments of step (a), especially if the sequence is homologous to the sequence of the fragment of step (a). Such supplementary fragments may, for example, introduce one or more direct mutations. Further, it may be composed of a synthetic sequence.
[0055]
Fragmentation may occur before or after denaturation of the sequence to be fragmented. Fragmentation may be controlled or random. In a random case, enzymes or mechanical means known to those skilled in the art, such as digestion with DNase I or sonication, can be used to randomly cut DNA. Controlling fragmentation facilitates control of the degree, speed, efficiency and / or location of recombination. A preferred embodiment involves hydrolyzing the parent nucleopolynucleotide with a restriction enzyme to produce a restriction donor piece. Restriction enzymes provide control over the degree, rate and efficiency of recombination by controlling the number of fragments generated per sequence. For example, the number may be increased by using a restriction enzyme having many cleavage sites, or by using several different restriction enzymes. The greater the number of generated fragments per sequence, the greater the number of fragments (n) that must be reassembled to form a recombinant sequence. n is preferably 3 or more.
[0056]
By controlling the nature and location of the fragment ends, the restriction enzyme provides further control not only on the degree and speed, but also on the location where recombination occurs. For example, fragmentation can be designed such that cleavage occurs in a region of the parent nucleus sequence that is homologous to a region in the reference sequence or assembly template.
[0057]
Preferably, fragments are about 15 to 500 residues in length. Where fragmentation is performed non-randomly, fragments are advantageously at least 15 residues in length, but more preferably about 15 to 40 residues in length. The phrase "at least 15 residues" means between about 15 residues and one residue less than the full length of the longest polynucleotide used. If fragmentation is performed randomly, it is more preferred that the length be at least 50 residues. The phrase "at least 50 residues" means between about 50 residues and one residue less than the full length of the longest polynucleotide.
[0058]
At least two ends of the fragment in step (a) must be capable of being hybridized adjacently and ligated. (In a ligation-only embodiment, all of the fragments that hybridize to form the final recombinant strand must have such ends.) In a preferred embodiment, the present invention employs a flap Using the trimming enzyme, fragments that would otherwise be unproductive are ligated to the ends. These enzymes identify and degrade or cleave the unhybridized ends of the fragments in a specific manner, and cover other hybridized fragments on the same template.
[0059]
A preferred enzyme is a flap endonuclease, which can be used in step (c) or during the hybridization of step (b). When a fragment is first made double-stranded, an embodiment of the invention consists in using a specific exonuclease that identifies and degrades single-stranded sequences, such as the unhybridized ends of the fragment. Such single-stranded exonucleases or flap endonucleases are preferably at a concentration that avoids the more common exonuclease activity that may, for example, degrade template or recombinant sequences (eg, about 1. 8 to 2.2 μg / ml flap endonuclease). These enzymes increase the number of fragment ends that can be ligated in step (c). It is particularly useful for cutting pieces randomly because of the tendency to have many overhang flaps. Use of such enzymes with low hybridization temperatures and / or long hybridization times (eg, 2 minutes) facilitates recombination between polynucleotides with low homology. For example, a preferred embodiment using random fragmentation involves the use of flap endonuclease and a wide range of hybridization temperatures (eg, 5 to 65 ° C.) in step (b). Step (b) can be decoupled from the ligation of step (c) with respect to temperature, especially if the hybridization temperature is below the high ligation temperature (eg, about 60 to 75 ° C.). Most preferably, the flap endonuclease concentration is about 2 μg / ml, the hybridization temperature is about 10 ° C., and the ligation temperature is about 65 ° C. When such a trimming enzyme is used, it is preferable that it is thermostable and thermostable like a ligase, and is active at a high temperature.
[0060]
Additional optional features of the method of the invention
[0061]
Unlike conventional shuffling methods, various embodiments of the present invention do not require the repetitive heating and cooling required for thermocycling, eg, sexual PCR. In various embodiments, the process may be used to generate gene length or short polynucleotides. Various embodiments may cause hybridization to occur under less stringent conditions. In various embodiments, the ratio of template to chimeric polynucleotide produced is about 1. Various embodiments do not use any DNase. In various embodiments, the initial library consists of single gene variants. In various embodiments, the initial library may include a polynucleotide having an artificially induced point mutation. In various embodiments, the present invention may be used for whole genome shuffling. In various embodiments, the steps may be performed in vivo rather than ex vivo. Finally, when amplifying fragments by, for example, PCR, the starting sequence can be designed to generate fragments with adjacent ends along all of the organization template.
[0062]
Example I
[0063]
The purpose of Example I is to generate a recombinant polynucleotide from the kanamycin resistance gene using isolated strand fragments.
[0064]
First, the resistance gene (1 kb) of pACYC184 is cloned into the polylinker of M13mp18 so that the isolated phagemid contains the non-coding strand of the gene.
[0065]
In parallel, the gene is amplified by PCR mutagenesis (error-prone PCR) with two initiators that complement the vector sequence M13mp18 on each side of the gene sequence. The initiator for the non-coding strand is phosphorylated, but the initiator for the coding strand is not. The products of the PCR mutagenesis are digested with lambda exonuclease to generate a coding strand library of mutants of the kanamycin resistance gene.
[0066]
This library of isolated strand sequences is digested with a restriction enzyme mixture, particularly HaeIII, HinfI and TaqI. The resulting isolated strand fragments are then hybridized with the isolated strand phagemid and ligated with thermostable ligase. This step is repeated several times until no small fragments are observed during precipitation on the agarose gel. In the meantime, the stripes corresponding to the isolated strands of the complete resistance gene are the major components of the "smear" visible on the gel.
[0067]
Stripes corresponding to the size of the gene are cut out of the gel and purified. It was hybridized with two complementary oligonucleotides (40-mer) of the M13mp18 sequence on each side of the gene, and this partial duplex was digested with EcoRI and SphI and then M13mp18 digested with the same enzymes. Ligate in the vector.
[0068]
The cells altered with the ligation product are then screened for increased resistance to kanamycin.
[0069]
The cloning of isolated strand recombinant molecules is optionally performed by PCR of the complete gene on two initiators and cloning of the double-stranded product of this amplification. To avoid unwanted mutations, this amplification should be performed with a Pfu-type polymerase and with a limited number of cycles.
[0070]
Purify the plasmid of a clone that is significantly more resistant to kanamycin than the initial stock and use it for PCR with the polymerizing enzyme Pfu under high fidelity conditions using a phosphorylated or non-phosphorylated initiator couple as defined above. . This results in a second generation of isolated strand fragments after treatment with lambda exonuclease and fragmentation with restriction enzymes. The enzymes used in this step may be different mixtures (eg, BstNI, TaqI and MnII).
[0071]
The recombination and selection steps are repeated several times until the resistance to kanamycin is substantially increased.
[0072]
Example II
[0073]
I.wrap up
[0074]
The starting library contains the code for PBP1b of E. coli (1), a 10 gene mutant of ponB. The sequence of each mutant differed from that of the original gene by 3 to 16 bases in length of the heterologous region. This results from the replacement of five initial codons with five alanine codons by the technique described by Lefevre et al. Described in (8) herein.
[0075]
This substitution represents a unique site for the restriction enzyme PvuII surrounded by two PstI enzyme sites and made the mutant identifiable by its digestion profile. FIG. 3 represents the location of the ten regions of the mutations associated with each mutant (PvuII and PstI).
[0076]
After PCR amplification of the mutant, the PCR product was purified and mixed in equimolar amounts to form a library. The polynucleotide sequence of this library was digested with the restriction enzymes HinfI and BsaI to generate a library of restriction fragments. Different amounts of restriction fragments were then incubated with different amounts of wild-type template in the presence of thermostable ligase. After several cycles of denaturation, hybridization and ligation, aliquots of the reaction mixture were made specific for the 5 'and 3' ends of the mutated gene and nonspecific for the 5 'and 3' ends of the wild-type template. Used to perform PCR amplification with a pair of primers. Amplification products were cloned and these clones were analyzed for digestion profile using PvuII or PstI restriction endonuclease. The profiles obtained indicated which fragments of the mutant were able to recombine with others to form the entire gene.
[0077]
II.Materials and methods
[0078]
A.Varieties and plasmids
[0079]
Varieties MC1061 (F "araD139, △ (ara-leu) 7696, galE15, galK16, △ (lac) X74, rpsL (Str^R), McrA mcrB1, hsdR2 (r_k ⁻m_k ⁺)) Is obtained from E. coli K12.
[0080]
The vector pARAPONB results from the vector pARA13 (3) into which the ponB gene has been introduced with a thrombin cleavage site (9) between the restriction sites NcoI and NarI. The vector pET26b + was developed by Studier and Moffatt (10) and is one of the pET vectors commercialized by NOVAGEN Corporation.
[0081]
B.Oligonucleotide
[0082]
Oligonucleotides were synthesized by ISOPRIM corporation (Toulouse). The oligonucleotide sequences are reported in Table I below.
[0083]
[Table I]

[0084]
C.reagent
[0085]
The restriction and modification enzymes cited in Table II below were used according to the supplier's recommendations.
[0086]
[Table II]

[0087]
The buffers used are reported in Table III below.
[0088]
[Table III]

[0089]
III.Preparing the template
[0090]
The wild-type ponB gene was amplified by a PCR reaction step using oligonucleotides M1 and M2 as primers (FIG. 4). Add 50 ng of the pPONBPBR plasmid carrying the wild-type gene (7) to a mixture of 10 μl of polymerization buffer, 10 μl of 2 mM dNTPs, 20 pmoles each of oligonucleotides M1 and M2, and 5 U of Taq DNA polymerase in a final volume of 100 μl. Thereby, five PCR reactions were prepared. These mixtures were incubated on a Perkin-Elmer 9600 thermocycler according to the following program. (94 ° C for 2 minutes)-(94 ° C for 15 seconds-60 ° C for 30 seconds-72 ° C for 1 minute) x 29 cycles-(72 ° C for 3 minutes).
[0091]
The five PCR products were mixed and loaded on a 1% TBE agarose gel. After electrophoresis and staining of the gel with ethidium bromide, a 2651 bp stripe pattern corresponding to the ponB gene amplification product surrounded by two 26 bp and 90 bp fragments was visualized by ultraviolet fluoroscopy. It was then cut off with a scalpel and purified with the QUIAquick system (QIAGEN). All purified DNA was eluted with 120 μl of buffer T. The concentration of this DNA was approximately 100 ng / μl from its absorbance measurement at 260 nm.
[0092]
IV.Prepare library
[0093]
A.Amplification of mutant gene
[0094]
The 10 mutant genes were individually amplified by PCR with oligonucleotides N and E. These oligonucleotides introduce restriction sites NcoI and EcoRI, respectively, which allow cloning of the product obtained at these two sites.
[0095]
By adding 50 ng of plasmid containing the mutated gene to a final volume of 100 μl of a mixture containing 10 μl of polymerization buffer, 10 μl of 2 mM dNTPs, 20 pmoles each of oligonucleotides N and E, and 5 U of Taq DNA polymerase. A PCR reaction was set up. The mixture was incubated on a Perkin Elmer 9600 thermocycler according to the following program. (94 ° C for 2 minutes)-(94 ° C for 15 seconds-60 ° C for 30 seconds-72 ° C for 1 minute) x 29 cycles-(72 ° C for 3 minutes).
[0096]
The peculiarities of gene amplification were demonstrated by incubating 5 μl of each PCR product for 1 hour at 37 ° C. in a mixture containing 3 μl of restriction buffer A and 5 U of PvuII enzyme in a final volume of 30 μl. Demonstrated by nuclease restriction profile. 15 μl of the digestion reaction was loaded on a TBE 1% agarose gel. After electrophoresis and staining with ethidium bromide, the gel was exposed to ultraviolet light. Visualization of the restriction fragments allowed confirmation that the gene amplification of each mutant gene was unique.
[0097]
In parallel, 3 μl of each PCR reaction was loaded on a TBE 1% agarose gel. After electrophoresis, the gel was processed as described above. The brightness of each stripe allowed an assessment that the gene amplification had the same yield.
[0098]
B.Generate restriction fragment library
[0099]
50 μl of each 10 PCR were mixed and loaded on a 1% TBE agarose gel. After electrophoresis and staining with ethidium bromide, a striped pattern at 2572 bp, corresponding to the amplification product of the 10 mutated genes, was collected with a scalpel and purified with the QUIAQUICK system (QIAGEN). All purified DNA was eluted with 120 μl of buffer T. The concentration of this DNA was about 100 ng / μl from its absorbance at 260 nm.
[0100]
To generate a library of restriction fragments, 1 hour at 50 ° C., 100 μl in a mixture containing 12 μl restriction buffer B, 1.2 μl BSA (10 mg / ml), 25 U enzyme BsaI, and 4 μl water. This DNA was incubated. Then, 2 μl of restriction buffer B, 2 μl BSA (1 mg / ml), 50 U of the enzyme HinfI, and 11.5 μl of water were added to the mixture, which was incubated at 37 ° C. for 1 hour. The digestion mixture was purified on a Qiaquick column (QIAGEN) and eluted with 30 μl of buffer T. One μl of the eluate was loaded on a 1% TBE agarose gel and the digestion was complete and six restriction fragments were generated, resulting in six 590 bp, 500 bp, 472 bp, 438 bp, 298 bp and 274 bp. It was demonstrated that a fragment library was generated. The concentration of this DNA was about 250 ng / μl from its absorbance at 260 nm.
[0101]
V.Recombination ligation reaction (RLR)
[0102]
In the presence of thermostable DNA ligase, the RLR reaction was initiated by incubating a predetermined amount of the restriction fragment HinfI-BsaI from the 10 mutant genes with the complete template (ie, the wild-type ponB gene). went. Table IV below reports the composition of the mixture for the RLR.
[0103]
[Table IV]

[0104]
Negative control is exactly the same as that of RLR4, but does not include thermostable DNA ligase. These different mixtures were covered with drops of mineral oil and incubated on a Perkin-Elmer 9600 thermocycler in 200 μl microtubes according to the following program. (94 ° C, 5 minutes)-(94 ° C, 1 minute -65 ° C, 4 minutes) x 35 cycles.
[0105]
Then, 10 μl of each RLR reaction was added to a final volume of 100 μl PCR reaction mixture containing 10 μl polymerization buffer, 10 μl 2 mM dNTPs, 40 pmoles each of oligonucleotides A1 and A2, and 5 U Taq DNA polymerase. The mixture was incubated on a Perkin Elmer 9600 thermocycler according to the following program. (94 ° C, 5 minutes)-(94 ° C, 30 seconds-46 ° C, 30 seconds-72 ° C, 1 minute) x 29 cycles-(72 ° C, 2 minutes). This PCR reaction allows specific amplification of the ligation product formed during the RLR reaction without amplifying the template, as oligonucleotides A1 and A2 cannot hybridize to the template, as shown in FIG. did.
[0106]
5 μl of each RLR reaction and 10 μl of each of the previous PCR reactions were loaded on a 1% TBE agarose gel. After staining with ethidium bromide, the gel was exposed to ultraviolet light, as shown in FIG.
[0107]
Analysis of this gel reveals that only the RLR4 reaction, as well as the negative control, contains the restriction fragment visible (tracks 4 and 5).
[0108]
The lack of a PCR product for the negative control (Track 10) is not only due to the fact that the PCR reaction is specific (no complete template amplification), but also because of the generation of impure PCR products under selected conditions. It also demonstrates that the restriction fragments present within cannot be replaced by primers. In parallel, the presence of a unique stripe at approximately 2500 bp in tracks 6, 7 and 8 indicates that the RLR product can be amplified by PCR for RLR 1, 2 and 3 reactions. Thus, these three RLR reactions allowed the regeneration of one or more complete genes, starting from the restriction fragments of the six libraries.
[0109]
VI.Analysis of amplification products
[0110]
A.Cloning
[0111]
The PCR amplification products of the RLR1, 2 and 3 reactions were purified on the Wizard PCR Preps System (Promega) and eluted in 45 μl of Buffer T. 6 μl of each purified PCR was incubated for 1 hour at 37 ° C. in a mixture containing 3 μl restriction buffer C, 3 μl BSA (1 mg / ml), 20 U EcoRI enzyme, 10 U NcoI enzyme, and 15 μl water. did.
[0112]
In parallel, two vectors (pARAPONB and pET26b +) were prepared for cloning. These vectors were transformed with 3 μg of plasmid in a mixture containing 3 μl restriction buffer C, 3 μl BSA (1 mg / ml), 20 U EcoRI enzyme, 10 U NcoI enzyme, and 19 μl water at 37 ° C. for 2 hours. It was linearized by incubation.
[0113]
The linearized vector and the digested PCR were purified on a TBE 1% agarose gel using the QUIAGEN system. Each vector or each digested PCR was eluted in 30 μl buffer T.
[0114]
Each ligation of the PCR digested with each vector was performed under the conditions described in Table V below and incubated for 16 hours at 16 ° C.
[0115]
[Table V]

[0116]
200 μl of chemically capable MC1061 cells (4) were transformed in 10 μl of each ligation by heat shock (5) and these transformed cells were spread on selection media.
[0117]
No clones were obtained after conversion of the ligation control TLpAR and TLpET. This indicates that the NcoI-EcoRI vectors pARAPONB and pET26b + cannot cause intramolecular ligation.
[0118]
B.Screening by PCR
[0119]
A first screening of the clones obtained after transformation of the ligation with the vector pARAPONB is performed by PCR. Forty-two 42 colonies from each of the ligated LpAR1, LpAR2 and LpAR3 were individually transferred to a final volume of 50 μl containing 5 μl polymerization buffer, 40 pmoles each of oligonucleotides A1 and A2, 5 μl 2 mM dNTPs, and 5 U Taq DNA polymerase. Was resuspended in the PCR mixture. Negative control was obtained by adding 50 ng of plasmid pBR322 instead of colonies to the PCR mixture. These 43 tubes were incubated on a Perkin-Elmer 9600 thermocycler according to the following program. (94 ° C, 5 minutes)-(94 ° C, 30 seconds-46 ° C, 30 seconds-72 ° C, 1 minute) x 29 cycles-(72 ° C, 2 minutes). Then, 5 μl of each of these PCR reactions was incubated for 1 hour at 37 ° C. in a final volume of 20 μl mixture containing 2 μl restriction buffer A, 2 μl BSA (1 mg / ml), and 5 U restriction enzyme PvuII. Was placed.
[0120]
10 μl of each of these digests were loaded on a TBE 1% agarose gel in parallel with 5 μl of each of the undigested PCRs (this disrupted the non-specific striping of the PCR with the fragments obtained by restriction digestion). Avoided occurrence). After electrophoresis and staining of the gel with ethidium bromide, stripes resulting from digestion with the enzyme PvuII were used to determine which fragments of the early mutant were relevant for others to reconstruct the entire gene. The pattern was analyzed. This screen demonstrates the presence of 27 genes with one mutation, seven genes with two mutations, and eight genes with no more mutations.
[0121]
C.Screening by plasmid DNA mini-preparation
[0122]
A second screening was performed by extracting plasmid DNA (5) from 21 clones resulting from the transformation of the ligation with the vector pET26b + (7 clones from each ligation). Five microliters of the plasmid DNA obtained for each clone is placed in a final volume of 10 μl containing 1 μl restriction buffer C, 6 U of enzyme PstI, 3 U of enzyme NcoI, and 6 U of enzyme EcoRI for 1 hour at 37 ° C. Incubated. 5 μl of each of these digests was loaded on a TBE 1% agarose gel. After electrophoresis and staining of this gel with ethidium bromide, arising from digestion with the PstI enzyme to determine which fragments of the early mutant were related to others in reconstructing the entire gene The stripe pattern was analyzed. This screen demonstrates the presence of 13 genes with one mutation, five genes with two mutations, and three genes with no more mutations.
[0123]
D.Statistical analysis of recombination
[0124]
Judging from the position of each mutation relative to the cleavage sites of the enzymes HinfI and BsaI (see FIG. 6), the probability of obtaining a gene with 0, 1, 2, 3, or 4 mutations of the initial gene via RLR was calculated. It is possible to do.
[0125]
Assuming that the RLR response is quite random, the probability P is:
[0126]
(Equation 1)

[0127]
The two screens performed yield results close to these statistical expectations, as reported in Table VI below. Thus, it indicates that the RLR reaction is quasi-random. Due to the loss of genes with zero mutations, it can be observed that the proportion of genes with one mutation is slightly higher. This phenomenon could be attributable to the previously observed weak toxicity of the ponB gene and to a slight omission of expression of the vectors pARAPONB and pET26b +, which favors the selection of genes with non-activating mutations.
[0128]
[Table IV]

[0129]
Example III
[0130]
Example III represents an embodiment of the invention using controlled digestion.
[0131]
I.Materials and methods
[0132]
A.Bacterial species, genome and plasmid DNA
[0133]
Standard techniques and procedures were used for all DNA manipulations. E. coli MC1061DE3 cells were used to propagate the expression plasmid pET26b + (Novagen).
[0134]
B.Oligonucleotide
[0135]
All synthetic oligonucleotide primers for PCR were synthesized by MWG Biotech. Sense primer 5'AGGAATTCCATATGCGAAAGAAAAGACGGGGGA3 ', and antisense primer 5'ATAAAGCTTTCACTTGATGAGCCTGAGATTTC3 'was used to amplify the Thermotoga neapolitana xylanase A gene to introduce NdeI and HindIII restriction sites (underlined). The NdeI site contained the initial codon (bold).
[0136]
C.enzyme
[0137]
Restriction enzymes, DNA polymerase and thermostable ligase were purchased from NEB and EPICENTRE and used according to the manufacturer's recommendations.
[0138]
D.DNA amplification, cloning and expression
[0139]
PCR amplification was performed on a PE 9600 thermocycler. The Thermotoga neapolitana xylanase A amplicon was digested with primer-specific restriction endonucleases, ligated into compatible sites on pET26b +, and converted to E. coli MC1061DE3. The MC1061DE3 clone containing the pET26b + XynA expression vector was propagated at 37 ° C. in LB containing kanamycin (60 μg / ml).
[0140]
E. FIG.Biochemical features
[0141]
A heat inactivation experiment was performed directly on Escherichia coli expressing XynA. After centrifugation at 6000 g for 5 minutes at 4 ° C., the cells were resuspended in 200 mM acetate buffer pH 5.6. Resuspension was performed in an appropriate volume to normalize the cell volume for each sample. Then 150 μl of cells were incubated for different times at the appropriate temperature. 100 μl of these cells were added to 100 μl of 0.5% (w / v) xylan in 200 mM acetate buffer pH 5.6 and incubated at 80 ° C. for 10 minutes. Then, 200 μl of 3,5-dinitrosalicylic acid was added, boiled for 5 minutes, cooled on ice for 5 minutes and centrifuged at 12000 g for 5 minutes. 150 μl was transferred into a μ titer plate and the OD was measured at 540 nm.
[0142]
For optimal temperature experiments, 100 μl of 0.5% (w / v) xylan in 200 mM acetate buffer pH 5.6 was added to 100 μl of resuspended cells and incubated at different temperatures for 10 minutes. Then 200 μl of 3,5-dinitrosalicylic acid was added, boiled for 5 minutes, cooled on ice for 5 minutes and centrifuged at 12000 g for 5 minutes. 150 μl was transferred into a μ titer plate and the OD was measured at 540 nm.
[0143]
II.result
[0144]
A.Generation of less thermostable mutants of XynA
[0145]
To generate a mutant with low thermostability of the XynA protein, error-prone PCR was performed on the WT XynA gene using a 1% agarose gel as shown in FIG. The product was digested with primer-specific restriction endonucleases, ligated into compatible sites on pET26b + and converted to E. coli MC1061DE3 to generate an error-prone library.
[0146]
One clone from this error-prone library (mutant 33) appeared to have much lower thermostability compared to the WT protein. Rapid biochemical analysis, including determination of optimal temperature and heat inactivation, was performed and compared to that of the WT. With respect to the optimal temperature, mutant 33 had an optimal temperature around 78 ° C. compared to WT (above 90 ° C.), but after incubation at 82 ° C. for 30 minutes or 95 ° C. for 1 minute. No residual activity was detected from mutant 33. Thus, the inactivation constant was calculated from FIG. The heat inactivation of mutant 33 at 82 ° C was estimated to be 0.120 / min at 82 ° C. At these temperatures, there was no or low detection of heat inactivation for the WT protein.
[0147]
B.Shuffling experiment
[0148]
Mutant 33 and the WT gene were then recombined using L-Shuffling ™ technology to generate mutants with different thermostability. Different mutants were expected. Mutants with WT optimal temperature, mutants with lower thermostability than WT, and mutants with higher thermostability than mutant 33 with optimal temperature.
[0149]
1)Fragment library
[0150]
After PCR amplification of WT and mutant 33, the product was digested with a mixture of six restriction enzymes, HincII, BamHI, XhoI, SphI, EcoRI and EcoRV, to generate eight fragments (from 120 to 700 pb). . Fragmentation of the PCR product with a mixture of six restriction endonucleases using a 3% agarose gel, see FIG.
[0151]
2)Shuffling experiment
[0152]
The RLR (recombinant ligation reaction) is performed by repeating the denaturation and ligation or hybridization steps for several cycles to form a standardized fragment (shown in FIG. 9) together with a thermostable ligase, and pET26 + XynA digested with NdeI / HindIII as a template. It was carried out using.
[0153]
Negative control was performed under the same conditions without thermostable ligase (B). The results are shown in FIG. 10, an L-Shuffling ™ experiment using 1% agarose gel. FIG. 10 shows that in the absence of thermostable ligase, fragments are not used for recombination. Then, selective digestion of the template was performed by adding DpnI to the reaction mixture.
[0154]
3)Cloning products
[0155]
PCR Pfu amplification (FIG. 11, PCR Pfu to L-Shuffling ™ product using 1% agarose gel) was performed using 5 ′ sense and 5 ′ antisense synthetic primers and the above protocol to obtain A and B (FIG. 9, negative control) was performed on both DpnI digested L Shuffling ™ products. Despite obtaining large amounts of amplified L-Shuffling ™ product for cloning, no template amplification occurred. Thus, the L Shuffling ™ product is digested with primer-specific restriction endonucleases, ligated into compatible sites on pET26b +, and converted to E. coli MC1061DE3 to generate an L Shuffling ™ library did.
[0156]
4)Biochemical features
[0157]
Several clones were selected from the L-Shuffling ™ library for activity remaining after incubation at 82 ° C. for 30 minutes.
[0158]
Clones 24, 41 and 56 (FIG. 12, heat inactivation of mutants at 95 ° C.) have the optimal temperature of mutant 33 and clone 6 has the optimal temperature of WT xylanase. Under these experimental conditions, WT xylanase maintained 100% activity after incubation at 95 ° C. for 120 minutes. In contrast, no residual activity was detected for mutant 33 after 1 minute at 95 ° C. FIG. 13 shows four mutants from the L Shuffling ™ library that showed different properties than the two parent nuclei.
[0159]
Example IV
[0160]
Example IV represents an embodiment of the invention that uses random digestion.
[0161]
I.Materials and methods
[0162]
A.Bacterial species, genome and plasmid DNA
[0163]
Standard techniques and procedures were used for all DNA manipulations. The expression plasmid pET26b + (Novagen) was propagated using Escherichia coli MC1061DE3 cells.
[0164]
B.Oligonucleotide
[0165]
All synthetic oligonucleotide primers for PCR were synthesized by MWG Biotech. Sense primer 5'AGGAATTCCATATGCGAAAGAAAAGACGGGGGA3 ', and antisense primer 5'ATAAAGCTTThe Thermotoga neapolitana xylanase A gene was amplified using TCACTTGATGAGCCTGAGATTTC3 ', and NdeI and HindIII restriction sites (underlined) were introduced. Sense primer 5'GGAATTCCATATGGCGGCGGCAGCCGGCA3 ', and antisense primer 5'GGAATTCUsing CTACTGCCGCTCCGATTGTGG3 ', the Acidobacterium capsulatum xylanase gene was amplified and NdeI and EcoRI restriction sites (underlined) were introduced. The NdeI site contained the initial codon (bold).
[0166]
C.enzyme
[0167]
Restriction enzymes, DNA polymerase and thermostable ligase were purchased from Neb and Epicenter and used according to the manufacturer's recommendations.
[0168]
II.result
[0169]
The Thermotoga neapolitana gene (3.2 kb) and the Acidobacter capsulatatum gene (1.2 kb) recombined.
[0170]
A.Fragment library
[0171]
PCR amplification was performed on the Thermotoga neapolitana and Acidobacteria capsulatum genes followed by DNase I digestion.
[0172]
See FIG. 13, DNase I fragmentation of Thermotoga neapolitana (A) and Acidobacteria capsulatum (B) genes using 1% agarose gel.
[0173]
B.Shuffling experiment
[0174]
RLR was performed with several cycles of denaturation and hybridization or ligation with fragments standardized with thermostable ligase and thermostable flap (shown in FIG. 13).
[0175]
Negative control was performed under the same conditions, but without thermostable ligase and / or thermostable flap (A, B and C). The results are shown in FIG. 14, an L-Shuffling ™ experiment using 1% agarose gel. FIG. 14 shows that in the absence of a thermostable ligase and a thermostable flap, the fragments do not recombine. In FIG. 14, A represents a fragment having no ligase and flap activity, B represents a fragment having only ligase, C represents a fragment having only flap, and D represents a shuffling state.
[0176]
Example V
[0177]
Example V used the materials and methods of Example III, but was run with a different number of cycles of steps (b) and (c). See FIG. 15A, L Shuffling ™ using steps (b) and (c) of n cycles, and FIG. 15B, PCR amplification of the corresponding L Shuffling ™ product. As shown in FIGS. 15A and B, at least one cycle (n = 1) is required to obtain a recombinant polynucleotide.
[0178]
Example VI
[0179]
Example VI used the materials and methods of Example III, but
Experiments were performed with the following seven quantities of fragments.
1: 1x
2: 2x
3: 3x
4: 4x
5: 11x
6: 14x
7: 17x
[0180]
FIG. 16, L-Shuffling ™ experiment with increasing amounts of fragments shows results for these seven amounts.
[0181]
The above presentation is not intended to limit the scope of the invention. While embodiments of the present invention have been described in detail with reference to the accompanying drawings, it will be apparent to one skilled in the art that modifications to the method described herein are possible. Those skilled in the art can make these and various other changes and embodiments without departing from the scope of the invention. The scope of the invention should be determined with reference to the claims, and equivalents, in view of the prior art.
[0182]
(References)

[Brief description of the drawings]
[0183]
Reference is made to the accompanying drawings.
FIG. 1A schematically depicts conventional DNA shuffling.
FIG. 1B schematically illustrates a conventional StEP (FIG. 1B).
FIG. 2-1 schematically represents an embodiment of the process of the invention, and variations and uses thereof.
FIG. 2-2 schematically represents an embodiment of the process of the invention and its variants and uses.
FIG. 2-3 schematically represents an embodiment of the process of the invention and its variants and uses.
FIG. 3 shows the location of 10 regions (PvuII and PstI) of mutations by each mutant of the ponB gene.
FIG. 4 shows the positions of primers used for comparison with the ponB gene sequence.
FIG. 5 shows the migration of RLR of RLR reaction and PCR reaction product on agarose gel.
FIG. 6 represents the location of the mutation relative to the restriction fragment.
FIG. 7 shows that errors in PCR results for the WT XynA gene using 1% agarose gel are likely to occur.
FIG. 8 shows the heat inactivation of mutant 33 at 82 ° C.
FIG. 9 depicts the results of fragmentation of the PCR product with six restriction endonucleases using a 3% agarose gel.
FIG. 10 depicts the results of an L-Shuffling ™ experiment using 1% agarose gel.
FIG. 11 shows the results of using PCR Pfu on L Shuffling ™ products using 1% agarose gel.
FIG. 12 shows the heat inactivation of mutants at 95 ° C.
FIG. 13 shows the results of DNase I fragmentation of Thermotoga neapolitana (A) and Acidobacterium capsulatum (B) genes using 1% agarose gel.
FIG. 14 depicts the results of an L-Shuffling ™ experiment using 1% agarose gel.
FIG. 15A shows the results of L Shuffling ™ using steps (b) and (c) for n cycles.
FIG. 15B shows the PCR amplification of the corresponding L Shuffling ™ product.
FIG. 16 depicts the results of an L-Shuffling ™ experiment with increasing amounts of fragments.

Claims (85)

For shuffling the polynucleotide non-randomly, a method of template adjustment ligation direction localization,
a) obtaining, directly or indirectly, single-stranded fragments of at least two homologous polynucleotides from a polynucleotide library;
b) forming at least one partially double-stranded polynucleotide by hybridizing said fragment to one or more devised organized templates until at least two fragments hybridize adjacently Note that at least one of the templates shares at least one homology region with the homologous polynucleotide,
c) treating said partially double-stranded polynucleotide to form at least one recombinant polynucleotide, said treatment comprising, independent of order,
(I) ligating the nick, and (ii) performing any or a combination of techniques that satisfy the following gaps, as needed:
Filling the gap by further hybridizing the fragment to the template to increase the number of adjacent hybridizing fragments;
Filling short gaps by trimming overhanging flaps of partially hybridized fragments, and filling short gaps through polymerization. 2. The method of claim 1, wherein the step occurs in vitro. 2. The method of claim 1, wherein said homologous polynucleotide is double-stranded. The method of claim 1, wherein steps (b) and (c) are performed simultaneously. 2. The method of claim 1, wherein the recombinant polynucleotide is formed after one cycle of the method. The method of claim 1, wherein the recombinant polynucleotide is formed after more than one iteration of steps (a), (b) and / or (c). The method of claim 6, wherein the recombinant polynucleotide is formed after more than three repetitions of steps (a), (b) and / or (c). 2. The method of claim 1, wherein after more than three repetitions of steps (b) and (c), the recombinant polynucleotide is formed. The method of claim 1, wherein the template or homologous polynucleotide used in the next cycle of the method is a recombinant polynucleotide generated in a previous cycle of the method. The method of claim 1, wherein the template is initially double stranded. The method of claim 1, further comprising treating the template strand of the recombinant polynucleotide to eliminate, separate, or degrade the template strand. 12. The method of claim 11, wherein the recombinant polynucleotide is separated from the template strand for labeling on the template strand or on the recombinant strand. The method of claim 11, wherein the template comprises uracil. 14. The method according to claim 13, wherein the template is an mRNA sequence. 2. The method of claim 1, wherein the fragment is initially double stranded. 2. The method of claim 1, wherein in steps (a), (b) or (c), supplemental or surrogate fragments are added. 2. The method of claim 1, wherein step (a) comprises fragmenting the homologous polynucleotide with at least one restriction enzyme having multiple cleavage sites on the homologous polynucleotide, or with a plurality of different restriction enzymes. the method of. 18. The method of claim 17, wherein the fragment in step (a) is at least 15 residues in length. 19. The method of claim 18, wherein the fragment in step (a) is about 15 to 40 residues in length. Step (a) comprises obtaining at least two populations of fragments from separate polynucleotide libraries or using at least two different populations of fragments from the same polynucleotide library using different restriction enzymes. 2. The method of claim 1, comprising obtaining. 2. The method of claim 1, wherein step (a) comprises fragmenting the homologous polynucleotide randomly with DNase I, wherein the homologous polynucleotide, or a fragment obtained therefrom, is initially double-stranded. the method of. 22. The method of claim 21, wherein the fragment in step (a) is at least 50 residues in length. 2. The method of claim 1, wherein the fragment of step (a) is amplified. 24. The method of claim 23, wherein the fragment of step (a) is amplified using oligonucleotide primers that generate fragments with adjacent ends along the entire length of the template. 2. The method of claim 1, wherein step (a) comprises fragmenting the homologous polynucleotide into three or more fragments. The method according to claim 1, wherein a flap endonuclease is added for use in step (b) and / or step (c). 27. The method of claim 26, wherein the flap endonuclease has the same thermostability and high temperature activity as the ligase used in step (c). 28. The flap endonuclease concentration of about 1.8 to 2.2 [mu] g / ml, hybridization occurs at a temperature of about 5 to 20C, and ligation occurs at a temperature of about 60 to 75 [deg.] C. the method of. Polynucleotide libraries can be derived from native genes by continuous orientation mutagenesis, by error-prone PCR, by random chemical mutagenesis, by random mutagenesis in vivo, or the same. The method of claim 1, wherein the sequence is produced by combining genes from gene families within different species, resulting in different sequences within the polynucleotide library. 2. The method of claim 1, wherein the polynucleotide library consists of a synthetic sequence, or wherein a synthetic fragment is added in step (a), (b) or (c). 2. The method of claim 1, wherein the template is obtained from a polynucleotide library or from a consensus sequence of the library. The method of claim 1, wherein a polynucleotide that complements the 3 'end of one fragment and the 5' end of an adjacent fragment is used as a template. 2. The method according to claim 1, wherein the recombinant polynucleotide is obtained without using a polymerizing enzyme. 2. The method of claim 1, wherein the recombinant polynucleotide is obtained without inducing crossover or strand crossover. 2. The method of claim 1, wherein the fragments and the recombinant polynucleotide are obtained without size fractionation. 2. The method of claim 1, wherein step (b) or (c) requires multiple hybridization events. The method of claim 1, wherein step (b) requires a single hybridization event. The method of claim 1, wherein step (b) requires a single hybridization event and step (c) does not require any hybridization events. 2. The method of claim 1, wherein the polynucleotide library or template is a recombinant polynucleotide. The method according to claim 1, wherein the single-stranded fragment is an isolated strand fragment. The method of claim 1, wherein the template is an isolated strand template. 2. The method of claim 1, wherein said fragments and said template are isolated strands. 43. The method of claim 42, wherein the isolated strand fragment is from the top strand of the homologous polynucleotide and the isolated strand template is from the bottom strand of the homologous polynucleotide. 43. The method of claim 42, wherein the isolated strand fragment is from the bottom strand of a previous homologous polynucleotide and the isolated strand template is from the top strand of the homologous polynucleotide. 2. The method of claim 1, wherein all, or substantially all, of the template is longer than the fragment of step (a). 2. The method of claim 1, wherein the template strand of the recombinant polynucleotide is excluded, separated, or degraded. 2. The method of claim 1, wherein the homologous polynucleotides are 30 to 90% homologous to each other. 2. The method of claim 1, wherein all, or substantially all, of the template is longer than the fragment of step (a). 2. The method of claim 1, wherein the template is approximately equally homologous to each of the homologous polynucleotides. 2. The method of claim 1, wherein at least half of the homologous polynucleotides differ from each other by more than two residues. 2. The method of claim 1, wherein at least half of the homologous polynucleotides are about 20 to 45% homologous to each other. 2. The method of claim 1, further comprising translating the recombinant polynucleotide in vitro and expressing the protein. The method of claim 1, further comprising selecting (d) at least one of said recombinant polynucleotides having desired properties. 54. The method of claim 53, wherein the step occurs in vitro. 54. The method of claim 53, wherein said single-stranded fragment is an isolated strand fragment. 54. The method of claim 53, wherein the template is an isolated strand template. 54. The method of claim 53, wherein short gaps are filled by trimming overhang flaps of partially hybridized fragments. 54. The method of claim 53, wherein all, or substantially all, of the template is longer than the fragment of step (a). 54. The method of claim 53, further comprising translating the recombinant polynucleotide in vitro to express the protein. 54. The method of claim 53, further comprising, before step (d), amplifying the recombinant polynucleotide. 54. The method of claim 53, wherein before step (d), the template strand is eliminated, separated, or degraded. Further, prior to step (d), but after eliminating, separating or degrading the template strand, regenerating the double-stranded recombinant polynucleotide from the recombinant strand and cloning said double-stranded recombinant polynucleotide 62. The method of claim 61, comprising: For non-random shuffling of polynucleotides in vitro, a method of template-adjusted ligation orientation,
a) obtaining, directly or indirectly, an isolated strand restriction fragment of at least two homologous polynucleotides from a polynucleotide library;
b) forming at least one partially double-stranded polynucleotide by hybridizing said fragment to one or more devised organized templates until at least two fragments hybridize adjacently Wherein still, or substantially all, of the template is isolated, transient, longer than the fragment, and shares at least one homology region with the homologous polynucleotide;
c) treating said partially double-stranded polynucleotide to form at least one recombinant polynucleotide, said treatment comprising, irrespective of order,
(I) ligating the nick, and (ii) performing one or a combination of techniques, as needed, to fill the following gaps:
Filling the gap by further hybridizing the fragment to the template to increase the number of adjacent hybridizing fragments, and trimming the short gap by trimming the overhang flap of the partially hybridized fragment. And d) selecting at least one of said recombinant polynucleotides having the desired properties. 64. The method of claim 63, wherein the short gap is filled by trimming the overhang flap of the partially hybridized fragment. A method of template-adjusted ligation-only for in vitro non-random shuffling of polynucleotides, comprising:
a) obtaining, directly or indirectly, an isolated strand restriction fragment of at least two homologous polynucleotides from a polynucleotide library;
b) repeatedly hybridizing the fragment to one or more devised organized templates until all of the fragments that hybridize to the template hybridize adjacently, wherein the template is an isolated strand and transient At least one of said templates shares at least one region of homology with said homologous polynucleotide;
c) ligating a nick to form at least one recombinant polynucleotide, and d) selecting at least one of said recombinant polynucleotides having desired properties. A method of template-adjusted ligation orientation for in vitro non-random shuffling of a region containing a polynucleotide mutation, comprising:
a) locating the restriction site relative to the region containing the mutation between the polynucleotide allelic units;
c) obtaining, directly or indirectly, a fragment corresponding to the restriction site from the allelic unit;
b) forming at least one partially double-stranded polynucleotide by hybridizing said fragment to one or more devised organized templates until at least two fragments hybridize adjacently; Note that at least one whole or a part of the template is homologous to a region containing the mutation in the allelic unit,
c) treating said partially double-stranded polynucleotide to form at least one recombinant polynucleotide, said treatment comprising, irrespective of order,
(I) ligating the nick, and (ii) performing one or a combination of techniques that fill the following gaps:
Fill gaps by further hybridizing the fragments to the template to increase the number of adjacent hybridizing fragments, and fill short gaps by trimming the overhanging flaps of the partially hybridized fragments. And d) selecting at least one of said recombinant polynucleotides having desired properties. A method of template-adjusted ligation direction localization for in vitro non-random low homology shuffling of gene strains,
a) obtaining, directly or indirectly, an isolated strand restriction fragment consisting of at least two polynucleotides of the gene family from the gene family library;
b) forming at least one partially double-stranded polynucleotide by hybridizing said fragment to one or more devised organized templates until at least two fragments hybridize adjacently Wherein the template is an isolated strand and transient, and at least one of the templates shares at least one region of homology with the genotype polynucleotide;
c) treating said partially double-stranded polynucleotide to form at least one recombinant polynucleotide, said treatment comprising, in any order,
(I) ligating the nick, and (ii) performing one or a combination of techniques that fill the following gaps:
Fill gaps by further hybridizing the fragments to the template to increase the number of adjacent hybridizing fragments, and fill short gaps by trimming the overhang flaps of the partially hybridized fragments. And d) selecting at least one of said recombinant polynucleotides having desired properties. A recombinant polynucleotide obtained by the method according to claim 1. A vector comprising the polynucleotide of claim 68. 69. A cell host converted by the recombinant polynucleotide of claim 68. 70. A protein encoded by the recombinant polynucleotide of claim 68. 69. A library consisting of the recombinant polynucleotide of claim 68. 74. A library comprising the vector of claim 69, the cell host of claim 70, or the protein of claim 71. A physical array capable of performing the method of claim 1. A logical array simulating the method of claim 1. 75. A logical array simulating the physical array of claim 74. 64. The method of claim 1, 53 or 63, wherein at least two of the adjacently hybridizing fragments hybridize adjacently in step (b) before ligating or filling the gap occurs. 54. The method of claim 1 or 53, wherein the step occurs in vivo. For shuffling the polynucleotide non-randomly, a method of template adjustment ligation direction localization,
a) by hybridizing a single-stranded fragment of at least two homologous polynucleotides to one or more devised organized templates until at least two fragments hybridize adjacently, Forming a single-stranded polynucleotide, wherein at least one of the templates shares at least one homology region with the homologous polynucleotide;
b) treating said partially double-stranded polynucleotide to form at least one recombinant polynucleotide, said treatment comprising, in any order,
(I) ligating the nick, and (ii) performing one or a combination of techniques, as needed, to fill the following gaps:
Filling the gap by further hybridizing the fragment to the template to increase the number of adjacent hybridizing fragments;
Filling short gaps by trimming overhanging flaps of partially hybridized fragments, and filling short gaps through polymerization. A polynucleotide shuffling reaction mixture comprising a single-stranded fragment of at least two homologous polynucleotides and at least one devised organized template to which at least two fragments can hybridize adjacently. At least two isolated strand restriction fragments of a homologous polynucleotide, and at least one devised organized template to which at least two restriction fragments can hybridize adjacently, wherein the template is a transient, isolated strand. An in vitro polynucleotide shuffling reaction mixture that is and is longer than most or all of the fragments. At least one partially double-stranded polynucleotide comprising a free single-stranded fragment of at least two homologous polynucleotides and a strand of the devised assembling template and a partial strand of an opposite hybridizing fragment. A polynucleotide shuffling reaction mixture wherein at least two hybridizing fragments are adjacently hybridized. At least one partially duplicated strand consisting of a free isolated strand restriction fragment of at least two homologous polynucleotides, and a devised isolated strand assembly template strand, and an opposing hybridized restriction fragment partial strand; An in vitro polynucleotide comprising at least two hybridizing restriction fragments, wherein the template strand is transient and longer than most or all of the free restriction fragments. -Shuffling reaction mixture. 20. The method of claim 19, wherein the fragment in step (a) is about 15 residues in length. 23. The method of claim 22, wherein the fragment in step (a) is about 50 to 500 residues in length.

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