背景技術(shù)：
：合成生物學(xué)依賴于由組分部分構(gòu)建新的dna的能力。雙鏈(ds)dna分子已通過(guò)在第一dna雙鏈的兩端產(chǎn)生交錯(cuò)末端得以組裝。使用限制性核酸內(nèi)切酶、或者通過(guò)使用核酸外切酶消化、或者通過(guò)t4dna聚合酶，然后雜交，以及任選地將第二dna雙鏈與第一雙鏈連接，已經(jīng)實(shí)現(xiàn)這一點(diǎn)。在反應(yīng)混合物中使用核酸外切酶和連接酶的技術(shù)還指定使用非鏈置換聚合酶。技術(shù)實(shí)現(xiàn)要素：除了其它，本公開提供一種組合物，其包括：i.5’-3’核酸外切酶；ii.鏈置換聚合酶；iii.單鏈(ss)dna結(jié)合蛋白質(zhì)；和iv.非天然存在的緩沖劑，其中組合物不包括擁擠劑和/或非鏈置換聚合酶。該組合物可以用于將多核苷酸組裝到合成子(synthon)中。取決于出于在包含其自身連接酶的細(xì)菌細(xì)胞中克隆的目的進(jìn)行組裝，還是出于不包括細(xì)菌中克隆步驟的目的進(jìn)行組裝，組合物的實(shí)施方式任選地包含連接酶。在先描述的組裝方法需要非鏈置換聚合酶，并進(jìn)一步額外地需要擁擠劑(crowdingagent)(例如，參見(jiàn)us8,968,999)。與現(xiàn)有技術(shù)相反，本發(fā)明的實(shí)施方式表明，當(dāng)與5’-3’核酸外切酶一起使用時(shí)，鏈置換聚合酶相對(duì)于非鏈置換聚合酶具有優(yōu)勢(shì)。相對(duì)于包括擁擠劑，這一組合傾向于包括ss結(jié)合蛋白質(zhì)，這與聲稱擁擠劑的使用比包括使用單鏈結(jié)合蛋白質(zhì)的替代方案要有效4倍的現(xiàn)有技術(shù)教導(dǎo)相反。本發(fā)明的實(shí)施方式提供組合物、方法和試劑盒，其在單步方法中和/或在單一反應(yīng)容器中提供由包括ds和/或ss核酸分子的寡核苷酸和多核苷酸來(lái)組裝和克隆功能性基因和合成子的效率得以增加。這些實(shí)施方式不依賴于擁擠劑，也不需要非鏈置換聚合酶用于填充兩分子退火之后存在的間隙。例如，當(dāng)作用于dsdna的5’-3’核酸外切酶產(chǎn)生可以與來(lái)自另一分子的3’ssdna突出端有效退火的3’ssdna突出端時(shí)，鏈置換聚合酶可以填充分子退火之后留下的間隙。鏈置換dna聚合酶和5’-3’核酸外切酶活性的組合產(chǎn)生在接合位點(diǎn)處或附近包含切口的雙鏈合成子。這一切口可以通過(guò)連接酶在體外密封，或者通過(guò)內(nèi)源性細(xì)胞連接酶在體內(nèi)密封。此外，在反應(yīng)混合物中包括ssdna結(jié)合蛋白質(zhì)使得能夠有效地組裝相對(duì)低濃度的核酸片段，從而在不損失效率或者不損失接合準(zhǔn)確性的情況下節(jié)約成本。在一些組合物實(shí)施方式中，鏈置換聚合酶是b族聚合酶。鏈置換聚合酶應(yīng)當(dāng)優(yōu)選地在相同反應(yīng)條件下(例如，使用比如圖1a～1e中所述的試驗(yàn))鏈置換活性大于以聚合酶(thermofisher，waltham，ma)(通常描述為非鏈置換的)所觀察者。在本發(fā)明的組合物、方法和試劑盒中，主要使用鏈置換聚合酶的鏈置換活性。在一些實(shí)施方式中，鏈置換聚合酶可以是非天然存在的，例如，鏈置換聚合酶可以是突變體。突變體的實(shí)例包括具有一個(gè)或更多個(gè)氨基酸替換的聚合酶，非天然存在的聚合酶可以或者另外是具有不相關(guān)氨基酸序列的部分的融合蛋白，其中融合聚合酶在自然界不存在。優(yōu)選地，鏈置換聚合酶在50℃或更高下穩(wěn)定，并且因此可以稱作熱穩(wěn)定性鏈置換聚合酶。在一些情形中，鏈置換聚合酶是具有不相關(guān)或異源性dna結(jié)合結(jié)構(gòu)域的融合聚合酶。在一些實(shí)施方式中，聚合酶部分的氨基酸序列與seqidno:102的同一性可以為至少90％或95％或98％或99％。在另一個(gè)實(shí)施方式中，聚合酶的氨基酸序列與seqidno:1的同一性可以為至少90％或95％或98％或99％或100％，優(yōu)選至少90％。在另一個(gè)實(shí)施方式中，聚合酶的氨基酸序列與seqidno:33至seqidno:55中任意者的同一性可以為至少90％或95％或98％或99％或100％，優(yōu)選至少90％。在一些實(shí)施方式中，dna結(jié)合結(jié)構(gòu)域部分的氨基酸序列與seqidno:2的同一性可以為至少90％或95％或98％或99％。在另一個(gè)實(shí)施方式中，聚合酶的氨基酸序列與seqidno:1、seqidno:3、seqidno:56至seqidno:96或seqidno:102中任意者的同一性可以為至少90％或95％或98％或99％或100％，優(yōu)選至少90％。在另一個(gè)實(shí)施方式中，本文所述的任意聚合酶結(jié)構(gòu)域部分可以與本文所述的任意dna結(jié)合結(jié)構(gòu)域組合，其條件是聚合酶部分和dna結(jié)合結(jié)構(gòu)域是異源性的。例如，在其他實(shí)施方式中，融合蛋白的氨基酸序列與seqidno:1和seqidno:2的同一性可以為至少90％或95％或99％或100％，優(yōu)選至少90％。在其他實(shí)施方式中，融合蛋白與seqidno:3的序列同一性可以為至少90％或95％或98％或99％或100％，優(yōu)選至少90％。鏈置換聚合酶可以具有或不具有3’-5’核酸外切酶活性。鏈置換聚合酶具有3’-5’核酸外切酶活性時(shí)，多核苷酸接合可以通過(guò)使用包括本文例示者的條件來(lái)平衡3’-5’核酸外切酶活性、5’-3’聚合活性和鏈置換活性加以優(yōu)化。組裝的效率和準(zhǔn)確性可以使用本文所述的試驗(yàn)來(lái)確認(rèn)(例如，參見(jiàn)圖3a和3b)。在一些實(shí)施方式中，聚合酶不是phusion、9°n、pfu或vent或者氨基酸序列與phusion或野生型9°n、pfu或vent的同一性為至少90％的聚合酶。在一些實(shí)施方式中，聚合酶是熱穩(wěn)定性的，即，在至少40℃或至少50℃的溫度下有活性。與鏈置換聚合酶相反，一些聚合酶比如taqdna聚合酶經(jīng)由5’→3’核酸外切酶活性降解遇到的下游鏈。該活性用于切口翻譯方案。因此taqdna聚合酶不包括在鏈置換聚合酶的定義中。確定合成子形成的效率和準(zhǔn)確性的試驗(yàn)描述于實(shí)施例中，并示于圖3a～b。設(shè)計(jì)的組裝片段編碼lacl和lacz蛋白質(zhì)，如果dna片段組裝正確，其產(chǎn)生藍(lán)色菌落。因此，過(guò)夜板“藍(lán)色”菌落的數(shù)目指示組裝的效率和準(zhǔn)確性。在不存在藍(lán)色的情況下，可能發(fā)生有效的組裝，但接合/延伸區(qū)的錯(cuò)誤阻止了表達(dá)。當(dāng)合成子得以組裝、并且然后克隆到宿主細(xì)胞中，合成子形成的效率和準(zhǔn)確性轉(zhuǎn)化成每個(gè)克隆將包含正確組裝的合成子的置信度。借助該置信度，僅需要對(duì)一個(gè)或多個(gè)復(fù)制的克隆進(jìn)行測(cè)序，以確認(rèn)合成子的存在。這樣降低了對(duì)可能包含錯(cuò)誤的克隆進(jìn)行測(cè)序的成本和不便。在一個(gè)實(shí)施方式中，至少80％或者另外可選地至少90％的克隆將會(huì)包含正確組裝的合成子。在一些實(shí)施方式中，使用本文所述組合物的方法能夠達(dá)到基本上超過(guò)最小需求的產(chǎn)率。例如，在單一轉(zhuǎn)化事件中可以制備至多5,000或10,000個(gè)克隆。如果組裝的目的不是制備合成子文庫(kù)，而是制備單例合成子，則可以使用更低的核酸片段和試劑起始量，甚至其低于本文提供的范圍。適合用于組裝混合物的濃度范圍的實(shí)例包括以下：0.02nm～100nm的dna片段或者例如0.2nm～10nmdna可以加入到反應(yīng)容器中的試劑混合物中。在一個(gè)實(shí)施方式中，盡管可以使用更高或更低的比例，載體dna與dna片段以1:1的比例包括在內(nèi)。與對(duì)于dsdna所選擇的濃度相比較，可以優(yōu)選更高的ssdna濃度。反應(yīng)容器中的試劑混合物還可以包括0.0004u/μl～0.064u/μl的5’-3’核酸外切酶(例如0.0004u/μl～0.01u/μl)；0.5u/μl～32u/μl任選的連接酶(例如1u/μl～10u/μl)；0.0025u/μl～0.25u/μl鏈置換聚合酶(例如0.005u/μl～0.1u/μl)；和0.001μg/μl～0.1μg/μl的ss結(jié)合蛋白質(zhì)(例如0.01μg/μl～0.5μg/μl)(單位對(duì)應(yīng)于制造商(newenglandbiolabs,ipswich,ma)所指定者)。5’-3’核酸外切酶的量能夠根據(jù)核酸片段重疊長(zhǎng)度和每個(gè)片段的大小加以進(jìn)一步優(yōu)化。例如，5’-3’核酸外切酶的量可以在核酸片段長(zhǎng)度大于80個(gè)核苷酸的范圍內(nèi)增加。指定范圍內(nèi)鏈置換聚合酶的絕對(duì)濃度并非關(guān)鍵。盡管已知有許多其他的ssdna結(jié)合蛋白質(zhì)，并且其可以用于組合物中，組合物中使用的ssdna結(jié)合蛋白質(zhì)可以是大腸桿菌(e.coli)reca、t7基因2.5產(chǎn)物、redb(來(lái)自噬菌體λ)或rect(來(lái)自rac原噬菌體)、etssb(極端熱穩(wěn)定的單鏈dna結(jié)合蛋白質(zhì))或者與seqidno:100的序列同一性為90％的ss結(jié)合蛋白質(zhì)。如通過(guò)菌落數(shù)目所測(cè)量，比起在否則不存在ss結(jié)合蛋白質(zhì)的情況下所發(fā)生的，包括ss結(jié)合蛋白質(zhì)提高了特別是對(duì)于具有更長(zhǎng)重疊序列(例如，至少20個(gè)核苷酸)的核酸片段的組裝效率。任選的連接酶可以是依賴于nad+的連接酶，比如taq連接酶，或者依賴于atp的連接酶，比如t4連接酶。然而，對(duì)于pcr，由于atp能夠抑制后續(xù)的合成子擴(kuò)增中使用的taq聚合酶，使用依賴于nad+的連接酶是方便的。合適的連接酶的實(shí)例包括與seqidno:101的序列同一性為至少90％的蛋白質(zhì)。在此使用的5’-3’核酸外切酶可以是具有5’-3’核酸外切酶活性以及ss核酸內(nèi)切酶活性的酶(參見(jiàn)，例如，garforth等人,pnas,96,38-43(1999))。具有核酸外切酶和ss核酸內(nèi)切酶活性的5’-3’核酸外切酶的實(shí)例包括t5核酸外切酶、以及其同源物和變體。在一個(gè)實(shí)施例中，5’-3’核酸外切酶與seqidno:98的氨基酸序列同一性為至少90％。沒(méi)有要求在用鏈置換聚合酶接合多核苷酸之前使5’-3’核酸外切酶變性。因此，在實(shí)施例中描述了使用熱穩(wěn)定性5’-3’核酸外切酶。在一些實(shí)施方式中，組合物還可以包括dntp(即，dgtp、datp、dgtp和dttp的混合物)，并且，在一些實(shí)施方式中，當(dāng)使用t55’-3’核酸外切酶時(shí)，組合物還可以包括鉀鹽，比如kcl(例如，濃度范圍7mm～150mm)。概括地講，提供一種制備合成子的方法。在一些實(shí)施方式中，該方法可以包括在合適的反應(yīng)條件下將本文所述組合物的一種實(shí)施方式與一組多核苷酸和/或寡核苷酸一起孵育，上述組合物的實(shí)施方式包括本文所述的鏈置換聚合酶和5’-3’核酸外切酶和任選的連接酶(如果反應(yīng)在體外或者在不含連接酶的細(xì)胞或有機(jī)體中的體內(nèi))，并且還可以包含ss結(jié)合蛋白質(zhì)，在上述的一組多核苷酸和/或寡核苷酸中至少一種或一些組成員具有與一種或一些其他組成員重疊的序列。在一些實(shí)施方式中，多核苷酸或寡核苷酸可以是dsdna，例如，重疊pcr產(chǎn)物或重疊限制片段。在其他實(shí)施方式中，多核苷酸可以是ssdna或rna。在一些實(shí)施方式中，該多核苷酸組可以包括ssdna或rna。在一些實(shí)施方式中，該多核苷酸組可以包括ds多核苷酸。在一些實(shí)施方式中，該多核苷酸組可以包括至少一種ds多核苷酸和至少一種ss多核苷酸。在一些實(shí)施方式中，該多核苷酸組可以包括除了在亞群成員之間變化的子序列以外具有相同序列的多核苷酸亞群。在其他實(shí)施方式中，該多核苷酸組可以包括ss或ds多核苷酸，或者在其用于接合目的的末端具有重疊區(qū)、但形成合成子的內(nèi)部序列不同的多核苷酸。因此，在本發(fā)明的方法的一個(gè)實(shí)施方式中，該多核苷酸組中的多核苷酸為ds；比如，其中ds多核苷酸是重疊pcr產(chǎn)物或重疊限制片段或者由ss多核苷酸組裝。在本發(fā)明的方法的一另外可選的實(shí)施方式中，合成子由該多核苷酸組中ss的多核苷酸組裝。在本發(fā)明的方法的另一另外可選的實(shí)施方式中，合成子由包括至少一種ds多核苷酸和至少一種ss寡核苷酸的混合物的一組多核苷酸組裝。在本發(fā)明的方法的實(shí)施方式中，合成子由包括在除亞群成員之間變化的子序列以外彼此相同的多核苷酸亞群的一組多核苷酸組裝。方法的實(shí)施方式可以用于制備各種合成子，包括編碼序列、載體、用于基因工程和表達(dá)組件的引導(dǎo)分子(guidemolecule)。在組裝之前，初始ds多核苷酸的長(zhǎng)度可以在100堿基至30kb的范圍內(nèi)，盡管該范圍以外的多核苷酸也可以在某些情形中使用。例如，在一些實(shí)施方式中，單個(gè)片段大小可以至多20kb～30kb或更長(zhǎng)，或者短至30堿基～500堿基。而且，在一些實(shí)施方式中，不同大小的片段可以在組裝反應(yīng)中接合。在一個(gè)實(shí)施例中，將長(zhǎng)多核苷酸(例如，長(zhǎng)度5kb～20kb的片段)與短核苷酸(例如，長(zhǎng)度100堿基～500堿基的片段)接合。新組裝的合成子可以使用單分子測(cè)序方法直接進(jìn)行測(cè)序，或者在克隆或擴(kuò)增之后進(jìn)行測(cè)序。在一個(gè)實(shí)施方式中，該組成員可以包含長(zhǎng)度小于2kb的重疊序列，例如，在15～200個(gè)核苷酸的范圍內(nèi)，例如，20～100個(gè)核苷酸。在一個(gè)實(shí)施方式中，提供一種組合物，其中組合物具有5’-3’核酸外切酶；鏈置換聚合酶；和包含濃度范圍7mm～150mm(例如20mm～50mm)的鉀鹽(比如kcl)的緩沖液。除鉀鹽以外，還可以使用范圍10mm～100mm(比如20mm)的鈉鹽(例如氯化鈉)。組合物中可以包括ss結(jié)合蛋白質(zhì)。在一些實(shí)施方式中，組合物不含擁擠劑，比如聚乙二醇(peg)、ficoll或葡聚糖。在一些實(shí)施方式中，組合物不含非鏈置換聚合酶。在另一個(gè)實(shí)施方式中，用于形成合成子的組合物中包括多核苷酸和/或寡核苷酸片段。在方法的另一個(gè)實(shí)施方式中，一組寡核苷酸可以在不存在非鏈置換聚合酶的情況下使用一種組合物來(lái)接合，上述組合物包括除ss結(jié)合蛋白質(zhì)以外或者代替ss結(jié)合蛋白質(zhì)的擁擠劑比如聚乙二醇(peg)、ficoll或葡聚糖，和至少7mm鉀鹽比如kcl，連同鏈置換聚合酶和5’-3’核酸外切酶。在一個(gè)實(shí)施方式中，鉀鹽濃度低于150mm，例如，20mm～50mm。另外提供一種用于多核苷酸組裝的試劑盒，其包括：i.5’-3’核酸外切酶；ii.任選的連接酶；iii.鏈置換聚合酶：和iv.ssdna結(jié)合蛋白質(zhì)。在某些實(shí)施方式中，例如，試劑盒還可以包括dntp和/或緩沖劑。試劑盒的組分可以在單獨(dú)的容器中(例如，一個(gè)或多個(gè)不同的反應(yīng)管)，或者，試劑盒的組分可以在單一容器中。組分可以是凍干的，或者是溶液狀態(tài)的，或者一部分凍干并且一部分為溶液狀態(tài)。組分可以部分或全部固定化在固體表面比如球珠(bead)上或者反應(yīng)室表面上，或者是溶液狀態(tài)的。可以將組分加入到部分或全部固定化的或者溶液狀態(tài)的靶標(biāo)多核苷酸中。在一些實(shí)施方式中，試劑盒可以包含一種或多種試劑盒組分混合物。在一些實(shí)施方式中，試劑盒不包含非鏈置換聚合酶或擁擠劑。在一個(gè)實(shí)施方式中，提供與seqid:1的序列同一性為至少80％、85％、90％或95％的聚合酶用于組裝混合物。在另一個(gè)實(shí)施方式中，提供結(jié)合結(jié)構(gòu)域與seqid:2的序列同一性為至少80％、85％、90％或95％的聚合酶用于組裝組合物。在另一個(gè)實(shí)施方式中，提供與seqid:3的序列同一性為至少80％、85％、90％或95％的聚合酶用于組裝混合物。這些組合物可以在其中聚合酶為鏈置換性的反應(yīng)條件下使用。組合物可以在其中與聚合酶活性有關(guān)的任何3’核酸外切酶活性呈活性的反應(yīng)條件下使用。組裝反應(yīng)可以使用ss或ds核酸發(fā)生?？梢越M裝任意數(shù)目的片段，例如2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20或更多個(gè)片段?？梢詫⒒瘜W(xué)合成的ss多核苷酸組裝到ds重疊片段中，并且與線性化載體的末端雜交并連接，以形成適合于克隆的合成子。另外可選地，可以將雙鏈片段組裝到合成子中，并插入到用于克隆或pcr或恒溫?cái)U(kuò)增的載體中(參見(jiàn)，例如，圖2a～2c)。通過(guò)將核酸片段與線性化ds載體的3’ss末端雜交，還可以將ss核酸片段直接插入到載體中(參見(jiàn)，例如，圖7)。ss核酸可以在ss核酸片段與線性化載體的3’ss末端雜交的同時(shí)、在其之前或之后通過(guò)重疊互補(bǔ)末端來(lái)組裝。在插入到載體中用于克隆之前，組裝的片段可以通過(guò)pcr或恒溫方法進(jìn)行擴(kuò)增。核酸片段可以包含隨機(jī)化核苷酸序列或簡(jiǎn)并密碼使得能夠形成包含在可變區(qū)的每個(gè)核苷酸位置處的代表性變體的文庫(kù)。隨機(jī)序列可以位于每個(gè)末端處的給定序列之間。在一個(gè)實(shí)施方式中，位于指定序列之間的隨機(jī)序列可以用于與第二核酸片段(例如第二ss基因組多核苷酸)或者線性化載體的末端雜交。在一個(gè)實(shí)施例中，隨機(jī)序列與用于將cas9蛋白質(zhì)引導(dǎo)至用于基因編輯的靶標(biāo)核酸的靶標(biāo)基因組序列雜交(參見(jiàn)，例如，圖9a～9c)。因此，根據(jù)本發(fā)明這一方面，該方法可以用于將一組多核苷酸的至少一種成員與第二核酸片段(比如第二ss基因組多核苷酸)或者與線性化載體末端雜交。例如，該方法可以用于將該多核苷酸組的至少一種成員與靶標(biāo)基因組序列雜交，以在基因編輯方法中將cas核酸內(nèi)切酶比如cas9引導(dǎo)至靶標(biāo)基因組核酸。在一些實(shí)施方式中，提供一種將多種多核苷酸組裝到合成子的方法，其包括：將多種多核苷酸與包括5’-3’核酸外切酶、鏈置換聚合酶、任選的連接酶、ss結(jié)合蛋白質(zhì)和緩沖劑的組合物合并，其中每種多核苷酸均在一條鏈上具有3’ss末端多核苷酸序列：其可以與第二多核苷酸上的互補(bǔ)性ss互補(bǔ)序列雜交，并且可以任選地在單一容器中在恒溫條件下接合以形成連續(xù)的雙鏈多核苷酸。合成子可以進(jìn)一步在其末端與用于擴(kuò)增和/或克隆的線性化質(zhì)粒的末端接合。在一些實(shí)施方式中，整個(gè)組裝方法可以作為“一步”反應(yīng)(在單一管中，其不必在反應(yīng)開始之后的期間打開)進(jìn)行。在一個(gè)實(shí)施例中，組分在反應(yīng)容器中混合在一起，并在40℃至60℃的溫度下孵育一段時(shí)間，例如，5分鐘至12小時(shí)，從而制備合成子。在一方面，該方法包括在要通過(guò)聚合酶接合的多核苷酸中進(jìn)行鏈置換的步驟，上述聚合酶與seqidno:1或seqidno:102和/或seqidno:2或seqidno:3的氨基酸序列同一性為至少90％。在方法實(shí)施方式中，不要求有額外的3’-5’核酸外切酶回切(chewback)步驟。另一方面還包括通過(guò)在反應(yīng)混合物中包括最小濃度為7mm的鉀鹽提高組裝反應(yīng)效率，其中鉀鹽的實(shí)例有kcl。在一方面，提供一種方法，其中多核苷酸包含給定的序列末端之間的隨機(jī)序列。在另一方面，該方法還包括篩選具有與基因組dna雜交的活性的隨機(jī)序列以及鑒定具有雜交活性的隨機(jī)序列。在另一方面，該方法還包括通過(guò)轉(zhuǎn)錄具有雜交活性的隨機(jī)序列以形成rna，以及在cas核酸內(nèi)切酶的存在下使用用于基因編輯的rna，從而進(jìn)行基因編輯。在本發(fā)明的組合物、試劑盒或方法的一個(gè)實(shí)施方式中，本發(fā)明的組合物、試劑盒或方法中使用的鏈置換聚合酶可以是非天然存在的，比如，突變體或融合蛋白。在本發(fā)明的組合物、試劑盒或方法中，非天然鏈置換融合蛋白酶的特征可以在于聚合酶部分的氨基酸組成與seqidno:33至seqidno:55、或seqidno:1中任意者的同一性為至少90％或95％或99％或100％，或者dna結(jié)合部分的氨基酸組成與seqidno:56至seqidno:98或seqidno:2中任意者的同一性為至少90％或95％或99％或100％。在一個(gè)實(shí)施方式中，氨基酸序列與seqidno:102的同一性可以為90％或95％或98％或99％的聚合酶部分，其與選自氨基酸序列與seqidno:56至seqidno:98中任意者的同一性為至少90％或95％或99％或100％的多肽中的異源性dna結(jié)合部分融合。概括來(lái)講，在一方面，提供一種制劑，其包括氨基酸序列與seqidno:3的序列同一性為至少90％的組合物。該制劑還可以包括5’-3’核酸外切酶，例如，t5核酸外切酶。該制劑還可以包括ssdna結(jié)合蛋白質(zhì)，例如，etssb、大腸桿菌reca、t7基因2.5產(chǎn)物、噬菌體λredb或rac原噬菌體rect，更具體地，包括熱穩(wěn)定性ss結(jié)合蛋白質(zhì)，比如etssb。該制劑還可以包括連接酶。在一方面，包括組合物的制劑還可以包括ss結(jié)合結(jié)構(gòu)域和5’-3’核酸外切酶，其中該制劑不包括擁擠劑和/或非鏈置換聚合酶。該制劑還可以包括鉀鹽。在一方面，包括組合物的制劑還可以包括一組中的多種多核苷酸，其中所述組中的至少一種多核苷酸的序列與所述組中另一種多核苷酸重疊；并且其中多核苷酸選自：(i)ds多核苷酸；(ii)ss寡核苷酸；(iii)至少一種ds多核苷酸和至少一種ss寡核苷酸；和(iv)除了在亞群成員之間變化的序列以外，否則彼此相同的多核苷酸亞群。在一方面，多核苷酸組具有至少3種成員或至少4種成員或至少5種成員。通常，用于制備合成子的方法包括：在合適的反應(yīng)條件下將還包括5’-3’核酸外切酶以及任選的連接酶和ssdna結(jié)合蛋白質(zhì)的權(quán)利要求1的組合物與形成一組的多種多核苷酸一起孵育，其中所述組的成員具有重疊序列；以及將至少兩種多核苷酸接合，以制備合成子。在一方面，該多核苷酸組包含至少3種成員或至少4種成員或至少5種成員。在一方面，該制劑還包括連接酶。一方面，該制劑還包括ssdna結(jié)合蛋白質(zhì)。在一方面，多核苷酸為ds，并且ds多核苷酸是重疊pcr產(chǎn)物、重疊限制片段或者由ss寡核苷酸組裝。在一方面，多核苷酸是ss寡核苷酸。在一方面，多核苷酸組包括至少一種ds多核苷酸和至少一種ss寡核苷酸。概括來(lái)講，本發(fā)明提供一種試劑盒，其包括根據(jù)權(quán)利要求1的制劑和5’-3’核酸外切酶，如例如t5核酸外切酶。在一方面，試劑盒還可以包括ss結(jié)合蛋白質(zhì)。在另一方面，試劑盒可以包括連接酶。在另一方面，試劑盒可以包括緩沖劑。在一方面，試劑盒不包括擁擠劑。在一方面，組合物和5’-3’核酸外切酶在相同容器中。在另一方面，組合物和5’-3’核酸外切酶在不同容器中，任選地是在適合于合并在單一容器中的緩沖劑中。概括來(lái)講，本發(fā)明提供一種用于組裝合成子的組合物，其包括：5’-3’核酸外切酶，其在一方面具有ss核酸內(nèi)切酶活性，例如，與seqidno:98的序列同一性為90％；鏈置換聚合酶，其包括b族鏈置換聚合酶，其優(yōu)選為非天然存在的，比如源自天然存在的聚合酶的突變體或融合蛋白，其可以另外地為熱穩(wěn)定性的；任選的ssdna結(jié)合蛋白質(zhì)，例如，ssdna結(jié)合蛋白質(zhì)為etssb、大腸桿菌reca、t7基因2.5產(chǎn)物、噬菌體λredb或rac原噬菌體rect；和非天然存在的緩沖劑，其中組合物不包括擁擠劑和/或非鏈置換聚合酶。在一方面，組合物還包括連接酶和/或ss結(jié)合結(jié)構(gòu)域。在一方面，組合物包括一組至少兩種多核苷酸(多種多核苷酸)。在一方面，組合物不包括非鏈置換聚合酶。在另一方面，組合物不包括9°n、phusion、vent或pfudna聚合酶。在一方面，組合物中的鏈置換聚合酶是融合蛋白，其中聚合酶部分的氨基酸序列與seqidno:1或seqidno:102或seqidno:33至seqidno:55中任意者的同一性為至少90％。例如，融合蛋白的氨基酸序列與seqidno:1或seqidno:102和seqidno:2的同一性可以為至少90％。例如，鏈置換聚合酶與seqidno:3的序列同一性可以為至少90％。在一方面，組合物可以包括濃度至少7mm的鉀鹽。在一方面，組合物可以包括一組多核苷酸，其中所述組中的至少一種多核苷酸具有與所述組中另一種多核苷酸重疊的序列，并且其中多核苷酸選自：(i)ds多核苷酸；(ii)ss寡核苷酸；(iii)至少一種ds多核苷酸和至少一種ss寡核苷酸；和(iv)除了在亞群成員之間變化的序列以外，否則彼此相同的多核苷酸亞群。在一方面，該多核苷酸組中至少一種成員包含位于每個(gè)末端處給定序列之間的用于與第二ss基因組多核苷酸雜交的隨機(jī)序列，其中，例如，隨機(jī)序列為ss，并且其能夠與用于將cas蛋白質(zhì)引導(dǎo)至用于基因編輯的靶標(biāo)基因組核酸的靶標(biāo)基因組序列雜交。概括來(lái)講，本發(fā)明提供用于形成合成子的方法，其包括在合適的反應(yīng)條件下孵育包含一組具有重疊序列的多核苷酸的任意上述組合物；以及將至少一些多核苷酸與其他多核苷酸接合，以制備合成子。在該方法的在一方面，所述組中全部或部分多核苷酸為ds。在另一方面，ds多核苷酸是重疊pcr產(chǎn)物；重疊限制片段或者由互補(bǔ)ss寡核苷酸組裝的合成ds分子，其中這些寡核苷酸可以在合成器中制備。在一方面，所述組中的全部或部分多核苷酸是ss寡核苷酸。在一方面，該多核苷酸組包括至少一種ds多核苷酸和至少一種ss寡核苷酸。在一方面，該多核苷酸組包括除了在亞群成員之間變化的序列以外，否則彼此相同的多核苷酸亞群。在一方面，多核苷酸的重疊序列長(zhǎng)度小于2千堿基。在該方法的一方面，鏈置換聚合酶包括與seqidno:1、seqidno:2、seqidno:3、seqidno:33至seqidno:96或seqidno:102中任意者的同一性為至少90％的氨基酸序列。在該方法的一方面，該多核苷酸組中至少一種成員包含給定的序列末端之間的隨機(jī)序列。該方法的另一方面包括篩選具有與基因組dna雜交的活性的隨機(jī)序列以及鑒定具有雜交活性的隨機(jī)序列。該方法的另一方面包括通過(guò)轉(zhuǎn)錄具有雜交活性的隨機(jī)序列以形成rna，以及在cas蛋白質(zhì)的存在下使用用于基因編輯的rna，從而進(jìn)行基因編輯。概括來(lái)講，本發(fā)明提供一種用于多核苷酸組裝的試劑盒，其包括：5’-3’核酸外切酶；鏈置換聚合酶；和任選的ssdna結(jié)合蛋白質(zhì)，其中該試劑盒任選地不包括擁擠劑和/或非鏈置換聚合酶。在一方面，該試劑盒包括連接酶。在另一方面，該試劑盒包括dntp。在另一方面，該試劑盒包括緩沖劑。在另一方面，該試劑盒的單個(gè)組分可以在相同的容器或單獨(dú)的容器中，比如一個(gè)或多個(gè)不同的儲(chǔ)存或反應(yīng)容器。概括來(lái)講，本發(fā)明提供一種組合物，其包括聚合酶融合蛋白，其中聚合酶融合蛋白包括與seqidno:2、seqidno:56至seqidno:96中任意者的同一性為至少90％的氨基酸序列和異源性聚合酶結(jié)構(gòu)域。在一方面，聚合酶融合蛋白包括與seqidno:2中任意者的同一性為至少90％的氨基酸序列；和異源性聚合酶結(jié)構(gòu)域。概括來(lái)講，本發(fā)明提供一種包括聚合酶融合蛋白的組合物，其中聚合酶融合蛋白包括氨基酸序列與seqidno:1、seqidno:33至seqidno:55或seqidno:102中任意者的同一性為至少90％的聚合酶結(jié)構(gòu)域；和異源性dna結(jié)合結(jié)構(gòu)域。在一方面，聚合酶融合蛋白包括氨基酸序列與seqidno:1的同一性為至少90％的聚合酶結(jié)構(gòu)域；和異源性dna結(jié)合結(jié)構(gòu)域。在一方面，聚合酶融合蛋白具有氨基酸序列與seqidno:102的同一性為至少90％的聚合酶結(jié)構(gòu)域；和異源性dna結(jié)合結(jié)構(gòu)域。在一方面，聚合酶融合蛋白的氨基酸序列與seqidno:3的同一性為至少90％。在一方面，上述組合物還包括5’-3’核酸外切酶，比如t5核酸外切酶。在一方面，組合物還包括單鏈dna結(jié)合蛋白質(zhì)，例如選自etssb、大腸桿菌reca、t7基因2.5產(chǎn)物、噬菌體λredb或rac原噬菌體rect中的單鏈結(jié)合蛋白質(zhì)。在一方面，組合物可以包括連接酶。在一方面，連接酶是熱穩(wěn)定性的。在一方面，組合物不包括擁擠劑和/或非鏈置換聚合酶。在另一方面，組合物還包括dntp。在另一方面，組合物還包括濃度至少7mm的鉀鹽。組合物的一方面包括一組多核苷酸，其中所述組中的至少一種多核苷酸的序列與所述組中另一種多核苷酸重疊；并且其中多核苷酸選自：(i)ds多核苷酸；(ii)ss寡核苷酸；(iii)至少一種ds多核苷酸和至少一種ss寡核苷酸；和(iv)除了在亞群成員之間變化的序列以外，否則彼此相同的多核苷酸亞群。概括來(lái)講，本發(fā)明提供一種用于制備合成子的方法，其包括將一組多核苷酸與特征在于上文所述的包括聚合酶的組合物一起孵育，其中單個(gè)多核苷酸包含與其他多核苷酸中的序列重疊的序列，其中不同多核苷酸的重疊序列能夠在合適的反應(yīng)條件下交叉雜交，并且，例如其中重疊區(qū)小于2千堿基，其中組合物還包括5’-3’核酸外切酶和任選的連接酶和ssdna結(jié)合蛋白質(zhì)；以及將多核苷酸接合，以制備合成子。在不同的方面，組合物包括連接酶；和/或ssdna結(jié)合蛋白質(zhì)。在另一方面，所述組中一種或多種多核苷酸為ds，其中ds多核苷酸是pcr產(chǎn)物、重疊限制片段或者由ss寡核苷酸組裝；和/或一種或多種多核苷酸是ss寡核苷酸；和/或該多核苷酸組包括至少一種ds多核苷酸和至少一種ss寡核苷酸。概括來(lái)講，本發(fā)明提供一種用于多核苷酸組裝的試劑盒，其包括上述聚合酶融合蛋白，和5’-3’核酸外切酶；和ssdna結(jié)合蛋白質(zhì)。在一方面，該試劑盒可以包括任意或全部的連接酶、dntp和緩沖劑，其中試劑盒組分可以在相同容器中或不同容器中。附圖說(shuō)明技術(shù)人員將會(huì)理解到，下述附圖僅僅出于說(shuō)明目的。附圖無(wú)意以任何方式對(duì)本發(fā)明教導(dǎo)的范圍加以限定。圖1a～1e示出實(shí)施例1所述的試驗(yàn)如何區(qū)分鏈置換和非鏈置換聚合酶。該試驗(yàn)確認(rèn)，t4dna聚合酶是非鏈置換的，并在封閉寡核苷酸(blockingoligonucleotide)(2)處在44個(gè)核苷酸長(zhǎng)度時(shí)終止模板dna的合成(圖1c)，而bst聚合酶(圖1d)和非天然聚合酶(圖1e)是鏈置換的，并可以通過(guò)置換封閉寡核苷酸(長(zhǎng)度27個(gè)核苷酸)繼續(xù)fam引物(1)的dna合成。圖1a示出以下酶試驗(yàn)中使用的dna模板上引物(1)和封閉寡核苷酸(2)的序列和位置。圖1b示出毛細(xì)管電泳之后得到的樣品中觀察到的熒光，其中不加入酶，并且起始原料在與24個(gè)核苷酸的fam引物對(duì)應(yīng)的位置處形成峰。圖1c示出加入t4dna聚合酶的結(jié)果。引物延長(zhǎng)至最終長(zhǎng)度44個(gè)核苷酸(24個(gè)核苷酸加上20個(gè)核苷酸)，但由封閉寡核苷酸終止。圖1d示出，bstdna聚合酶(大的片段)對(duì)封閉寡核苷酸進(jìn)行鏈置換，并通過(guò)將引物延長(zhǎng)至總長(zhǎng)度72個(gè)核苷酸(24+20+27+da)對(duì)模板進(jìn)行拷貝。圖1e示出作為校對(duì)聚合酶的b族鏈置換dna聚合酶對(duì)封閉寡核苷酸進(jìn)行鏈置換，并通過(guò)將引物延長(zhǎng)71個(gè)核苷酸(24+20+27)對(duì)模板進(jìn)行拷貝。圖2a～2c示出dna組裝方法中的步驟。圖2a示出將5種片段中每一個(gè)的擴(kuò)增子加入到具有氨芐青霉素抗性標(biāo)志物的5種質(zhì)粒中。用產(chǎn)生具有重疊區(qū)以及兩側(cè)為noti限制性位點(diǎn)的擴(kuò)增子的引物對(duì)5種片段進(jìn)行初始擴(kuò)增。noti切割產(chǎn)生粘性末端。noti限制性位點(diǎn)(3)允許由載體釋放出每種擴(kuò)增子。限制性酶切片段與相鄰片段具有80個(gè)堿基對(duì)重疊區(qū)(4)。在圖2c中，出于方便及降低成本，第一片段和相鄰的試劑載體末端以及最后片段和相鄰的試劑載體末端之間的重疊為15～25核苷酸，例如，20核苷酸，但這并不意在是限制性的。圖2b示出noti切割的任選測(cè)序的片段(5)(由載體回收到的擴(kuò)增子)，然后將其在單個(gè)反應(yīng)容器中用包括t5/5’-3’核酸外切酶、具有3’-5’核酸外切酶活性的dna聚合酶、ss結(jié)合蛋白質(zhì)etssb(newenglandbiolabs,ipswich,ma)和dna連接酶的酶混合物處理(6)～(8)。盡管在此使用noti，取決于方便性，也可以使用其他的限制性核酸內(nèi)切酶以用于切割。可以用兩種或更多種限制性核酸內(nèi)切酶進(jìn)行雙消化。例如，已經(jīng)發(fā)現(xiàn)，用兩種限制性核酸內(nèi)切酶對(duì)載體dna進(jìn)行雙消化降低來(lái)自未切割載體的本底。將重疊ssdna序列與相鄰片段雜交。t5核酸外切酶在每個(gè)片段上將dna鏈從5’到3’回切(chewback)，以暴露3’ss區(qū)(6)，這促使片段在ss結(jié)合蛋白質(zhì)的存在下一起退火(7)。通過(guò)與鏈置換聚合酶相關(guān)的3’-5’核酸外切酶活性的手段，達(dá)到2個(gè)堿基翼(flap)的除去，然后通過(guò)鏈置換聚合酶進(jìn)行延長(zhǎng)，以充填組裝產(chǎn)物中的間隙(8)。任何殘余的切口或5’翼可以通過(guò)連接酶和/或t5核酸外切酶修復(fù)。圖2c示出現(xiàn)在按順序接合并插入到用于轉(zhuǎn)化到細(xì)菌宿主中的攜帶氯霉素抗性基因(cam)的第二質(zhì)粒中的5個(gè)片段(片段(frag)1至片段5)。圖3a和3b示出，使用氯霉素板以選擇在板上生長(zhǎng)的菌落，并且在iptg和x-gal的存在下，包含laclz基因的那些菌落產(chǎn)生藍(lán)色菌落。試驗(yàn)提供了對(duì)其中基因以功能性形式有效組裝的克隆的定量評(píng)價(jià)。圖3a示出僅有氯霉素。圖3b示出氯霉素+iptg+xgal。圖4示出質(zhì)粒事實(shí)上確實(shí)包含整個(gè)基因。對(duì)圖2b所示的組裝產(chǎn)物進(jìn)行pcr擴(kuò)增，以確認(rèn)所有片段在轉(zhuǎn)化之前得以接合和連接。條帶1和2是重復(fù)的pcr結(jié)果。條帶m是來(lái)自newenglandbiolabs,ipswich,ma的2-logdna梯狀帶(ladder)。圖5示出通過(guò)菌落數(shù)目測(cè)定的組裝混合物中kcl的影響。緩沖液中使用的kcl濃度增加，表現(xiàn)出組裝混合物中使用鏈置換聚合酶的組裝的準(zhǔn)確性/效率增加。左邊的柱狀圖(t26)不含kcl，而右邊的柱狀圖(t26k)包含25mmkcl，其示出效率提高1.5倍。無(wú)論組裝條件如何，這一提高均發(fā)生。如果在不存在ss結(jié)合蛋白質(zhì)的情況下使用peg或其他擁擠劑，預(yù)計(jì)有類似的相對(duì)效率提高。圖6示出根據(jù)制造商所提供的方案，實(shí)施例2中所述的混合物(鏈置換聚合酶/ss結(jié)合蛋白質(zhì)/5’-3’核酸外切酶/連接酶)(mix1)和市售gibsonmix(gamm)(非鏈置換聚合酶和聚乙二醇)(syntheticgenomics,lajolla,ca/newenglandbiolabs,ipswich,ma)之間的比較。mix1產(chǎn)生顯著更高的dna組裝和轉(zhuǎn)化效率。圖7示出ssdna寡核苷酸和dsdna片段之間的dna組裝的概略圖。將ss靶標(biāo)dna寡核苷酸插入到dna載體中。已合成出ss靶標(biāo)dna寡核苷酸，以在每個(gè)末端上具有20～30個(gè)核苷酸的與3’載體末端的重疊區(qū)。然而，該寡核苷酸的尺寸可以具有小于20個(gè)核苷酸的重疊區(qū)，例如小于15個(gè)核苷酸或小于10個(gè)核苷酸，或者另外可選地大于30個(gè)核苷酸，例如，至少40個(gè)或50個(gè)或60個(gè)核苷酸或更多。在重疊區(qū)之外，該寡核苷酸優(yōu)選地具有位于末端之間的不重疊的1個(gè)或更多個(gè)核苷酸。將包含5’-3’核酸外切酶、鏈置換聚合酶、連接酶和ss結(jié)合蛋白質(zhì)的組裝主混合物加入到ss寡核苷酸和載體的混合物中，以使得dsdna載體的5’末端得以回切(chewedback)，以產(chǎn)生ss突出端(9)。然后ssdna的3’末端能夠退火至載體的5’末端，然后dna聚合酶對(duì)ss模板進(jìn)行復(fù)制，以填充空隙，并產(chǎn)生平端dsdna。將切口通過(guò)連接酶密封(10)。再次，核酸外切酶(在此為t5核酸外切酶)這次在靶標(biāo)dna的平端回切5’末端，產(chǎn)生3’ss區(qū)，以使得互補(bǔ)序列退火，并完成靶標(biāo)dnads整合到dna載體中(11)。片段可以借助填充空隙的dna聚合酶和密封切口的連接酶退火(12)以制備合成子。圖8a～8c示出由短ss寡核苷酸橋接dsdna的工作流程。(方案描述于圖7中)。圖8a提供用于整合到在此示出為具有ofp報(bào)告子的crispr核酸酶載體的dsdna載體中的短ss寡核苷酸序列的實(shí)例。圖8b示出的工作流程起始于ss寡核苷酸和dscrispr核酸酶載體(9424bp)，其用5’-3’核酸外切酶、鏈置換聚合酶、連接酶和ss結(jié)合蛋白質(zhì)處理(13)，以產(chǎn)生完整的ds環(huán)狀dna。將該dna轉(zhuǎn)化到感受態(tài)細(xì)胞中(14)。過(guò)夜孵育之后，通過(guò)微制備對(duì)菌落進(jìn)行分析，然后進(jìn)行質(zhì)粒測(cè)序(15)。圖8c示出插入的序列和相鄰序列、u6啟動(dòng)子序列(載體)、設(shè)計(jì)的ss寡核苷酸(71聚體(mer))的序列和骨架(scaffold)模板特異性序列(載體)。將在每個(gè)末端包括25個(gè)核苷酸的重疊區(qū)(黑體示出的靶標(biāo)dna的21核苷酸)的ss寡核苷酸(71聚體)適當(dāng)?shù)卣系剿拗骷?xì)胞中的載體中。圖9a～9c示出，與圖8中類似的工作流程可以用于在重疊端之間具有簡(jiǎn)并堿基的ss寡核苷酸。同樣，圖9a中的起始序列示出于工作流程(圖9b)之上，并且來(lái)自組裝庫(kù)的菌落的sanger測(cè)序結(jié)果示出于下方(圖9c)，實(shí)線是指sgrna靶標(biāo)序列庫(kù)。sgrna靶標(biāo)序列包含21個(gè)可變核苷酸位置，提供421種變體。該庫(kù)包含每種可能的變體，并且每種變體適于克隆反映重疊末端和載體之間序列的簡(jiǎn)并性。圖9a示出包含簡(jiǎn)并堿基的ss寡核苷酸的序列。將圖9a的sgrna靶標(biāo)序列插入在載體的u6啟動(dòng)子序列和骨架模板特異性序列之間(16)，將其轉(zhuǎn)化到宿主細(xì)胞中(17)，并通過(guò)微制備和測(cè)序進(jìn)行合成子分析(18)，如上文及在此所述。對(duì)來(lái)自組裝庫(kù)的克隆進(jìn)行sanger測(cè)序。圖9c提供序列的實(shí)例。圖10示出將組裝反應(yīng)產(chǎn)物轉(zhuǎn)化到大腸桿菌中之后選自一板的187個(gè)菌落的結(jié)果。對(duì)每個(gè)菌落進(jìn)行pcr擴(kuò)增和測(cè)序，以確認(rèn)ssdna的插入，并對(duì)簡(jiǎn)并堿基的分布進(jìn)行分析。在此示出的結(jié)果確認(rèn)，不同的菌落確實(shí)包含不同的簡(jiǎn)并序列。未檢測(cè)到偏性。首先通過(guò)將序列轉(zhuǎn)化成fastq文件，然后在github上使用來(lái)自fastx工具包的fastx_quality_stats工具，進(jìn)行分析。使用來(lái)自berkeley的weblogo產(chǎn)生序列標(biāo)識(shí)。術(shù)語(yǔ)說(shuō)明除非另外指出，本文使用的所有科技術(shù)語(yǔ)的含義與本發(fā)明所屬領(lǐng)域普通技術(shù)人員所通常理解的相同。singleton等人，dictionaryofmicrobiologyandmolecularbiology，第二版，johnwileyandsons,newyork(1994)和hale&markham,theharpercollinsdictionaryofbiology,harperperennial,n.y.(1991)為技術(shù)人員提供了許多本文所用術(shù)語(yǔ)的通常含義。為清楚和便于參考起見(jiàn)，以下仍對(duì)某些術(shù)語(yǔ)加以定義。如本文所用，如基因合成領(lǐng)域所用的術(shù)語(yǔ)“合成子”是指多核苷酸組裝體。多核苷酸組裝可以包括組裝一定大小的重疊片段，其可以在寡核苷酸合成器上制備，對(duì)于每個(gè)合成的多核苷酸，目前通常為2000～3000個(gè)堿基。另外可選地，重疊片段可以由連接有接頭(adaptor)以提供重疊序列的天然存在的核酸通過(guò)pcr獲得。出于組裝目的，對(duì)每個(gè)片段的大小沒(méi)有限定。依賴于末端處的重疊序列，可以將許多片段端到端地組裝，使得能夠準(zhǔn)確、有效地產(chǎn)生具有任意所需長(zhǎng)度的構(gòu)建體。優(yōu)選地，合成子是由較短的多核苷酸的組裝體形成的不含間隙和切口的較長(zhǎng)的連續(xù)多核苷酸。然而，由核酸片段組裝體產(chǎn)生的合成子的長(zhǎng)度不限定于任何具體的大小。如本文所用，術(shù)語(yǔ)“5’-3’核酸外切酶”是指從5’末端，即以5’至3’方向降解dna的核酸外切酶。所關(guān)注的5’-3’核酸外切酶可以在平端處以及在某些實(shí)施方式中在3’和/或5’突出端處從dsdna鏈的5’末端除去核苷酸。t5核酸外切酶、λ核酸外切酶和t7核酸外切酶均為5’-3’核酸外切酶的實(shí)例。在某些實(shí)施方式中，優(yōu)選t5核酸外切酶。t5核酸外切酶額外地具有ss核酸內(nèi)切酶活性。如本文所用，術(shù)語(yǔ)“連接酶”是指可以具體地在切口處將dna分子的3’末端與另一dna分子的5’末端共價(jià)接合的酶。盡管已知有許多其他的連接酶并可以在本文使用，連接酶的實(shí)例包括t7連接酶、t4dna連接酶、大腸桿菌dna連接酶和taq連接酶。如本文所用，術(shù)語(yǔ)“鏈置換聚合酶”是指能夠置換酶下游的一個(gè)或多個(gè)核苷酸比如至少10個(gè)或100個(gè)或更多個(gè)核苷酸的聚合酶。鏈置換聚合酶可以與phusion區(qū)分，其中phusion在本領(lǐng)域公認(rèn)的定義是非鏈置換聚合酶。在一些實(shí)施方式中，在至少50℃或至少55℃的溫度下，鏈置換聚合酶是穩(wěn)定、有活性的(包括鏈置換活性)。taq聚合酶是切口翻譯聚合酶，因此，其不是鏈置換聚合酶。如本文所用，術(shù)語(yǔ)“單鏈(ss)dna結(jié)合蛋白質(zhì)”是指與ssdna結(jié)合、防止早熟退火、保護(hù)ssdna不被核酸酶和聚合酶消化、和/或去除dna二級(jí)結(jié)構(gòu)以使其他酶對(duì)其有效發(fā)揮功能的蛋白質(zhì)。優(yōu)選在本文所述的組合物中包括ss結(jié)合蛋白質(zhì)，以優(yōu)化合成子形成的效率。盡管已知有許多其他者，并可以在本文使用，例如λ噬菌體redb、rac原噬菌體的rect和下文所列的序列，ssdna結(jié)合蛋白質(zhì)的實(shí)例有t4基因32蛋白質(zhì)、大腸桿菌ssb、t7gp2.5ssb、和噬菌體phi29ssb、以及etssb。在一些情形中可以使用在50℃下穩(wěn)定的熱穩(wěn)定性ssdna結(jié)合蛋白質(zhì)。因此，在本發(fā)明的組合物、試劑盒或方法的一個(gè)實(shí)施方式中，ssdna結(jié)合蛋白質(zhì)為t4基因32蛋白質(zhì)、大腸桿菌ssb、t7gp2.5ssb、噬菌體phi29ssb、etssb、λ噬菌體redb或rac原噬菌體rect。在一個(gè)實(shí)施方式中，ssdna結(jié)合蛋白質(zhì)是etssb。在本發(fā)明的組合物、試劑盒或方法的一個(gè)實(shí)施方式中，ssdna結(jié)合蛋白質(zhì)是熱穩(wěn)定性的(即，在40℃～60℃下穩(wěn)定)。如本文所用，術(shù)語(yǔ)“緩沖劑”是指當(dāng)向溶液中加入酸或堿時(shí)使得溶液抵抗ph改變的試劑。本發(fā)明的組合物、試劑盒和方法中可以使用的合適的非天然存在的緩沖劑的實(shí)例包括，例如，tris、hepes、taps、mops、兩性離子緩沖劑(tricine)或mes。術(shù)語(yǔ)“非天然存在的”是指組合物在自然界中不存在。本文所述的任何蛋白質(zhì)均可以是非天然存在的，其中術(shù)語(yǔ)“非天然存在的”是指蛋白質(zhì)具有與其天然狀態(tài)下不同的氨基酸序列和/或翻譯后修飾方式。例如，非天然存在的蛋白質(zhì)可以在蛋白質(zhì)的n-末端、c-末端和/或在n-末端和c-末端之間具有一個(gè)或多個(gè)氨基酸替換、缺失或插入?！胺翘烊淮嬖诘摹钡鞍踪|(zhì)的氨基酸序列可以與天然存在的氨基酸序列不同(即，與天然存在的蛋白質(zhì)的氨基酸序列的同一性小于100％)，但與天然存在的氨基酸序列的同一性為至少80％、至少85％、至少90％、至少95％、至少97％、至少98％或至少99％。在某些情形中，如果由不同的(例如，細(xì)菌)細(xì)胞制備非天然存在的蛋白質(zhì)，其可以包含n-末端甲硫氨酸或者可以缺少一個(gè)或多個(gè)翻譯后修飾(例如，糖基化、磷酸化等)。“突變的”蛋白質(zhì)相對(duì)于野生型蛋白質(zhì)可以具有一個(gè)或多個(gè)氨基酸替換，并且可以包括“融合”蛋白。術(shù)語(yǔ)“融合蛋白”是指由多種在天然狀態(tài)下未接合的多肽組分組成的蛋白質(zhì)。融合蛋白可以是2個(gè)、3個(gè)或甚至4個(gè)或更多個(gè)不同蛋白質(zhì)的組合。術(shù)語(yǔ)多肽包括融合蛋白，包括，但不限于，兩個(gè)或更多個(gè)異源性氨基酸序列的融合，多肽與以下的融合：異源性靶標(biāo)序列、連接序列(linker)、免疫學(xué)標(biāo)簽、可檢測(cè)的融合配偶體比如熒光蛋白、β-半乳糖苷酶、熒光素酶等，等等。融合蛋白可以具有一個(gè)或多個(gè)加在蛋白質(zhì)n-末端、c-末端和/或中間部分的異源性結(jié)構(gòu)域。如果融合蛋白的兩部分是“異源性”的，則它們?cè)谄涮烊粻顟B(tài)下不是相同蛋白質(zhì)的一部分。在上下文述及核酸時(shí)，術(shù)語(yǔ)“非天然存在的”是指核酸包含：a)與天然狀態(tài)下的核酸不同的核苷酸序列(即，與天然存在的核酸序列的序列同一性小于100％)，b)一個(gè)或多個(gè)非天然存在的核苷酸單體(其可以產(chǎn)生不是g、a、t或c的非天然骨架或糖)，和/或c)可以在核酸的5’-末端、3’-末端和/或在5’-和3’-末端之間包含一個(gè)或多個(gè)其他修飾(即，加入的標(biāo)記或其他部分)。在上下文述及制劑時(shí)，術(shù)語(yǔ)“非天然存在的”是指：a)例如由于組分在不同的位置、在不同的細(xì)胞中或在不同的細(xì)胞區(qū)室中而非通過(guò)自然組合，這樣組分的組合物；b)其相對(duì)濃度在自然界未發(fā)現(xiàn)的組分的組合物；c)缺少某些通常在自然界中與組分之一關(guān)聯(lián)的組分的組合物；d)自然界中未發(fā)現(xiàn)的形式例如干燥、凍干、結(jié)晶或含水形式的組合物；和/或e)包含自然界中未發(fā)現(xiàn)的組分的組合物。例如，制劑可以包含自然界中未發(fā)現(xiàn)的“非天然存在的”緩沖劑(例如，tris、hepes、taps、mops、兩性離子緩沖劑或mes)、洗滌劑、染料、反應(yīng)促進(jìn)劑或抑制劑、氧化劑、還原劑、溶劑或防腐劑。可能需要使用具有3’核酸外切酶活性的鏈置換聚合酶。盡管無(wú)意拘泥于理論，但需要3’核酸外切酶，以除去雙鏈3’末端上的側(cè)翼序列(flapsequence)，其中側(cè)翼序列可以是酶切的產(chǎn)物，以便從其置于的質(zhì)粒中提取靶標(biāo)多核苷酸。這是如實(shí)施例中所述的使用noti的情形。然而，如果使用在切除的片段上產(chǎn)生平端的限制性核酸內(nèi)切酶，可以不要求3’核酸外切酶活性。3’核酸外切酶活性可以通過(guò)使用標(biāo)準(zhǔn)dna模板和引物加以常規(guī)測(cè)定，其中引物具有或不具有非雜交3’核苷酸。如果聚合酶具有3’核酸外切酶活性，則使用任一引物對(duì)將會(huì)檢測(cè)到擴(kuò)增子。如果聚合酶缺少3’核酸外切酶活性，則使用具有非雜交3’核苷酸的引物將檢測(cè)不到擴(kuò)增子。如本文所用，術(shù)語(yǔ)“鉀鹽”是指包括但不限于kcl的鉀鹽。術(shù)語(yǔ)“鈉鹽”是指包括但不限于nacl的鈉鹽。如本文所用，術(shù)語(yǔ)“多核苷酸”包括寡核苷酸，并且其是指任意長(zhǎng)度的核酸。多核苷酸可以是dna或rna。除非指定，多核苷酸可以為ss或ds。多核苷酸可以是合成的，例如，在dna合成器中合成，或者是天然存在的，例如，提取自天然的來(lái)源，或者源自克隆或擴(kuò)增的材料。本文所指的多核苷酸可以包含修飾的堿基。如本文所用，術(shù)語(yǔ)“多核苷酸組”或“一組多核苷酸”是指至少兩種多核苷酸的集合。在一些實(shí)施方式中，一組多核苷酸可以包括至少5種、至少10種、至少12種、或至少15種或更多種多核苷酸。如本文所用，術(shù)語(yǔ)“重疊序列”是指在兩個(gè)多核苷酸中互補(bǔ)的序列，并且其中重疊序列為ss，在一種多核苷酸上，其可以與另一種多核苷酸上另一重疊互補(bǔ)ss區(qū)雜交。例如，在一組多核苷酸中，重疊序列可以在至少5、10、15或更多種多核苷酸中互補(bǔ)。重疊序列可以在兩個(gè)不同分子的3’末端處(例如，兩個(gè)ss寡核苷酸的3’末端，或者第一ds多核苷酸頂部鏈的3’末端和第二ds分子底部鏈的3’末端)或者與其接近(例如，大約5、10、20個(gè)核苷酸以內(nèi))，其中，如果非重疊序列在3’末端處，則非重疊序列可以使用聚合酶的3’-5’核酸外切酶活性去除。重疊序列的長(zhǎng)度可以變化，在一些情形中，長(zhǎng)度可以為至少12個(gè)核苷酸(例如，長(zhǎng)度至少15個(gè)、20個(gè)或更多個(gè)核苷酸)，和/或長(zhǎng)度可以至多100個(gè)核苷酸(例如，長(zhǎng)度至多50個(gè)、至多30個(gè)、至多20個(gè)或至多15個(gè)核苷酸)。另外可選地，多核苷酸組中的重疊序列可以為2kb或更小，或者1kb或更小，或者小于900堿基、800堿基、700堿基、600堿基、500堿基、400堿基、300堿基、200堿基或100堿基。優(yōu)選地，重疊序列長(zhǎng)度在15個(gè)核苷酸至80個(gè)核苷酸范圍內(nèi)，例如，至多20個(gè)、至多25個(gè)、至多30個(gè)、至多35個(gè)、至多40個(gè)、至多45個(gè)、至多50個(gè)、至多55個(gè)、至多60個(gè)、至多65個(gè)、至多70個(gè)、至多75個(gè)或至多80個(gè)核苷酸。重疊的最小長(zhǎng)度可以通過(guò)tm定義，其優(yōu)選地等于或大于48℃。如本文所用，術(shù)語(yǔ)“多核苷酸組裝”是指2個(gè)或更多個(gè)、4個(gè)或更多個(gè)、6個(gè)或更多個(gè)、8個(gè)或更多個(gè)、10個(gè)或更多個(gè)、12或更多個(gè)、15或更多個(gè)多核苷酸，例如4個(gè)或更多個(gè)多核苷酸彼此接合產(chǎn)生更長(zhǎng)的多核苷酸的反應(yīng)。在許多實(shí)施方式中，多核苷酸組裝反應(yīng)的產(chǎn)物，即“組裝的多核苷酸”或“合成子”應(yīng)當(dāng)包含一個(gè)拷貝的每種重疊序列。如本文所用，術(shù)語(yǔ)“在合適的反應(yīng)條件下孵育”是指保持反應(yīng)在合適的溫度和時(shí)間，以實(shí)現(xiàn)所需的結(jié)果，即多核苷酸組裝。適合用于本發(fā)明方法的酶和試劑的反應(yīng)條件是已知的(例如，如本文實(shí)施例中所述)，因此，本發(fā)明方法的合適的反應(yīng)條件可以很容易確定。這些反應(yīng)條件可以根據(jù)所用的酶而改變(例如，取決于其最佳溫度等)。如本文所用，術(shù)語(yǔ)“等溫”是指對(duì)于組裝進(jìn)行不要求主動(dòng)調(diào)節(jié)溫度的溫度條件。水浴或加熱塊溫度無(wú)關(guān)緊要的變化在術(shù)語(yǔ)等溫的含義范圍之內(nèi)。例如，術(shù)語(yǔ)“等溫”可以指反應(yīng)開始之后不要求熱變性步驟的反應(yīng)條件。更具體地，等溫方法不涉及熱循環(huán)，即在高于90℃的變性溫度和退火/延長(zhǎng)溫度之間的循環(huán)。等溫條件通常涉及在低于90℃的溫度下孵育一段時(shí)間(例如，5分鐘至12小時(shí)或更長(zhǎng)時(shí)間)。在一個(gè)實(shí)施方式中，等溫?cái)U(kuò)增反應(yīng)在30℃～75℃，例如40℃～60℃范圍內(nèi)的溫度下進(jìn)行。如本文所用，術(shù)語(yǔ)“接合”是指在兩個(gè)序列之間產(chǎn)生共價(jià)連接。如本文所用，術(shù)語(yǔ)“組合物”是指除了列出的那些之外，可以包含其它試劑例如甘油、鹽、dntp等的試劑的組合。組合物可以是任意形式的，例如，含水的或凍干的，并且可以是任意狀態(tài)的(例如，冷凍的或液體形式)。如本文所用，“載體”是片段或合成子可以整合到其中使得可以在宿主細(xì)胞中復(fù)制改造載體的合適的dna。線性化載體可以通過(guò)環(huán)狀載體的限制性核酸內(nèi)切酶消化或者通過(guò)pcr產(chǎn)生。片段和/或線性化載體的濃度可以通過(guò)凝膠電泳或其他方式測(cè)定。本文使用的任何一種或多種蛋白質(zhì)(例如，連接酶、ssbp、5’-3’核酸外切酶或聚合酶等)可以是溫度敏感的或熱穩(wěn)定的，其中，如本文所用，術(shù)語(yǔ)“溫度敏感”是指酶在65℃的溫度下10分鐘之后喪失其活性的至少95％，并且術(shù)語(yǔ)“熱穩(wěn)定”是指酶在65℃的溫度下10分鐘之后保持其活性的至少95％。具體實(shí)施方式在對(duì)各種實(shí)施方式進(jìn)行更詳細(xì)的說(shuō)明之前，應(yīng)當(dāng)理解到，本公開的教導(dǎo)不限于所述的具體實(shí)施方式，因此，其當(dāng)然可以變化。還應(yīng)當(dāng)理解到，本文所用的術(shù)語(yǔ)僅出于說(shuō)明具體實(shí)施方式的目的，而無(wú)意于進(jìn)行限定，因?yàn)楸景l(fā)明教導(dǎo)的范圍將僅由其權(quán)利要求書進(jìn)行限定。盡管本發(fā)明的教導(dǎo)結(jié)合各種實(shí)施方式進(jìn)行說(shuō)明，但無(wú)意于將本發(fā)明的教導(dǎo)限制于這些實(shí)施方式。相反，如本領(lǐng)域技術(shù)人員將會(huì)理解到，本發(fā)明的教導(dǎo)包括各種替代方式、修改方式和等同方式。在提供數(shù)值范圍時(shí)，應(yīng)當(dāng)理解到，位于該范圍上限和下限之間的每個(gè)中間值(每個(gè)中間值精確到下限的單位的十分之一，除非上下文中有清楚地相反表示)，以及任何其他所述的或在其所述范圍內(nèi)的中間值均包括在本公開以內(nèi)。盡管在實(shí)施或測(cè)試本發(fā)明教導(dǎo)內(nèi)容時(shí)也可以使用與本文所述的那些類似或等同的任何方法和材料，但是現(xiàn)對(duì)一些示例性方法和材料加以說(shuō)明。任何出版物的引用是出于其公開早于申請(qǐng)日，不應(yīng)當(dāng)理解成認(rèn)可本發(fā)明的權(quán)利要求由于在先發(fā)明而無(wú)權(quán)早于這些出版物。而且，所提供出版物的日期可能與實(shí)際的公開日期不同，其需要獨(dú)立地確認(rèn)。必須注意到，如本文和其權(quán)利要求中所用的，單數(shù)形式“一個(gè)”、“一種”和“該”包括復(fù)數(shù)的指示物，除非上下文中有清楚地相反表示。還注意到，權(quán)利要求可以撰寫成排除任何任選的要素。這樣，該聲明意在用作結(jié)合權(quán)利要求要素的引用使用這些排除性術(shù)語(yǔ)如“僅僅”、“僅有”等、或者使用“否定”限定的前提基礎(chǔ)。如閱讀本公開時(shí)對(duì)本領(lǐng)域技術(shù)人員將會(huì)顯而易見(jiàn)的是，本文說(shuō)明和例示的每個(gè)單獨(dú)的實(shí)施方式均具有獨(dú)立的組分和特征，其可以在不脫離本發(fā)明教導(dǎo)的范圍或精神的前提下很容易地與任何其他若干實(shí)施方式的特征分開或組合。任何所引用的方法均可以以所引用事件的順序或者以任何其他邏輯上可能的順序?qū)嵤Ｔ诙嗪塑账峤M中重疊的序列可以是任何合適的長(zhǎng)度，比如，2kb或更小，或者1kb或更小，或者小于900堿基、800堿基、700堿基、600堿基、500堿基、400堿基、300堿基、200堿基或100堿基。重疊區(qū)可以少至8個(gè)核苷酸。優(yōu)選地，重疊序列長(zhǎng)度在15個(gè)核苷酸～80個(gè)核苷酸范圍內(nèi)，例如，至多20、至多25、至多30、至多35、至多40、至多45、至多50、至多55、至多60、至多65、至多70、至多75或者至多80個(gè)核苷酸。例如，最小重疊長(zhǎng)度可以由tm定義，其優(yōu)選等于或大于48℃。合成的寡核苷酸和多核苷酸在其用于合成子組裝之前可以包含其合成過(guò)程中產(chǎn)生的錯(cuò)誤。為了在組裝之前校正這些錯(cuò)誤，期望進(jìn)行錯(cuò)配修復(fù)步驟。就此而言，已經(jīng)說(shuō)明了多種在組裝之前實(shí)現(xiàn)合成核酸的錯(cuò)配修復(fù)的方法。合成核酸的種群可以具有隨機(jī)錯(cuò)誤，使得制劑的變性和復(fù)性可以顯示錯(cuò)配。已經(jīng)從自然界分離出的蛋白質(zhì)，比如muthls、cel-1核酸酶、t7endo1、uvrd、t4endovii、大腸桿菌endov(參見(jiàn)us7,851,192和us8,048,664)，可以選擇性地與包含錯(cuò)配的dna雙鏈結(jié)合；在錯(cuò)配堿基處切割核酸；并任選地基于模板核苷酸序列用正確堿基替代。盡管本領(lǐng)域中有教導(dǎo)非鏈置換聚合酶必須與ss結(jié)合蛋白質(zhì)、5’-3’核酸外切酶和連接酶一起使用以組裝dna片段，本文出人意料地顯示，鏈置換聚合酶可以在發(fā)生鏈置換的條件下使用，并且在起始多核苷酸片段出人意料的低濃度下有效，以有效地由多個(gè)片段產(chǎn)生單個(gè)核酸。可以用于本發(fā)明的組裝混合物、組合物、試劑盒或方法的實(shí)施方式中的鏈置換聚合酶的實(shí)例包括b族聚合酶成員，比如(但不限于)表1中所確認(rèn)的任意者(seqidno:33至seqidno:55)。此外，可以使用這些聚合酶的融合，例如，在多種聚合酶和/或ss結(jié)合結(jié)構(gòu)域(比如，如表2所示)(seqidno:56至seqidno:97)之間的融合。在實(shí)施方式中，表1中的任何聚合酶部分或者與表1中任意這些蛋白質(zhì)部分的氨基酸序列同一性為至少80％、85％、90％、95％、98％、99％或100％的蛋白質(zhì)均可以在n-末端處或c-末端處與表2中所述的任何dna結(jié)合結(jié)構(gòu)域或者與表2中任意dna結(jié)合部分的氨基酸序列同一性為至少80％、85％、90％、95％、98％、99％或100％的蛋白質(zhì)的部分來(lái)融合，以形成本文使用的鏈置換融合聚合酶。dna結(jié)合結(jié)構(gòu)域可以任選地融合在聚合酶的n-末端或c-末端。也可以使用如通過(guò)本文提供的試驗(yàn)(參見(jiàn)，例如圖1a～1e和實(shí)施例1)確定顯示為鏈置換性的其他聚合酶變體或新的分離物。這些來(lái)源發(fā)現(xiàn)的聚合酶的序列通過(guò)genbank很容易獲得。由于鏈置換序列的高度保守性，任何與這些野生型聚合酶的氨基酸序列同一性為80％、85％、90％或95％的變體可以預(yù)計(jì)具有鏈置換特性，其可以在預(yù)選擇的緩沖劑中在實(shí)施例1提供的試驗(yàn)中快速并容易地驗(yàn)證，而無(wú)需過(guò)度實(shí)驗(yàn)。在一個(gè)實(shí)施方式中，本發(fā)明的反應(yīng)混合物、組合物、試劑盒或方法包括或使用與seqidno:1或seqidno:102的序列同一性為至少80％、85％、90％、95％、98％、99％或100％(例如，與seqidno:1或seqidno:102的序列同一性為100％)的鏈置換聚合酶。在另一個(gè)實(shí)施方式中，本發(fā)明的反應(yīng)混合物、組合物、試劑盒或方法包括或使用結(jié)合結(jié)構(gòu)域與seqidno:2的序列同一性為至少80％、85％、90％、95％、98％、99％或100％(例如，與seqidno:2的序列同一性為100％)的聚合酶。在另一個(gè)實(shí)施方式中，本發(fā)明的反應(yīng)混合物、組合物、試劑盒或方法包括或使用與seqidno:1或seqidno:102并且與seqidno:2或seqidno:3或seqidno:33至seqidno:97中任意者的序列同一性為至少80％、85％、90％、95％、98％、99％或100％(例如，與seqidno:1或seqidno:102并且與seqidno:2或seqidno:3或seqidno:33至seqidno:97中任意者的序列同一性為100％)的聚合酶。這些組合物可以在其中聚合酶為鏈置換性的反應(yīng)條件下使用。組合物可以在其中任何與聚合酶活性有關(guān)的3’-5’核酸外切酶活性是有活性的反應(yīng)條件下使用。當(dāng)在反應(yīng)中使用限制性酶比如noti時(shí)這一點(diǎn)會(huì)有幫助。在該情形中，3’-5’核酸外切酶可以除去雙鏈3’-末端上的側(cè)翼序列。然而，如果使用在切除的片段上產(chǎn)生平端的限制性核酸內(nèi)切酶，則可以不要求有3’-5’核酸外切酶活性。組裝反應(yīng)可以在等溫條件下進(jìn)行。在一個(gè)實(shí)施方式中，等溫條件為50℃。表1.聚合酶列表methanocaldococcusvulcaniusm7sp-13gi|502573182seqidno:33archaeoglobusfulgidusdsm4304sp-16gi|499180464seqidno:34archaeoglobusprofundusdsm5631sp-17gi|502704426seqidno:35caldicellulosiruptorhydrothermalis108sp-19gi|503168530seqidno:36desulfurococcusmucosusdsm2162sp-27gi|503328138seqidno:37pyrolobusfumariisp-29gi|503791850seqidno:38pyrobaculumoguniensechsp-30gi|379003208seqidno:39staphylothermusmarinusf1sp-33gi|500164563seqidno:40pyrococcusyayaosiich1sp-42gi|503672202seqidno:41thermococcussp.am4-delsp-43gi|503888003seqidno:42thermococcushydrothermalissp-44gi|17375628seqidno:43thermococcusthioreducenssp-45gi|117958105seqidno:44thermococcuswaiotapuensissp-46gi|378813034seqidno:45thermococcussibiricusmm739sp-47gi|506329477seqidno:46pyrococcusglycovoranssp-48gi|7288074seqidno:47pyrococcussp.na2sp-49gi|503513858seqidno:48ferroglobusplacidusdsm10642sp-61gi|502730992seqidno:49palaeococcusferrophilusdsm13482sp-5gi|851288004seqidno:50thermococcusgammatoleransej3sp-50gi|506339349seqidno:51thermococcuscelericrescenssp-51gi|332308985seqidno:52vulcanisaetadistributadsm14429sp-60gi|503101260seqidno:53methanopyruskandleriav19sp-7gi|20094475seqidno:54thermoproteusneutrophilusv24stasp-9gi|171185774seqidno:55表2.dna結(jié)合蛋白質(zhì)在本發(fā)明的實(shí)施方式中，反應(yīng)混合物、組合物、試劑盒或方法可以包括或使用5’-3’核酸外切酶，比如t5/5’-3’核酸外切酶，其對(duì)溫度敏感，并可以通過(guò)將溫度升高到50℃以上失活。在一個(gè)實(shí)施方式中，5’-3’核酸外切酶具有核酸外切酶活性以及ss核酸內(nèi)切酶活性。在一些實(shí)施方式中，反應(yīng)混合物還可以包括連接酶，例如要求nad+的連接酶和/或熱穩(wěn)定性連接酶，例如taq連接酶。在優(yōu)選的實(shí)施方式中，反應(yīng)混合物可以包括ss結(jié)合蛋白質(zhì)。ss結(jié)合蛋白質(zhì)可以是熱穩(wěn)定性的，例如，etssb。組裝反應(yīng)可以在等溫條件下進(jìn)行。在某些實(shí)施方式中，任選使用連接酶。例如，當(dāng)將組裝的片段直接導(dǎo)入到載體中用于宿主細(xì)胞轉(zhuǎn)化時(shí)，不需要有連接酶，因?yàn)樗拗骷?xì)胞比如大腸桿菌能夠在體內(nèi)修復(fù)切口。然而，如果出于在轉(zhuǎn)化之前確認(rèn)正確組裝的目的對(duì)組裝的片段進(jìn)行擴(kuò)增，則期望使用連接酶來(lái)封閉切口，并且使聚合酶能夠擴(kuò)增整個(gè)靶標(biāo)dna。單個(gè)片段的克隆可以使用其序列得自任何數(shù)據(jù)庫(kù)或出版物的化學(xué)合成的多核苷酸片段，其中多核苷酸片段具有重疊序列。通過(guò)將多核苷酸插入到質(zhì)粒中鄰近適用于切割所插入多核苷酸的限制性內(nèi)切酶位點(diǎn)的位點(diǎn)中，可以在質(zhì)粒中克隆這些片段?？梢允褂萌魏钨|(zhì)粒。本實(shí)施例使用包含氯霉素基因作為選擇標(biāo)志物的市售pacyc184?？梢允褂萌魏芜x擇標(biāo)志物代替氯霉素抗性基因。類似地，可以選擇任何切割酶的特異性識(shí)別位點(diǎn)，只要該切割酶能夠在寡核苷酸末端特異性切割以產(chǎn)生交錯(cuò)末端或平端，其中除了鄰近所關(guān)注的片段的末端的工程化的位置以外，特異性切割位點(diǎn)不發(fā)生在所關(guān)注的片段中。在本實(shí)施例中，已經(jīng)通過(guò)dna合成的方法將產(chǎn)生交錯(cuò)末端的8堿基切割者noti的識(shí)別位點(diǎn)(cgccggcg)引入鄰近于所關(guān)注的多核苷酸。但是，該位點(diǎn)可以存在于所選擇的質(zhì)粒中，或者通過(guò)擴(kuò)增引物加在所關(guān)注的合成寡核苷酸上。特異性切割酶的實(shí)例包括限制性核酸內(nèi)切酶和歸巢(homing)核酸內(nèi)切酶。一旦所關(guān)注的寡核苷酸或dna片段已經(jīng)化學(xué)合成或由現(xiàn)有dna克隆或擴(kuò)增并克隆到具有選擇標(biāo)志物的載體中，則優(yōu)選通過(guò)酶切割將其切除。然后將已經(jīng)合成或擴(kuò)增以便包括與其要接合的相鄰片段或寡核苷酸的重疊序列的片段或寡核苷酸在組裝反應(yīng)中組裝。在所選擇的雜交條件下，反應(yīng)混合物中的5’-3’核酸外切酶(例如，濃度范圍0.004-0.016u/μl)回切(chewback)片段或寡核苷酸5’末端處的任意ss區(qū)，并繼續(xù)回切經(jīng)過(guò)重疊序列區(qū)，并可以進(jìn)一步繼續(xù)有限的距離(例如，至少100堿基)，以提供3’ss區(qū)(參見(jiàn)，例如，圖2a-2c和圖7)。同時(shí)，通過(guò)圖1a-1e和實(shí)施例1的試驗(yàn)確定的鏈置換聚合酶(例如，濃度范圍0.005u/μl-0.5u/μl)修復(fù)雜交的ds區(qū)和任何殘余ss區(qū)之間殘留的間隙。由于聚合酶是鏈置換性的，其可以置換額外的下游序列，以形成ss側(cè)翼。但是，t5核酸外切酶的ss核酸內(nèi)切酶活性將會(huì)除去該側(cè)翼，并且任何相關(guān)的切口均可以通過(guò)連接酶(例如，濃度范圍0.001u/μl-20u/μl)修復(fù)。片段一經(jīng)組裝到更大一條dna中，將其在選擇性壓力下在宿主細(xì)胞菌落中克隆，來(lái)自這些菌落的dna可以從載體中釋放，并再次與其他片段組裝，并轉(zhuǎn)化到宿主細(xì)胞中，從而多次延長(zhǎng)dna的大小。宿主細(xì)胞可以是感受態(tài)細(xì)菌細(xì)胞，或者可以是酵母細(xì)胞或其它真核細(xì)胞。本文所述的組裝方法經(jīng)發(fā)現(xiàn)非常有效。例如，可以使用0.02nm-100nm寡核苷酸(ss)或dna片段(ds)來(lái)組裝更大的片段，其中反應(yīng)中使用的ss寡核苷酸的濃度可以比類似組裝反應(yīng)中使用的dsdna片段的量高至多大約50倍。類似地，可以使用等摩爾濃度的包含單一片段和選擇標(biāo)志物的質(zhì)粒和類似量的包含組裝片段的具有不同選擇標(biāo)志物的載體。這些量意在用于指導(dǎo)，但無(wú)論組裝效率是否提高，這些量均可以降低。例如，如通過(guò)使用lac1z作為指示物的組裝體的菌落數(shù)目所測(cè)定，加入鉀鹽kcl能夠?qū)a(chǎn)生組裝體的效率增加1.5倍(參見(jiàn)，例如，圖5)。用于兩個(gè)dsdna分子之間或者導(dǎo)入線性化載體的ss靶標(biāo)寡核苷酸的組裝過(guò)程也是非常高效的。本文提供了一個(gè)無(wú)意于限定的實(shí)例，其使用特定/隨機(jī)序列來(lái)識(shí)別可引入到細(xì)胞中以確定表型改變的crispr-cas基因編輯方案的向?qū)na。最初，什么序列可以適合于實(shí)現(xiàn)該目的可能并不知曉。包含簡(jiǎn)并序列的文庫(kù)的產(chǎn)生使得該類型的分析成為可能?；赾rispr/cas-9的基因編輯在基因組編輯領(lǐng)域迅速普及。由于最常用的包含cas9的質(zhì)粒的尺寸，將sgrna或sgrna文庫(kù)構(gòu)建至cas9/sgrna表達(dá)載體中會(huì)很繁瑣。使用ssdna寡核苷酸，該方法解決了這一問(wèn)題。在單獨(dú)的實(shí)施方式中，可以將表2的任何dna結(jié)合結(jié)構(gòu)域與bst聚合酶、bst大片段或其突變體融合(參見(jiàn)，例如，us8,993,298和us2015/0152396，其包括本文描述并要求保護(hù)的所有bst變體)。試劑盒本公開另外提供用于實(shí)施上述方法的試劑盒。在某些實(shí)施方式中，試劑盒可以包含：i.5’-3’核酸外切酶，ii.任選的連接酶，iii.鏈置換聚合酶：和iv.ssdna結(jié)合蛋白質(zhì)。試劑盒組分可以合并在一容器中，或者每個(gè)組分可以在其各自的容器中。例如，試劑盒組分可以合并在單個(gè)反應(yīng)管中、或者一個(gè)或多個(gè)不同的反應(yīng)管中。上文描述了該試劑盒組分進(jìn)一步的細(xì)節(jié)。根據(jù)如何實(shí)施所述方法，試劑盒還可以包含上文和下文中所述的可以用于該方法的其他試劑，例如，錯(cuò)配修復(fù)酶，比如muthls、cel-1核酸酶、t7endo1、uvrd、t4endovii、大腸桿菌endov、緩沖劑、dntp、向其中插入合成子的質(zhì)粒和/或接收質(zhì)粒的感受態(tài)細(xì)胞、對(duì)照物等。在一些實(shí)施方式中，試劑盒不含非鏈置換聚合酶和/或擁擠劑。除上述組分以外，試劑盒還包括使用試劑盒組分以實(shí)施本方法的說(shuō)明書。用于實(shí)施本方法的說(shuō)明書通常記錄在合適的記錄介質(zhì)上。例如，說(shuō)明書可以打印在比如紙或塑料等的介質(zhì)上。這樣，說(shuō)明書可以作為包裝說(shuō)明書存在于試劑盒中、在試劑盒或其組分的容器標(biāo)簽中(即，與包裝或分包裝關(guān)聯(lián))等。在其他實(shí)施方式中，說(shuō)明書作為電子存儲(chǔ)數(shù)據(jù)文件存在，存在于合適的計(jì)算機(jī)可讀存儲(chǔ)介質(zhì)中，例如，cd-rom、軟盤等。在又一其他的實(shí)施方式中，實(shí)際的說(shuō)明書不存在于試劑盒中，而是提供用于從遠(yuǎn)程資源例如經(jīng)由互聯(lián)網(wǎng)獲得的方法。該實(shí)施方式的實(shí)例為包括可以瀏覽說(shuō)明書和/或可以從其下載說(shuō)明書的網(wǎng)絡(luò)地址的試劑盒。就說(shuō)明書而言，獲取說(shuō)明書的這一方法記錄在合適的基質(zhì)上。本文所述的用于組裝片段并形成合成子的組合物、試劑盒和方法產(chǎn)生ds完全密封dna的產(chǎn)物，其可以用作pcr、rca的模板、或許多種其他分子生物學(xué)應(yīng)用，包括直接轉(zhuǎn)化感受態(tài)細(xì)菌或轉(zhuǎn)染真核宿主細(xì)胞。為了進(jìn)一步說(shuō)明本發(fā)明，提供以下具體實(shí)施例，應(yīng)理解到其提供是為了對(duì)本發(fā)明進(jìn)行說(shuō)明，而不應(yīng)理解成以任何方式限定其范圍。本文引用的所有參考文獻(xiàn)包括2014年8月27日提交的美國(guó)臨時(shí)申請(qǐng)第62/042,527號(hào)、2015年7月7日提交的第62/189,599號(hào)加上2015年7月16日提交的第62/193,168號(hào)，將其并入以供參考。實(shí)施例實(shí)施例1：確定聚合酶鏈置換特性的試驗(yàn)開發(fā)一種區(qū)分鏈置換和非鏈置換聚合酶的試驗(yàn)——制備包含10nmfam-引物/模板/封閉寡核苷酸、緩沖液(newenglandbiolabs,ipswich,ma)(圖1a)和0.1mmdntp的10μl反應(yīng)物。圖1b是作為對(duì)照的不存在任何聚合酶的fam標(biāo)記引物。將鏈置換dna聚合酶加入到反應(yīng)物中，并在50℃下與稀釋10倍的1μl樣品一起孵育30分鐘，并通過(guò)毛細(xì)管電泳進(jìn)行分析，fam引物通過(guò)被置換的封閉寡核苷酸得以延長(zhǎng)。結(jié)果示出于圖1d-1e中。圖2d中確定的鏈置換聚合酶bst聚合酶的峰位置與非天然聚合酶spb49f觀察到的峰對(duì)應(yīng)。少量遷移的原因在于3’-5’核酸外切酶活性引起的spb49f產(chǎn)生的平端，這在bst聚合酶中并不存在，使得bst聚合酶復(fù)制的產(chǎn)物具有3’da。圖1c示出了非鏈置換聚合酶-t4dna聚合酶的產(chǎn)物，其中合成在封閉引物處終止。實(shí)施例2：使用鏈置換聚合酶由6片段合成大的dna分子并使用鏈置換聚合酶確認(rèn)組裝有效使用pcr克隆試劑盒(newenglandbiolabs,ipswich,ma)分別由pcr產(chǎn)物(片段(frags)1、2、3、4、5，其一起覆蓋lacl-和lacz基因區(qū))構(gòu)建質(zhì)粒a、b、c、d和e。在該實(shí)驗(yàn)中，使用以下濃度的整合到單獨(dú)質(zhì)粒中的5個(gè)不同片段：50ng的各pcr(“片段”來(lái)源)，和25ng的包含氨芐青霉素的質(zhì)粒pminittm載體(neb#e1202)。首先使用pcr對(duì)5種用于組裝的片段進(jìn)行擴(kuò)增。制備lacl-laczdna片段組裝系統(tǒng)中使用的引物如下：5placiz-pacyc184vf1ttggtctggtgtcaaaaatgaatcgtcacggcgatttatg(seqidno：4)5placiz-pacyc184vr1gggtcattttcggcgaggactgcatcaacgcatatagcg(seqidno：5)not-izf1gcggccgcgtcctcgccgaaaatgacccagag(seqidno：6)not-izr1gcggccgctggtgtcgatggtagaacgaagcg(seqidno：7)not-izf2gcggccgccccactgacgcgttgcgcgagaag(seqidno：8)not-izr2gcggccgcggctgcgcaactgttgggaagggc(seqidno：9)not-izf3gcggccgctgcagcacatccccctttcgccag(seqidno：10)not-izr3gcggccgcatgatgctcgtgacggttaacgcc(seqidno：11)not-izf4gcggccgcaggtgcggattgaaaatggtctgc(seqidno：12)not-izr4gcggccgctcaccgcttgccagcggcttacca(seqidno：13)not-izf5gcggccgcgaatacctgttccgtcatagcgat(seqidno：14)not-izr5gcggccgctcatttttgacaccagaccaactgg(seqidno：15)將擴(kuò)增的片段克隆并測(cè)序，以確認(rèn)擴(kuò)增過(guò)程中沒(méi)有引入錯(cuò)誤。pcr擴(kuò)增的片段1的序列(seqidno：16)：gcggccgcgtcctcgccgaaaatgacccagagcgctgccggcacctgtcctacgagttgcatgataaagaagacagtcataagtgcggcgacgatagtcatgccccgcgcccaccggaaggagctgactgggttgaaggctctcaagggcatcggtcgagatcccggtgcctaatgagtgagctaacttacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgccagggtggtttttcttttcaccagtgagacgggcaacagctgattgcccttcaccgcctggccctgagagagttgcagcaagcggtccacgctggtttgccccagcaggcgaaaatcctgtttgatggtggttaacggcgggatataaccaacgcgcagcccggactcggtaatatcccactaccgagatatccgcaccaacgcgcagcccggactcggtaatggcgcgcattgcgcccagcgccatctgatcgttggcaaccagcatcgcagtgggaacgatgccctcattcagcatttgcatggtttgttgaaaaccggacatggcactccagtcgccttcccgttccgctatcggctgaatttgattgcgagtgagatatttatgccagccagccagacgcagacgcgccgagacagaacttaatgggcccgctaacagcgcgatttgctggtgacccaatgcgaccagatgctccacgcccagtcgcgtaccgtcttcatgggagaaaataatactgttgatgggtgtctggtcagagacatcaagaaataacgccggaacattagtgcaggcagcttccacagcaatggcatcctggtcatccagcggatagttaatgatcagcccactgacgcgttgcgcgagaagattgtgcaccgccgctttacaggcttcgacgccgcttcgttctaccatcgacaccagcggccgcpcr擴(kuò)增的片段2的序列(seqidno:17)：gcggccgccccactgacgcgttgcgcgagaagattgtgcaccgccgctttacaggcttcgacgccgcttcgttctaccatcgacaccaccacgctggcacccagttgatcggcgcgagatttaatcgccgcgacaatttgcgacggcgcgtgcagggccagactggaggtggcaacgccaatcagcaacgactgtttgcccgccagttgttgtgccacgcggttgggaatgtaattcagctccgccatcgccgcttccactttttcccgcgttttcgcagaaacgtggctggcctggttcaccacgcgggaaacggtctgataagagacaccggcatactctgcgacatcgtataacgttactggtttcacattcaccaccctgaattgactctcttccgggcgctatcatgccataccgcgaaaggttttgcgccattcgatggtgtccgggatctcgacgctctcccttatgcgactcctgcattaggaagcagcccagtagtaggttgaggccgttgagcaccgccgccgcaaggaatggtgcatgcaaggagatggcgcccaacagtcccccggccacggggcctgccaccatacccacgccgaaacaagcgctcatgagcccgaagtggcgagcccgatcttccccatcggtgatgtcggcgatataggcgccagcaaccgcacctgtggcgccggtgatgccggccacgatgcgtccggcgtagaggatcgagatctcgatcccgcgaaattaatacgactcactataggggaattgtgagcggataacaattcccctctagaaataattttgtttaactttaagaaggagatatacatatgaccatgattacggattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagccgcggccgcpcr片段3的序列(seqidno:18)：gcggccgctgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgctttgcctggtttccggcaccagaagcggtgccggaaagctggctggagtgcgatcttcctgaggccgatactgtcgtcgtcccctcaaactggcagatgcacggttacgatgcgcccatctacaccaacgtgacctatcccattacggtcaatccgccgtttgttcccacggagaatccgacgggttgttactcgctcacatttaatgttgatgaaagctggctacaggaaggccagacgcgaattatttttgatggcgttaactcggcgtttcatctgtggtgcaacgggcgctgggtcggttacggccaggacagtcgtttgccgtctgaatttgacctgagcgcatttttacgcgccggagaaaaccgcctcgcggtgatggtgctgcgctggagtgacggcagttatctggaagatcaggatatgtggcggatgagcggcattttccgtgacgtctcgttgctgcataaaccgactacacaaatcagcgatttccatgttgccactcgctttaatgatgatttcagccgcgctgtactggaggctgaagttcagatgtgcggcgagttgcgtgactacctacgggtaacagtttctttatggcagggtgaaacgcaggtcgccagcggcaccgcgcctttcggcggtgaaattatcgatgagcgtggtggttatgccgatcgcgtcacactacgtctgaacgtcgaaaacccgaaactgtggagcgccgaaatcccgaatctctatcgtgcggtggttgaactgcacaccgccgacggcacgctgattgaagcagaagcctgcgatgtcggtttccgcgaggtgcggattgaaaatggtctgctgctgctgaacggcaagccgttgctgattcgaggcgttaaccgtcacgagcatcatgcggccgcpcr片段4的序列(seqidno:19)：gcggccgcaggtgcggattgaaaatggtctgctgctgctgaacggcaagccgttgctgattcgaggcgttaaccgtcacgagcatcatcctctgcatggtcaggtcatggatgagcagacgatggtgcaggatatcctgctgatgaagcagaacaactttaacgccgtgcgctgttcgcattatccgaaccatccgctgtggtacacgctgtgcgaccgctacggcctgtatgtggtggatgaagccaatattgaaacccacggcatggtgccaatgaatcgtctgaccgatgatccgcgctggctaccggcgatgagcgaacgcgtaacgcgaatggtgcagcgcgatcgtaatcacccgagtgtgatcatctggtcgctggggaatgaatcaggccacggcgctaatcacgacgcgctgtatcgctggatcaaatctgtcgatccttcccgcccggtgcagtatgaaggcggcggagccgacaccacggccaccgatattatttgcccgatgtacgcgcgcgtggatgaagaccagcccttcccggctgtgccgaaatggtccatcaaaaaatggctttcgctacctggagagacgcgcccgctgatcctttgcgaatacgcccacgcgatgggtaacagtcttggcggtttcgctaaatactggcaggcgtttcgtcagtatccccgtttacagggcggcttcgtctgggactgggtggatcagtcgctgattaaatatgatgaaaacggcaacccgtggtcggcttacggcggtgattttggcgatacgccgaacgatcgccagttctgtatgaacggtctggtctttgccgaccgcacgccgcatccagcgctgacggaagcaaaacaccagcagcagtttttccagttccgtttatccgggcaaaccatcgaagtgaccagcgaatacctgttccgtcatagcgataacgagctcctgcactggatggtggcgctggatggtaagccgctggcaagcggtgagcggccgcpcr片段5的序列(seqidno:20)：gcggccgcgaatacctgttccgtcatagcgataacgagctcctgcactggatggtggcgctggatggtaagccgctggcaagcggtgaagtgcctctggatgtcgctccacaaggtaaacagttgattgaactgcctgaactaccgcagccggagagcgccgggcaactctggctcacagtacgcgtagtgcaaccgaacgcgaccgcatggtcagaagccgggcacatcagcgcctggcagcagtggcgtctggcggaaaacctcagtgtgacgctccccgccgcgtcccacgccatcccgcatctgaccaccagcgaaatggatttttgcatcgagctgggtaataagcgttggcaatttaaccgccagtcaggctttctttcacagatgtggattggcgataaaaaacaactgctgacgccgctgcgcgatcagttcacccgtgcaccgctggataacgacattggcgtaagtgaagcgacccgcattgaccctaacgcctgggtcgaacgctggaaggcggcgggccattaccaggccgaagcagcgttgttgcagtgcacggcagatacacttgctgatgcggtgctgattacgaccgctcacgcgtggcagcatcaggggaaaaccttatttatcagccggaaaacctaccggattgatggtagtggtcaaatggcgattaccgttgatgttgaagtggcgagcgatacaccgcatccggcgcggattggcctgaactgccagctggcgcaggtagcagagcgggtaaactggctcggattagggccgcaagaaaactatcccgaccgccttactgccgcctgttttgaccgctgggatctgccattgtcagacatgtataccccgtacgtcttcccgagcgaaaacggtctgcgctgcgggacgcgcgaattgaattatggcccacaccagtggcgcggcgacttccagttcaacatcagccgctacagtcaacagcaactgatggaaaccagccatcgccatctgctgcacgcggaagaaggcacatggctgaatatcgacggtttccatatggggattggtggcgacgactcctggagcccgtcagtatcggcggaattccagctgagcgccggtcgctaccattaccagttggtctggtgtcaaaaatgagcggccgc按設(shè)計(jì)的最終組裝的順序(片段1和2之間、2和3之間、3和4之間、4和5之間)，5個(gè)片段各自與相鄰片段具有80bp的重疊區(qū)。片段1和5還與載體末端共享20bp重疊。可以使用任何可用的載體，例如，pacyc184(newenglandbiolabs,ipswich,ma)。通過(guò)反向pcr制備pacyc184載體，其使得可以在用(newenglandbiolabs,ipswich,ma)處理和熱滅活之后在上述組裝混合物的存在下進(jìn)行片段1-5的組裝(參見(jiàn)圖2a-2c)。在組裝過(guò)程中，從陰影區(qū)域延伸的核苷酸通過(guò)t5核酸外切酶降解，同時(shí)灰色顯示的核苷酸通過(guò)聚合酶除去。片段組裝并轉(zhuǎn)化到大腸桿菌中之后，在具有iptg和x-gal的平板上記錄通過(guò)藍(lán)色/白色選擇確定的富有成效組裝。將t5核酸外切酶、taq連接酶、鏈置換dna聚合酶和ss結(jié)合蛋白質(zhì)(etssb)在緩沖液中合并在反應(yīng)混合物中，以形成mix1。這些酶均獲自newenglandbiolabs,ipswich,ma。將5種150ngnoti-hf-消化的質(zhì)粒(質(zhì)粒a、b、c、d和e)與105ng載體以及與mix1或gamm混合成總體積20μl。將反應(yīng)物在50℃下孵育60分鐘。使用2μl組裝的產(chǎn)物轉(zhuǎn)化到neb5-α(newenglandbiolabs,ipswich,ma)感受態(tài)細(xì)胞中。然后將細(xì)胞涂布在包含氯霉素的平板上。陽(yáng)性組裝體可以在具有氯霉素+iptg+x-gal的平板上作為藍(lán)色菌落識(shí)別出，并在37℃下孵育過(guò)夜。轉(zhuǎn)化之前確認(rèn)所有片段均接合并連接的組裝產(chǎn)物的pcr包括以下步驟：在確保5片段和載體連接在一起的pcr中使用1μl組裝產(chǎn)物。使用在載體上退火的成對(duì)pcr引物擴(kuò)增整個(gè)組裝的laclz基因(5.3kb)。條帶1和2是復(fù)制pcr結(jié)果。條帶m是來(lái)自newenglandbiolabs,ipswich,ma的2-logdna梯狀帶(參見(jiàn)圖4)。測(cè)序結(jié)果獲自選取的8個(gè)菌落，并且出于sanger測(cè)序的目的將質(zhì)粒dna提純。使用6個(gè)引物對(duì)4.8kb片段進(jìn)行測(cè)序。片段之間的接合序列以及從重疊區(qū)的延長(zhǎng)區(qū)均顯示出小于2％的序列錯(cuò)誤。用于對(duì)組裝的dna進(jìn)行測(cè)序的引物：實(shí)施例3：將單鏈寡核苷酸組裝到線性化載體或兩個(gè)不同的dsdna中與用于靶向智人(h.sapiens)的基因的sgrna對(duì)應(yīng)的寡核苷酸設(shè)計(jì)如下：1.篩選具有所需靶標(biāo)序列的pam序列。例如，以下中的ngg5’gcgaagaacctcttcccaagangg3’(seqidno:27)2.設(shè)計(jì)包含兩側(cè)為部分u6啟動(dòng)子序列和骨架rna序列的21核苷酸靶標(biāo)序列的71-堿基ssdna寡核苷酸。參見(jiàn)例如圖8a-c，其中ss寡核苷酸確定為：5’atcttgtggaaaggacgaaacaccggcgaagaacctcttcccaagagttttagagctagaaatagcaagtt3’(seqidno:28)或者圖9a-c，其中ss寡核苷酸設(shè)計(jì)成產(chǎn)生以下隨機(jī)文庫(kù)：5’atcttgtggaaaggacgaaacaccgn21gttttagagctagaaatagcaagtt3’(seqidno:29)3.在1×nebuffer2(newenglandbiolabs,ipswich,ma)中制備ssdna寡核苷酸，最終濃度為0.2μm。4.形成包含5μlssdna寡核苷酸(0.2μm)、30ng限制性內(nèi)切酶-線性化載體和ddh2o的10μl反應(yīng)混合物。5.適合用于上述方法的載體是來(lái)自lifetechnology的ds載體(具有ofp報(bào)告子的crispr核酸酶載體試劑盒，目錄號(hào)：a21174)。其他載體如質(zhì)粒#42230(px330-u6-chimeric_bb-cbh-hspcas9)由addgene提供(具體參見(jiàn)https://www.addgene.org/42230/)。另外可選地，可以使用在u6啟動(dòng)子的控制下包含sgrna骨架的任意質(zhì)粒。6.將包含ss結(jié)合蛋白質(zhì)、連接酶、核酸外切酶和聚合酶的10μl主體混合物加入到反應(yīng)混合物中，并將組裝反應(yīng)物在50℃下孵育1小時(shí)。7.將neb10-β感受態(tài)大腸桿菌遵照制制造商(newenglandbiolabs)的方案用2μl組裝產(chǎn)物轉(zhuǎn)化。8.將100μl轉(zhuǎn)化的細(xì)胞涂布在具有氨芐青霉素抗生素的平板上，并在37℃下孵育過(guò)夜。9.選取10個(gè)菌落使其生長(zhǎng)，并對(duì)質(zhì)粒dna進(jìn)行提純用于測(cè)序。與常規(guī)的必須合成兩種寡核苷酸并將其再退火的克隆方法不同，該實(shí)施例提供一種簡(jiǎn)單的方式來(lái)設(shè)計(jì)寡核苷酸并將其用所需載體進(jìn)行組裝，其相對(duì)于常規(guī)方法表現(xiàn)出顯著的改善，特別是在節(jié)約時(shí)間、便于使用和成本方面。seqidno:1mildadyitedgkpiirlfkkengrfkveydrnfrpyiyallkddsaiddvrkitserhgkvvrvidvekvkkkflgrpievwklyfehpqdvpamrdkirehpavidifeydipfakrylidkglipmegneeltflavdietlyhegeefgkgpiimisyadeegakvitwkkidlpyvevvaneremikrlikvirekdpdviityngdnfdfpyllkraeklgmklplgrdnsepkmqrlgdslaveikgrihfdlfpvirrtinlptytleavyeaifgkqkekvypheiaeawetgkglervakysmedakvtyelgkeffpmeaqlarlvgqplwdvsrsstgnlvewyllrkayernelapnkpdereyerrlresyeggyvkeperglwegivsldfrslypsiiithnvspdtlnkegcgeydeapevghrfckdfpgfipsllgslleerqkikkrmkeskdpverklldyrqraikilansfygyygyakarwyckecaesvtawgrqyielvrreleergfkvlyidtdglyatipgeknweeikrralefvnyinsklpgileleyegfytrgffvtkkkyalideegkivtrgleivrrdwseiaketqakvleailkhgnveeavkivkevteklsnyeipveklviyeqitrplneykaigphvavakrlaakgikikpgmvigyvvlrgdgpiskraiaieefdgkkhkydaeyyienqvlpaverilkafgykredlrwqktkqvglgawlkvkksseqidno:2iinpqarltpleleileiikqkksititeikeilserrkseyplslvseyisrlerkgyvkkiakgrkkfvealiseqidno:3mildadyitedgkpiirlfkkengrfkveydrnfrpyiyallkddsaiddvrkitserhgkvvrvidvekvkkkflgrpievwklyfehpqdvpamrdkirehpavidifeydipfakrylidkglipmegneeltflavdietlyhegeefgkgpiimisyadeegakvitwkkidlpyvevvaneremikrlikvirekdpdviityngdnfdfpyllkraeklgmklplgrdnsepkmqrlgdslaveikgrihfdlfpvirrtinlptytleavyeaifgkqkekvypheiaeawet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