固定化MutS介导的DNA合成错误的纠正及规模化基因合成
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摘要
应用芯片合成寡核苷酸进行基因的合成涉及成千上万条寡核苷酸。然而芯片合成的寡核苷酸文库中每条寡核苷酸的合成量很少,文库的合成精度相对较差,并且作为一混合物的合成文库成分很复杂。这些都限制了该技术的应用。因此,芯片合成寡核苷酸的广泛应用,需要在其合成准确度以及合成规模上有所推进。该研究中,我们建立了一种高通量纠错方法用于纠正芯片合成寡核苷酸文库及其组装基因中的合成错误。该方法利用固定化错配结合蛋白MutS的纤维素柱对寡核苷酸,尤其是芯片合成的寡核苷酸进行错误纠正,从而提高合成DNA的质量。
首先,我们建立了一种新的合成DNA错误纠正方法。该方法中,合成DNA中的错误合成的碱基通过变性退火形成错配,从而暴露合成错误的位点。含有错配的错误合成的DNA双链被固定在纤维素柱上的错配结合蛋白MutS特异性结合而滞留在柱子中,从而被移除。然后,以收集的穿透液为模板,通过PCR扩增出错误移除的DNA。重复该纠错过程可以进一步的提高合成DNA的精度。并且,为了提高固定化MutS的纤维素凝胶柱的错配结合能力,我们同时将具有不同错配结合能力的分别来源于水生栖热菌和大肠杆菌的MutS固定化到纤维素凝胶柱上,用于DNA合成错误的移除。在该研究中,为了构建一种更加高效,廉价,易于操作且高通量的DNA合成错误纠正方法。我们在寡核苷酸组装成基因片段之前,直接对芯片合成的寡核苷酸进行纠错。使用该种纠错方法,我们将合成的增强型绿色荧光蛋白基因表达菌的荧光克隆的比率由处理前的0.93%提高到处理后的83.22%,相应的合成基因的突变率降低了24.87倍,最终的突变率降低为1/2159bp。
另外,使用固定化MutS的纤维素凝胶柱,我们还设计验证了四种具有不同纠错规模的高通量错误纠正策略。前三种高通量纠错策略用于多基因的构建(如基因,代谢通路,或长链DNA构建物的构建模块)。为了降低下游基因组装过程的难度,合成的寡核苷酸文库首先通过在序列两端加入不同的引物对将寡核苷酸文库进行扩增分割成不同的寡核苷酸亚库。然后进行错误纠正。在策略1中,同时并行将用于组装单个组装片段的每个寡核苷酸亚库(11-32寡核苷酸)分别使用一个固定化MutS的纤维素凝胶柱进行纠错；在策略2中,同时并行将来源于同一个基因的多个寡核苷酸亚库(38-56寡核苷酸)分别使用一个固定化MutS的纤维素凝胶柱进行纠错；在策略3中,整个寡核苷酸文库的所有亚库(479寡核苷酸)使用一个固定化MutS的纤维素凝胶柱纠错。而在某些情况下,将寡核苷酸文库分割为亚库是不必要的,如使用芯片合成寡核苷酸进行重组文库的构建,整个文库的所有寡核苷酸在一起进行组装；在策略4中,使用一个固定化MutS的纤维素凝胶柱移除寡核苷酸文库(该寡核苷酸文库没有分割成亚库)中的合成错误,用于重组文库构建过程的纠错。
本文中,这些高通量策略通过合成可溶性甲烷单氧化酶(sMMO)基因簇,爱博霉素(Epo) A, B和C基因,番茄红素合成基因和红色荧光蛋白重组文库验证。使用这些策略,使用870芯片合成寡核苷酸为前体,我们共合成了21个基因(33.415kb)和一个红色荧光蛋白重组文库。将合成基因的错误率由～14/kb降低至0.56/kb。除此以外,使用一个标准的固定化MutS的纤维素凝胶柱进行纠错过程可以在1.5小时内完成,而一个固定化MutS的纤维素凝胶柱的价格也只需要$0.374。因此,该系统提供了一种使用芯片合成寡核苷酸进行大规模的基因合成的廉价高效的途径。
The application of microchip-synthesized oligonucleotides (MCp-oligos) for gene synthesis involving millions of oligos is limited by the minal amount of each oligos, low quality and high complex of the synthetic DNA pool. Consequently, its broad application requires advances in the accuracy and scale of synthetic DNA. In this study, a low-cost, effective and high-throughput (up to492oligos per run) error-removal method using an immobilized cellulose column containing the mismatch binding protein MutS was produced to generate high-quality DNA from oligos, particularly microchip-synthesized oligonucleotides.
At first, a new error correction system was constructed. In this error correction method, random errors in the synthetic DNA molecules are exposed through re-annealing as mismatches. Mismatch-containing duplexes are removed by the affinity binding with immobilized MutS on cellulose column. Then, the flow-through is serves as template for PCR to produce the error-depleted DNA. This process can be iterated for increased fidelity. And to improve the binding effective of MICC, MutS from Thermus aquaticus (TaqMutS) and MutS from E. coli (EcoMutS) which have different binding affinity to various mismatched were combinatorial immobilized on cellulose column to remove the errors in DNA molecules. In this study, to construct a more effective, low-cost, easy-manipulated, and higher throughput error-removal method for wide application of MCp-oligos. Error removal is conducted directly on the MCp-oligos before assembly. With this error-correction method, we were able to improved a population of synthetic enhanced green fluorescent protein (EGFP) clones from0.93%to83.22%fluorescent, and decreased errors24.87-fold to final values of1error per2159bp.
In additional, with this MutS immobilized cellulose column, four high-throughput error correction strategies corresponding to various scales were designed and evaluated. The first three stratrgies were applied for the synthesis of multi-gene construction (eg. genes, pathways or building blocks for larger DNA constructs). To simply the following gene assembly processing, the synthetic oligos pool were first devied into subpools by adding different pairs of priming site at each subpool oligos. Then error correction was performed. With the first strategy, each subpool (11-32oligos) from each fragment were parallel error correction simultaneously with corresponding MIC-Cs. With the second strategy, several subpool (38-56oligos) from each gene were parallel error correction simultaneously with corresponding MICCs. With the third strategy, all subpools of the entire oligos pool (479oligos) were error correced with only one MICC. In some case, the division of pool into subpool is not necessary, eg. construction of shuffling library with Mcp-oligos, all oligos of the entirl pool are assembled togather. The forth strategy which removes the errors of all oligos in one pool (the pool is not divided into subpools) with one MICC is applied for error correction in the construction of a shuffling library.
Here, these high-throughput error correction strategies demonstrate by proceeding the synthesis of sMMO gene cluster, Epo A, B and C genes, lycopene biosynthesis genes and a RFPs gene library. With this method,21genes encoding a total of33.415kb pairs of DNA and a RFP shuffling library from870MCp-oligos were constructed. And the error frequency can be reduced from-14/kb to as low as0.56/kb. Moreover, a standard MICC error correction process can be finished in1.5hour with a cost of$0.374/MICC. Thus, this system provids a low-cost and efficient approach for large scale de novo DNA synthesis with MCq-oligos.
首先,我们建立了一种新的合成DNA错误纠正方法。该方法中,合成DNA中的错误合成的碱基通过变性退火形成错配,从而暴露合成错误的位点。含有错配的错误合成的DNA双链被固定在纤维素柱上的错配结合蛋白MutS特异性结合而滞留在柱子中,从而被移除。然后,以收集的穿透液为模板,通过PCR扩增出错误移除的DNA。重复该纠错过程可以进一步的提高合成DNA的精度。并且,为了提高固定化MutS的纤维素凝胶柱的错配结合能力,我们同时将具有不同错配结合能力的分别来源于水生栖热菌和大肠杆菌的MutS固定化到纤维素凝胶柱上,用于DNA合成错误的移除。在该研究中,为了构建一种更加高效,廉价,易于操作且高通量的DNA合成错误纠正方法。我们在寡核苷酸组装成基因片段之前,直接对芯片合成的寡核苷酸进行纠错。使用该种纠错方法,我们将合成的增强型绿色荧光蛋白基因表达菌的荧光克隆的比率由处理前的0.93%提高到处理后的83.22%,相应的合成基因的突变率降低了24.87倍,最终的突变率降低为1/2159bp。
另外,使用固定化MutS的纤维素凝胶柱,我们还设计验证了四种具有不同纠错规模的高通量错误纠正策略。前三种高通量纠错策略用于多基因的构建(如基因,代谢通路,或长链DNA构建物的构建模块)。为了降低下游基因组装过程的难度,合成的寡核苷酸文库首先通过在序列两端加入不同的引物对将寡核苷酸文库进行扩增分割成不同的寡核苷酸亚库。然后进行错误纠正。在策略1中,同时并行将用于组装单个组装片段的每个寡核苷酸亚库(11-32寡核苷酸)分别使用一个固定化MutS的纤维素凝胶柱进行纠错；在策略2中,同时并行将来源于同一个基因的多个寡核苷酸亚库(38-56寡核苷酸)分别使用一个固定化MutS的纤维素凝胶柱进行纠错；在策略3中,整个寡核苷酸文库的所有亚库(479寡核苷酸)使用一个固定化MutS的纤维素凝胶柱纠错。而在某些情况下,将寡核苷酸文库分割为亚库是不必要的,如使用芯片合成寡核苷酸进行重组文库的构建,整个文库的所有寡核苷酸在一起进行组装；在策略4中,使用一个固定化MutS的纤维素凝胶柱移除寡核苷酸文库(该寡核苷酸文库没有分割成亚库)中的合成错误,用于重组文库构建过程的纠错。
本文中,这些高通量策略通过合成可溶性甲烷单氧化酶(sMMO)基因簇,爱博霉素(Epo) A, B和C基因,番茄红素合成基因和红色荧光蛋白重组文库验证。使用这些策略,使用870芯片合成寡核苷酸为前体,我们共合成了21个基因(33.415kb)和一个红色荧光蛋白重组文库。将合成基因的错误率由～14/kb降低至0.56/kb。除此以外,使用一个标准的固定化MutS的纤维素凝胶柱进行纠错过程可以在1.5小时内完成,而一个固定化MutS的纤维素凝胶柱的价格也只需要$0.374。因此,该系统提供了一种使用芯片合成寡核苷酸进行大规模的基因合成的廉价高效的途径。
The application of microchip-synthesized oligonucleotides (MCp-oligos) for gene synthesis involving millions of oligos is limited by the minal amount of each oligos, low quality and high complex of the synthetic DNA pool. Consequently, its broad application requires advances in the accuracy and scale of synthetic DNA. In this study, a low-cost, effective and high-throughput (up to492oligos per run) error-removal method using an immobilized cellulose column containing the mismatch binding protein MutS was produced to generate high-quality DNA from oligos, particularly microchip-synthesized oligonucleotides.
At first, a new error correction system was constructed. In this error correction method, random errors in the synthetic DNA molecules are exposed through re-annealing as mismatches. Mismatch-containing duplexes are removed by the affinity binding with immobilized MutS on cellulose column. Then, the flow-through is serves as template for PCR to produce the error-depleted DNA. This process can be iterated for increased fidelity. And to improve the binding effective of MICC, MutS from Thermus aquaticus (TaqMutS) and MutS from E. coli (EcoMutS) which have different binding affinity to various mismatched were combinatorial immobilized on cellulose column to remove the errors in DNA molecules. In this study, to construct a more effective, low-cost, easy-manipulated, and higher throughput error-removal method for wide application of MCp-oligos. Error removal is conducted directly on the MCp-oligos before assembly. With this error-correction method, we were able to improved a population of synthetic enhanced green fluorescent protein (EGFP) clones from0.93%to83.22%fluorescent, and decreased errors24.87-fold to final values of1error per2159bp.
In additional, with this MutS immobilized cellulose column, four high-throughput error correction strategies corresponding to various scales were designed and evaluated. The first three stratrgies were applied for the synthesis of multi-gene construction (eg. genes, pathways or building blocks for larger DNA constructs). To simply the following gene assembly processing, the synthetic oligos pool were first devied into subpools by adding different pairs of priming site at each subpool oligos. Then error correction was performed. With the first strategy, each subpool (11-32oligos) from each fragment were parallel error correction simultaneously with corresponding MIC-Cs. With the second strategy, several subpool (38-56oligos) from each gene were parallel error correction simultaneously with corresponding MICCs. With the third strategy, all subpools of the entire oligos pool (479oligos) were error correced with only one MICC. In some case, the division of pool into subpool is not necessary, eg. construction of shuffling library with Mcp-oligos, all oligos of the entirl pool are assembled togather. The forth strategy which removes the errors of all oligos in one pool (the pool is not divided into subpools) with one MICC is applied for error correction in the construction of a shuffling library.
Here, these high-throughput error correction strategies demonstrate by proceeding the synthesis of sMMO gene cluster, Epo A, B and C genes, lycopene biosynthesis genes and a RFPs gene library. With this method,21genes encoding a total of33.415kb pairs of DNA and a RFP shuffling library from870MCp-oligos were constructed. And the error frequency can be reduced from-14/kb to as low as0.56/kb. Moreover, a standard MICC error correction process can be finished in1.5hour with a cost of$0.374/MICC. Thus, this system provids a low-cost and efficient approach for large scale de novo DNA synthesis with MCq-oligos.
引文
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Tian, J. D., H. Gong, et al. (2004). "Accurate multiplex gene synthesis from programmable DNA microchips." Nature 432(7020):1050-1054.
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Xiong, A. S., Q. H. Yao, et al. (2006). "PCR-based accurate synthesis of long DNA sequences." Nat Protoc 1(2):791-797.
Xiong, A. S., Q. H. Yao, et al. (2004). "A simple, rapid, high-fidelity and cost-effective PCR-based two-step DNA synthesis method for long gene sequences." Nucleic Acids Res 32(12): e98.
Yang, B., X. Wen, et al. (2000). "Purification, cloning, and characterization of the CEL I nuclease." Biochemistry 39(13):3533-3541.
Yao, M. and Y. W. Kow (1996). "Cleavage of insertion/deletion mismatches, flap and pseudo-Y DNA structures by deoxyinosine 3'-endonuclease from Escherichia coli." Journal of Biological Chemistry 271(48):30672-30676.
Zhou, X., S. Cai, et al. (2004). "Microfluidic PicoArray synthesis of oligodeoxynucleotides and simultaneous assembling of multiple DNA sequences." Nucleic Acids Res 32(18): 5409-5417.