DNA assembly with error correction on a droplet digital microfluidics platform.
DNA assembly with error correction on a droplet digital microfluidics platform.
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DOI：
10.1186/s12896-018-0439-9
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发表时间：
2018-06-01
影响因子：
3.5
通讯作者：
Griffin PB
中科院分区：
文献类型：
--
作者：
Khilko Y;Weyman PD;Glass JI;Adams MD;McNeil MA;Griffin PB
定制合成DNA在合成生物学应用中的需求很高。然而，目前使用DNA寡核苷酸组装来生产这些序列的技术成本高昂，劳动密集型。微流控技术提供的自动化和减少的样本量可以显著降低与DNA合成相关的材料和劳动力成本。这项研究的目的是开发一种利用数字微流控设备的基因组装方案。为了实现这一目标，我们将实验室规模的寡核苷酸组装方法和酶误差校正方法应用到蒙德里安™数字微流控平台。我们优化了Gibson组装、聚合酶链式反应(PCR)和酶纠错反应，将12个寡核苷酸组装成一个339个碱基的双链DNA序列，编码部分人流感病毒血凝素(HA)基因。将反应缩小到0.6%-1.2%μL。初始的微流控组装方法是成功的，并且具有大约4个错误/kb的错误频率，错误源于原始的寡核苷酸合成。与传统的台式程序相比，PCR优化需要额外的氯化镁、Phusion聚合酶和PEG8000来实现组装和纠错产物的扩增。经过一轮纠错后，错误频率减少到平均1.8kb的− 1。我们证明了从寡核苷酸组装和纠错可以在数字微流控(DMF)平台上完全自动化。结果表明，液滴中的酶反应表现出对表面相互作用的强烈依赖，成功的芯片实现需要表面活性剂、分子排泄剂和过量的酶的补充。组装片段的酶纠错将序列保真度提高了2倍，这是一个显著的改进，但与台式分析相比略低于预期，这表明优化的能力有所增加。本文的在线版本(10.1186/s12896-0180439-9)包含向授权用户提供的补充材料。
Custom synthesized DNA is in high demand for synthetic biology applications. However, current technologies to produce these sequences using assembly from DNA oligonucleotides are costly and labor-intensive. The automation and reduced sample volumes afforded by microfluidic technologies could significantly decrease materials and labor costs associated with DNA synthesis. The purpose of this study was to develop a gene assembly protocol utilizing a digital microfluidic device. Toward this goal, we adapted bench-scale oligonucleotide assembly methods followed by enzymatic error correction to the Mondrian™ digital microfluidic platform. We optimized Gibson assembly, polymerase chain reaction (PCR), and enzymatic error correction reactions in a single protocol to assemble 12 oligonucleotides into a 339-bp double- stranded DNA sequence encoding part of the human influenza virus hemagglutinin (HA) gene. The reactions were scaled down to 0.6-1.2 μL. Initial microfluidic assembly methods were successful and had an error frequency of approximately 4 errors/kb with errors originating from the original oligonucleotide synthesis. Relative to conventional benchtop procedures, PCR optimization required additional amounts of MgCl2, Phusion polymerase, and PEG 8000 to achieve amplification of the assembly and error correction products. After one round of error correction, error frequency was reduced to an average of 1.8 errors kb− 1. We demonstrated that DNA assembly from oligonucleotides and error correction could be completely automated on a digital microfluidic (DMF) platform. The results demonstrate that enzymatic reactions in droplets show a strong dependence on surface interactions, and successful on-chip implementation required supplementation with surfactants, molecular crowding agents, and an excess of enzyme. Enzymatic error correction of assembled fragments improved sequence fidelity by 2-fold, which was a significant improvement but somewhat lower than expected compared to bench-top assays, suggesting an additional capacity for optimization. The online version of this article (10.1186/s12896-018-0439-9) contains supplementary material, which is available to authorized users.
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