基因合成技术研究进展

基因合成是生物学中一项最基本的、最常用的技术.对DNA调控元件、基因、途径乃至整个基因组的合成是验证生物学假设和利用生物学为人类服务的有力工具.合成生物学的快速发展对基因合成能力提出了日益迫切的需求.近年来,基于微芯片基因合成技术取得了很多令人振奋的新进展,正在向着高通量、高保真、自动化的方向发展.文中综述了DNA化学合成和基因组装及相关技术的最新研究进展和发展趋势,这些新技术正在推动着合成生物学向着更高的水平发展.     

Microfluidic PicoArray synthesis of oligodeoxynucleotides and simultaneous assembling of multiple DNA sequences

Large DNA constructs of arbitrary sequences can currently be assembled with relative ease by joining short synthetic oligodeoxynucleotides (oligonucleotides). The ability to mass produce these synthetic genes readily will have a significant impact on research in biology and medicine. Presently, high-throughput gene synthesis is unlikely, due to the limits of oligonucleotide synthesis. We describe a microfluidic PicoArray method for the simultaneous synthesis and purification of oligonucleotides that are designed for multiplex gene synthesis. Given the demand for highly pure oligonucleotides in gene synthesis processes, we used a model to improve key reaction steps in DNA synthesis. The oligonucleotides obtained were successfully used in ligation under thermal cycling conditions to generate DNA constructs of several hundreds of base pairs. Protein expression using the gene thus synthesized was demonstrated. We used a DNA assembly strategy, i.e. ligation followed by fusion PCR, and achieved effective assembling of up to 10 kb DNA constructs. These results illustrate the potential of microfluidics-based ultra-fast oligonucleotide parallel synthesis as an enabling tool for modern synthetic biology applications, such as the construction of genome-scale molecular clones and cell-free large scale protein expression.     

A scalable method for multiplex LED-controlled synthesis of DNA in capillaries

As research in synthetic biology and genomic sciences becomes more widespread, the need for diverse oligonucleotide populations has increased. To limit reagent cost, it would be advantageous to obtain high quality populations in minute amounts. Towards that end, synthesis of DNA strands in capillaries utilizing photolabile 3-nitrophenylpropyloxycarbonyl (NPPOC) chemistry and ultraviolet-light emitting diodes (UV-LEDs) was examined. Multiple oligonucleotides were made in single capillaries and were characterized by hybridization, sequencing and gene synthesis. DNA synthesized in capillaries was capable of being hybridized and signal intensities correlated with microarray data. Sequencing demonstrated that the oligonucleotides were of high quality (up to 44% perfect sequences). Oligonucleotides were combined and used successfully for gene synthesis. This system offers a novel, scalable method to synthesize high quality oligonucleotides for biological applications.     

Scalable gene synthesis by selective amplification of DNA pools from high-fidelity microchips

Development of cheap, high-throughput and reliable gene synthesis methods will broadly stimulate progress in biology and biotechnology. Currently, the reliance on column-synthesized oligonucleotides as a source of DNA limits further cost reductions in gene synthesis. Oligonucleotides from DNA microchips can reduce costs by at least an order of magnitude, yet efforts to scale their use have been largely unsuccessful owing to the high error rates and complexity of the oligonucleotide mixtures. Here we use high-fidelity DNA microchips, selective oligonucleotide pool amplification, optimized gene assembly protocols and enzymatic error correction to develop a method for highly parallel gene synthesis. We tested our approach by assembling 47 genes, including 42 challenging therapeutic antibody sequences, encoding a total of ～35 kilobase pairs of DNA. These assemblies were performed from a complex background containing 13,000 oligonucleotides encoding ～2.5 megabases of DNA, which is at least 50 times larger than in previously published attempts.     

Gene synthesis technology: recent developments and future prospects

Gene synthesis is a potentially powerful tool in molecular biology that has not yet reached widespread use because of the relatively high cost and labor-intensive nature of the process. This paper reviews some recent technological developments and current research activities of this laboratory which promise to greatly reduce the cost of gene synthesis and to increase the speed and efficiency of the process. We recently developed an improved device for "segmented" synthesis of oligonucleotides, which utilizes porous Teflon wafers containing derivatized controlled pore glass supports to simultaneously synthesize up to 100 different DNA sequences. The stepwise coupling efficiency with the "wafer synthesis device" is as high as that attained with current automated "gene machines" producing 1-4 oligonucleotides at a time, whereas the reagent usage is only 20-50% that of the current DNA synthesizers. At present, we are optimizing the conditions for rapid, efficient assembly of genes on a solid-phase support, wherein ordered, stepwise annealing/washing is performed to segmentally elongate a "starting" oligonucleotide attached to a solid-phase support. We expect that the wafer synthesis device (operated at reduced scale of synthesis), together with solid-phase gene assembly, will permit the synthesis and assembly of an average size gene (1 kb) in one week at a cost of less than $1000. These developments should make gene synthesis a routine and powerful tool in molecular biology.     

Parallel gene synthesis in a microfluidic device

The ability to synthesize custom de novo DNA constructs rapidly, accurately and inexpensively is highly desired by researchers, as synthetic genes and longer DNA constructs are enabling to numerous powerful applications in both traditional molecular biology and the emerging field of synthetic biology. However, the current cost of de novo synthesis—driven largely by reagent and handling costs—is a significant barrier to the widespread availability of such technology. In this work, we demonstrate, to our knowledge, the first gene synthesis in a microfluidic environment. The use of microfluidic technology greatly reduces reaction volumes and the corresponding reagent and handling costs. Additionally, microfluidic technology enables large numbers of complex reactions to be performed in parallel. Here, we report the fabrication of a multi-chamber microfluidic device and its use in carrying out the syntheses of several DNA constructs. Genes up to 1kb in length were synthesized in parallel at minute starting oligonucleotide concentrations (10–25nM) in four 500nl reactors. Such volumes are one to two orders of magnitude lower than those utilized in conventional gene synthesis. The identity of all target genes was verified by sequencing, and the resultant error rate was determined to be 1 per 560 bases.     

Parallel on-chip gene synthesis and application to optimization of protein expression

Low-cost, high-throughput gene synthesis and precise control of protein expression are of critical importance to synthetic biology and biotechnology. Here we describe the development of an on-chip gene synthesis technology, which integrates on a single microchip the synthesis of DNA oligonucleotides using inkjet printing, isothermal oligonucleotide amplification and parallel gene assembly. Use of a mismatch-specific endonuclease for error correction results in an error rate of ~0.19 errors per kb. We applied this approach to synthesize pools of thousands of codon-usage variants of lacZα and 74 challenging Drosophila protein antigens, which were then screened for expression in Escherichia coli. In one round of synthesis and screening, we obtained DNA sequences that were expressed at a wide range of levels, from zero to almost 60% of the total cell protein mass. This technology may facilitate systematic investigation of the molecular mechanisms of protein translation and the design, construction and evolution of macromolecular machines, metabolic networks and synthetic cells.     

从DNA合成技术角度阐述合成生物学

合成生物学是一个拥有巨大潜力的新兴学科,合成生物学技术的发展将会对未来生物、医药、农业、能源、材料和环保等方面产生巨大的推进作用。基因合成是合成生物学中最基本和使用最多的一种技术手段,合成生物学的快速发展对基因合成能力提出了空前需求。综述基因合成技术的发展历史、现状和未来趋势,探讨基因合成技术存合成生物学以及整个生命科学研究中的应用和重要意义。     

DNA synthesis, assembly and applications in synthetic biology

The past couple of years saw exciting new developments in microchip-based gene synthesis technologies. Such technologies hold the potential for significantly increasing the throughput and decreasing the cost of gene synthesis. Together with more efficient enzymatic error correction and genome assembly methods, these new technologies are pushing the field of synthetic biology to a higher level.     

工业生物技术中DNA合成发展现状及展望

DNA合成是生命科学领域的共性支撑技术和合成生物学的关键使能技术。以合成生物学为基础的工业生物技术持续快速发展,迫切需要更加便捷、经济、安全的DNA来源以满足其日益增长的大规模DNA合成需求。工业化DNA合成在通量、成本、速度等方面的优势日益凸显,有力推动了工业生物技术研发效率的提升和研发成本的下降。但是现有技术在生产过程中还存在着使用大量有机试剂、资源浪费等问题。随着DNA合成规模的持续快速提升,有毒化学品危害、成本负担、环境负担等问题日益突出。本文结合我们的工作实践,对工业生物技术中DNA合成需求、合成策略以及可持续发展面临的问题和解决方案研究进展进行探讨。     

DNA合成技术及应用

DNA从头合成技术是指以寡核苷酸链为起始的合成DNA片段的技术,其不断进步是合成生物学快速发展的基石之一。常规使用的连接介导的DNA合成技术和PCR介导的DNA合成技术日益成熟,精确合成长度已经达到0．5—1kb。微阵列介导的DNA合成技术不断发展,其低成本、高通量的特点吸引了人们的注意;而酵母体内DNA合成技术的成功探索也为体外DNA合成提供了一种补偿方法。DNA合成在优化密码子用于异源表达、构建异源代谢途径、合成人工基因组以及合成减毒病毒用于疫苗研制等方面有广泛应用。综述了DNA从头合成技术的研究进展,并介绍了DNA合成的前沿应用。     

High-fidelity gene synthesis by retrieval of sequence-verified DNA identified using high-throughput pyrosequencing

The construction of synthetic biological systems involving millions of nucleotides is limited by the lack of high-quality synthetic DNA. Consequently, the field requires advances in the accuracy and scale of chemical DNA synthesis and in the processing of longer DNA assembled from short fragments. Here we describe a highly parallel and miniaturized method, called megacloning, for obtaining high-quality DNA by using next-generation sequencing (NGS) technology as a preparative tool. We demonstrate our method by processing both chemically synthesized and microarray-derived DNA oligonucleotides with a robotic system for imaging and picking beads directly off of a high-throughput pyrosequencing platform. The method can reduce error rates by a factor of 500 compared to the starting oligonucleotide pool generated by microarray. We use DNA obtained by megacloning to assemble synthetic genes. In principle, millions of DNA fragments can be sequenced, characterized and sorted in a single megacloner run, enabling constructive biology up to the megabase scale.     

A computational framework for optimal masking in the synthesis of oligonucleotide microarrays

High-throughput genomic technologies are revolutionizing modern biology. In particular, DNA microarrays have become one of the most powerful tools for profiling global mRNA expression in different tissues and environmental conditions, and for detecting single nucleotide polymorphisms. The broad applicability of gene expression profiling to the biological and medical realms has generated expanding demand for mass production of microarrays, which in turn has created considerable interest in improving the cost effectiveness of microarray fabrication techniques. We have developed the computational framework for an optimal synthesis strategy for oligonucleotide microarrays. The problem was introduced by Hubbell et al. Here, we formalize the problem, obtain precise bounds on its complexity and devise several computational solutions.     

An alternate method for synthesis of double-stranded DNA segments

Recent progress in the chemical synthesis of DNA has now made it possible to rapidly synthesize single-stranded DNAs over 40 bases in length. We have taken advantage of these longer DNAs in assembling and cloning a 132-base pair gene segment coding for amino acids 126 through the stop codon of human leukocyte interferon alpha 2. The method used involves DNA polymerase I-mediated repair synthesis of synthetic oligonucleotide substrates having short stretches of complementary sequence at their 3' termini. In the presence of DNA polymerase I and the four deoxyribonucleoside triphosphates, those primer-templates are converted to full length double-stranded DNAs. The economy in chemical synthesis using this approach is substantial with a greater than 40% reduction in the amount of chemical synthesis required as compared with the conventional approach. We describe in detail this methodology for the biochemical assembly of long gene segments from synthetic oligodeoxyribonucleotides.     

利用DNA芯片技术高效合成OstreococcusTauri叶绿体基因组

目的：21世纪以来,随着合成生物学的高速发展及其所遇到的问题,开发下一代DNA合成技术已经成为了必然趋势。基因芯片技术和DNA大片段组装技术是建立下一代DNA合成平台的关键技术力量。方法：为了开发具有工业化标准的DNA芯片一基因组合成平台,我们首次利用电化学DNA芯片和DNA大片段组装技术合成了72kb的Ostreococcusmud的全叶绿体基因组。结果：首先,我们使用电化学DNA芯片合成仪合成了564条150bp的OligoMix,并成功扩增分离了其中96％的Oligo序列,剩下的基因组序列是通过传统的固相亚磷酰胺三脂合成法合成。在此基础上,我们利用DNA重组技术将564条150bpOligo片段分三步克隆到了一个pGSYN系统。通过高通量测序,我们证实叶绿体基因组被成功地人工合成。整个合成成本大约是目前传统基因合成成本的10％．20％。结论：研究证实基因芯片技术和DNA大片段组装技术的应用是能够明显的降低现阶段基因组合成工艺的成本。新技术的成熟推广和成本的有效控制也会进一步加速科学家对基因组功能的深入研究以及合成生物学的质的飞跃。     

合成基因组学：设计与合成的艺术

随着基因组相关技术(测序、编辑、合成等)和知识(功能基因组学)的日益成熟,合成基因组学在本世纪迎得了发展的契机。病毒、原核生物的全基因组相继被化学合成并支持生命的存活,第1个真核生物合成基因组计划已经完成过半,人类基因组编写计划提上日程。在基因组合成的实践过程中,研究者们不断探索对基因组进行重编和设计所应遵循的规则,提高从头合成、组装和替换基因组的技术手段。合成基因组在工业、环境、健康和基础研究领域有着广阔的应用前景,同时也带来了相应的伦理问题。结合在Sc2.0计划中的基因组合成研究和近期合成基因组学所取得的重大进展,本文综述了基因组设计和合成相关的科学、技术和伦理内容,并探讨了未来发展所面对的挑战。作为合成生物学最重要的领域之一,合成基因组学方兴未艾。     

Construction of synthetic genes using PCR after automated DNA synthesis of their entire top and bottom strands.

A new method is described for the direct construction of synthetic genes by applying a modified version of the polymerase chain reaction (PCR) to crude oligonucleotide mixtures made by automated solid phase DNA synthesis. Construction of the HIV-1 393 bp rev gene and the 655 bp nef gene by this method is illustrated. The sequences for the entire top and bottom strands of rev were each programmed into an automated DNA synthesizer. Following DNA synthesis, the two crude oligonucleotide solutions were mixed together, specific primers were added, and the target gene was amplified by a modified PCR technique. Although the longer (greater than 200 bases) strands comprise a very small percentage of the total DNA after solid phase synthesis, this method uses PCR to 'find' and amplify such strands to create the target gene. The rev gene constructed by this method was found to contain 4 sequence errors, which were subsequently corrected by site-directed mutagenesis. In order to evaluate the source of sequence errors, several nef genes were made from the top and bottom strand DNA synthesis solutions using independent PCR's. Results suggest that sequence errors arose from both DNA synthesis and PCR. The utility of this method in producing a functional gene is demonstrated by expression of rev in E.coli.     

An alternative method for the synthesis of tailor-made genes

Oligonucleotide synthesis was coupled with amplification by the polymerase chain reaction to generate an exact translational fusion between a plant signal sequence and an animal structural gene. A synthetic 111-mer oligonucleotide representing less than two percent of the reaction products was successfully amplified by using short primers containing restriction sites designed for ease of cloning and providing in-frame fusion. The method overcomes the length-versus-yield dilemma in oligonucleotide synthesis, and is generally adaptable to the construction of a translationally competent coding sequence from any two DNA fragments.     

A Method for Multiplex Gene Synthesis Employing Error Correction Based on Expression

Our ability to engineer organisms with new biosynthetic pathways and genetic circuits is limited by the availability of protein characterization data and the cost of synthetic DNA. With new tools for reading and writing DNA, there are opportunities for scalable assays that more efficiently and cost effectively mine for biochemical protein characteristics. To that end, we have developed the Multiplex Library Synthesis and Expression Correction (MuLSEC) method for rapid assembly, error correction, and expression characterization of many genes as a pooled library. This methodology enables gene synthesis from microarray-synthesized oligonucleotide pools with a one-pot technique, eliminating the need for robotic liquid handling. Post assembly, the gene library is subjected to an ampicillin based quality control selection, which serves as both an error correction step and a selection for proteins that are properly expressed and folded in E. coli. Next generation sequencing of post selection DNA enables quantitative analysis of gene expression characteristics. We demonstrate the feasibility of this approach by building and testing over 90 genes for empirical evidence of soluble expression. This technique reduces the problem of part characterization to multiplex oligonucleotide synthesis and deep sequencing, two technologies under extensive development with projected cost reduction.     

Meeting report: Synthetic DNA - Writing with the Letters of Life: January 24, 2012, Frankfurt, Germany

The one-day meeting on Synthetic DNA (January 24, 2012) organized by and held at the DECHEMA in Frankfurt attracted about 100 participants from academia and industry interested in synthesizing DNA and its applications in synthetic biology. In recent years the cost for synthetic DNA reduced from 7€/bp to 0.35€/bp which has opened up many new possibilities for molecular biology. You can purchase the gene, cDNA, oligo library or full vector specifically for a particular expression host and apply synthetic biology principles to produce or create new drugs, vaccines or any other biotechnological products. There are, however, great concerns within society to produce organisms that do not exist in nature, and the potential misuse of them. Adressing these concerns and to use a clear terminology that do not cause misunderstandings are important issues within the field, which were also discussed at this meeting.