Large-scale de novo DNA synthesis: technologies and applications

Kosuri, Sriram; Church, George M.

doi:10.1038/nmeth.2918

Cited by 736 publications

(645 citation statements)

References 139 publications

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“…However, despite countless advances in molecular biology, the assembly of DNA parts into new constructs remains a craft that is both time consuming and unpredictable. The decreasing costs of gene synthesis promises to alleviate these limitations by providing custom-made double-stranded DNA fragments typically between 200 and 2000 bp in length 1 . Nonetheless, gene synthesis does not eliminate the need for DNA assembly, which remains necessary for the production of constructs beyond one kilobase in size, both in research labs and at gene synthesis companies.…”

Section: Introductionmentioning

confidence: 99%

“…In order to tackle projects of increasing scale and complexity, researchers have invested significant effort into developing new tools for DNA assembly and into matching them with improved, lower-cost gene synthesis (for reviewes on gene synthesis see REFS. 1,5 1,5 ), as well as a suite of important new tools for genome editing (Box 1). With these combined advances the field is now at a point where gene synthesis and DNA assembly can empower even undergraduate students to construct entire eukaryotic chromosomes 6 .…”

Section: Introductionmentioning

confidence: 99%

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Bricks and blueprints: methods and standards for DNA assembly

Casini

Storch

Baldwin

et al. 2015

Nat Rev Mol Cell Biol

251

186

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PrefaceDNA assembly is a key part of constructing gene expression systems and even whole chromosomes. In the past decade a plethora of powerful new DNA assembly methods including Gibson assembly, Golden Gate and LCR have been developed. In this Innovation article we discuss these methods and standards such as MoClo, GoldenBraid, MODAL and PaperClip, which have been developed to facilitate a streamlined assembly workflow, aid material exchange, and the creation of modular, reusable DNA parts. IntroductionOur capacity to cut and paste DNA from different sources and to assemble it into gene constructs has been one of the key drivers of biological research and biotechnology over the past four decades. However, despite countless advances in molecular biology, the assembly of DNA parts into new constructs remains a craft that is both time consuming and unpredictable. The decreasing costs of gene synthesis promises to alleviate these limitations by providing custom-made double-stranded DNA fragments typically between 200 and 2000 bp in length 1 . Nonetheless, gene synthesis does not eliminate the need for DNA assembly, which remains necessary for the production of constructs beyond one kilobase in size, both in research labs and at gene synthesis companies. DNA assembly also enables carrying out projects with more complex experimental needs and is especially valuable for building diverse plasmid libraries and creating multicomponent systems, and has even been used to construct synthetic cells 2 .Addressing the limitations of DNA assembly methods has been one of the key goals of synthetic biology, a scientific discipline focused on the construction and testing of new or redesigned versions of genes, gene networks, pathways and cells 3,4 . In order to tackle projects of increasing scale and complexity, researchers have invested significant effort into developing new tools for DNA assembly and into matching them with improved, lower-cost gene synthesis (for reviewes on gene synthesis see REFS. 1,5 1,5 ), as well as a suite of important new tools for genome editing (Box 1). With these combined advances the field is now at a point where gene synthesis and DNA assembly can empower even undergraduate students to construct entire eukaryotic chromosomes 6 . This acceleration in the scale of DNA assembly enables construction projects too complex to be drawn out on the back of an envelope, which instead require an engineering approach. In the past decade important 1 assembly methods such as Gibson assembly and Golden Gate have been developed 7,8 , which define new protocols for joining together DNA parts. Alongside these methods, researchers have also developed various physical standards such as MODAL (Modular Overlap-Directed Assembly with Linkers) 9 and MoClo (Modular Cloning system) 12 that define rules for the format of DNA parts that can be used with them. These physical standards facilitate the re-use of parts between experiments, exchange of parts between research groups and importantly provide modularity in construction. ...

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Bricks and blueprints: methods and standards for DNA assembly

Casini

Storch

Baldwin

et al. 2015

Nat Rev Mol Cell Biol

251

186

View full text Add to dashboard Cite

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“…Although array-based platforms offer superior synthesis capabilities in terms of multiplexing and cost there are some challenges with using these platforms for making oligonucleotides for gene synthesis applications. One problem is that planar array synthesized oligonucleotides tend to be relatively low quality, which leads to more synthesis-related errors when compared with column-synthesized oligonucleotides (Kosuri and Church 2014;Wan et al 2014). Despite this, recent improvements in chip design and refinements to the synthesis process have led to an increase in the quality of array-synthesized oligonucleotides.…”

Section: Microarray-based Oligonucleotide Synthesismentioning

confidence: 99%

“…The cost of gene synthesis is typically directly tied to the cost of oligonucleotide synthesis from which the genes are made. The cost of oligonucleotide synthesis has not decreased appreciably in more than a decade, generally ranging from $0.05 to $0.17 per base depending on the synthesis scale, the length of the oligonucleotide, and the supplier (Kosuri and Church 2014). Traditionally, this cost floor has been carried through to the production of gene synthesis products.…”

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confidence: 99%

Synthetic DNA Synthesis and Assembly: Putting the Synthetic in Synthetic Biology

Hughes

Ellington

2017

Cold Spring Harb Perspect Biol

322

235

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The chemical synthesis of DNA oligonucleotides and their assembly into synthons, genes, circuits, and even entire genomes by gene synthesis methods has become an enabling technology for modern molecular biology and enables the design, build, test, learn, and repeat cycle underpinning innovations in synthetic biology. In this perspective, we briefly review the techniques and technologies that enable the synthesis of DNA oligonucleotides and their assembly into larger DNA constructs with a focus on recent advancements that have sought to reduce synthesis cost and increase sequence fidelity. The development of lower-cost methods to produce high-quality synthetic DNA will allow for the exploration of larger biological hypotheses by lowering the cost of use and help to close the DNA read -write cost gap.DNA neither cares nor knows. DNA just is. And we dance to its music.-Richard Dawkins River Out of Eden: A Darwinian Life

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“…These studies often rely on genetic manipulations such as deletions and insertions (a top-down approach), but recent advances in DNA synthesis provide a new optionwhole genome synthesis (a bottom-up approach). While several bacterial and viral genomes have been successfully synthesized, the yeast genome synthesis project (Sc2.0) aims to synthesize the first eukaryotic genome-all 16 chromosomes of Saccharomyces cerevisiae (Kosuri and Church, 2014). Due to its complexity and large scale, this project has been carried out by an international consortium that consists of approximately two hundred researchers from over 10 universities and institutions from five countries.…”

mentioning

confidence: 99%