Scientists at Duke University have received a grant from the National Institutes of Health to further develop a method of synthesizing genes using microarrays. The researchers believe their gene synthesis platform will prove "faster and cheaper" than alternative array-based approaches, as well as traditional methods of gene assembly.
The project, entitled "Automated Long DNA Synthesis Technology," was approved last week with $383,078 earmarked for the first year of the project. The official start date for the effort is May 27, 2011, and the project is anticipated to end on March 31, 2015.
Principal investigator Jingdong Tian told BioArray News this week that he hopes to receive subsequent grants of $383,000 per year for the remainder of the project. If the Duke team does receive that amount, the NIH will spend more than $1.5 million to support the effort by its end date.
Tian, a professor in Duke's department of biomedical engineering, said that the platform, which was described in a Nature Biotechnology paper last month, already exists as a "proof of concept."
In that paper, Tian and co-authors describe the development of the on-chip gene synthesis approach, which "integrates on a single microchip the synthesis of DNA oligonucleotides using inkjet printing, isothermal oligonucleotide amplification, and parallel gene assembly."
They applied the approach to synthesize pools of thousands of codon-usage variants of lacZα and 74 Drosophila protein antigens, which were then screened for expression in Escherichia coli. In one round of synthesis and screening, the authors reportedly obtained DNA sequences that were "expressed at a wide range of levels, from zero to almost 60 percent of the total cell protein mass." They concluded that the platform could "facilitate systematic investigation of the molecular mechanisms of protein translation and the design, construction, and evolution of macromolecular machines, metabolic networks, and synthetic cells."
Tian said this week that his goals in the newly funded project are to "explore new chip design and microarray fabrication process, improve DNA synthesis quality and throughput, and increase the automation and level of integration" of the system.
The main thrust of the work is toward developing a less costly and more efficient alternative to current methods of gene synthesis, where oligos are typically synthesized in columns.
As Tian and colleagues noted in the paper, the "integration of oligo synthesis and gene assembly on the same microchip facilitates automation and miniaturization," which can in turn reduce costs and increase throughput.
As described in the paper, Tian and colleagues synthesized oligos using a custom-made, inkjet DNA microarray synthesizer on embossed cyclic olefin copolymer chips. Tian said this week that the components of the internally developed printer have been purchased from various vendors.
Using the tools, gene-construction oligos were designed to be 48 or 60 bases long with a 25-base adaptor at the 3′ end, which provided a nicking site and anchored the oligo to the chip surface. COC slides were then prepatterned to form eight or 30 subarrays of silica thin-film spots. Each chamber in the 30-chamber design could print 361 spots and was used to synthesize only one gene or gene library up to 1 kilobase in length, according to the paper. Multiple spots were used to synthesize one oligo sequence.
The approach evidently provides a reduction in cost per oligo. The estimated cost of chip-oligonucleotide synthesis for 30 kilobase of sequence was less than $0.001 per base pair of final synthesized sequences, which Tian and colleagues said is one-tenth of the lowest reported cost for current methods. Including enzymatic processing and error correction, the average cost of integrated gene synthesis on a chip is less than $0.005 per basepair of final synthesized gene sequences.
"With multiplexing and more advanced chip design, greater throughput and lower costs are potentially achievable," Tian and co-authors noted in the paper.
They argue that their screening-based method is "faster and cheaper" than current methods and can be "performed on a large scale with high-throughput gene synthesis technology." They also believe it could "pave the way for systematic investigation of the molecular mechanisms of protein translation."
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'Filling a Gap'
Microarrays have been used to synthesize genes for years. As discussed in a recent Nature Methods technology feature, researchers have relied on array-synthesized oligos supplied by vendors like Agilent Technologies or worked with firms like Febit on gene synthesis.
Indeed, in 2007 Febit formed a synthetic biology subsidiary called Febit SynBio that sought to carve out a place for the German company in the nascent market, but the effort was largely wound up when the firm restructured to focus on microRNA research last year (BAN 6/29/2010).
According to the newly funded grant's abstract, Tian and his colleagues aim to create a system that is solely dedicated to synthesizing genes in the lab.
This de novo synthesis approach would give researchers "total freedom to test the effects of designed novel sequences or fine sequence variations on genome function," the Duke researchers noted in the abstract.
They argued that existing gene synthesis technology "still relies on costly and cumbersome macro-scale DNA oligonucleotide synthesis and manual gene assembly procedures." Ultimately, they aim to create an "integrated, microfluidics and microarray-based de novo gene synthesis technology platform to fill this gap."
Such an automated gene synthesis platform would be able to accurately write designed gene sequences into double-stranded DNA molecules, ready for cloning, expression and other downstream applications and could become ubiquitous in molecular biology labs, they predicted.
"Like thermal cyclers, automated gene synthesizers will be widely used and become an indispensable tool for life science researchers," the investigators wrote in the abstract.
"In addition to genome structure and function studies, the technology will enable many types of biomedical research … such as large-scale synthetic genomics, construction of genetic circuits and metabolic pathways, design, evolution and optimization of new proteins, enzymes, antibodies and other pharmaceuticals," they added.
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