Researchers' dreams of automated gene synthesis could soon become a reality, predict Jingdong Tian and colleagues at Duke University, Durham, US

Bulky commercial DNA synthesisers are currently used to synthesise short oligonucleotides, the building blocks for gene assembly

The emerging field of synthetic biology aims to re-engineer existing biological systems and create novel ones in order to achieve new functions and fashion new products, all through rewriting the genetic code, DNA. The applications are broad and significant, including biomolecular design and engineering; DNA nanotechnology and computing; gene circuits and networks construction; metabolic engineering; and genome synthesis. These improved or new biological systems could help solve our food, energy, material, pharmaceutical and environmental problems in the near future.

The traditional way of modifying DNA sequences is called genetic engineering and mainly relies on various enzymatic activities to manipulate DNA. The future way of doing business would be directly through de novo gene and genome synthesis. Therefore, the ability to rapidly, accurately and economically synthesise DNA constructs of any size or sequence is crucial. Scientists believe that as soon as the technology is automatic and cheap enough, it will replace conventional genetic engineering and transform biomedical research as we know it. Imagine the degree of freedom researchers will enjoy if, at the touch of a screen, they get all the DNA sequences they can dream about synthesised for them.

Today, DNA oligonucleotide synthesis (less than 200 bases) has been automated and is based on the phosphoramidite four-step cycle process, which couples acid-activated deoxynucleoside phosphoramidites to deoxynucleosides on a solid support. Methods for synthesising longer, gene-sized DNA molecules are being improved and mainly rely on the assembly of pre-synthesised oligonucleotides. However, the overall development of gene and genome synthesis technology has lagged far behind that of gene and genome sequencing. For instance, sequencing a small microbial genome consisting of a few megabases using today's automated genome sequencer would cost only a few thousand dollars and a couple of days. To synthesise the same genome from scratch using available commercial technology (which has yet to be accomplished) would cost tens of millions of dollars and take a whole research team several months. Even worse, the tons of chemical waste (mainly organic solvent) resulting from a small genome synthesis project would surely cause environmental concerns.

The main challenges in technology development for de novo gene synthesis include improving or inventing new DNA synthesis chemistry, reducing or eliminating synthesis errors, miniaturising the reaction apparatus, increasing the throughput of oligonucleotide synthesis and automating the gene assembly process. Synthesis errors accumulate rapidly in an elongating DNA molecule if the step-wise coupling efficiency is less than 100 per cent (it is currently 99.5 to 99.8 per cent). Identifying and correcting those errors is one of the most costly and time-consuming steps in gene synthesis and the hardest process to automate.

To increase throughput and reduce cost and chemical consumption, a current trend is to use DNA microchip and microfabrication techniques to miniaturise and massively parallelise the oligonucleotide synthesis process. Scientists are testing new technologies, such as digital photolithography, inkjet printing, electrochemical array and microfluidics. A few research groups have demonstrated the feasibility of using oligonucleotides synthesised and harvested from DNA microchips for gene assembly. Once scientists optimise the process, synthesising a whole microbial genome from a single microchip will become feasible.

Read more in issue 7 of  MolecularBioSystems, a synthetic biology theme issue.

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