Parallel gene synthesis in a microfluidic device

Abstract

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 1 kb in length were synthesized in parallel at minute starting oligonucleotide concentrations (10–25 nM) in four 500 nl 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.

Photoelectrochemical synthesis of DNA microarrays

“…The use of a high-loading support may become increasingly important as the complexity of the DNA pool increases, because the quantity of each sequence decreases with the spot area. It should be noted that parallel gene synthesis in microfluidic devices may also address issues associated with the minute quantities of DNA obtained from cleaved microarrays (42).…”

Open-source, community-driven microfluidics with Metafluidics

“…Microfluidics enables single-cell manipulation and analysis, reaction volumes of nano-or picoliters (thereby reducing costs), high-throughput execution of parallel experiments, automated routine liquid handling, integration of multiple biological processes in a single system, and programmability for complex protocols. Synthetic biologists have used microfluidics to miniaturize DNA assembly (for oligonucleotides 6 , genes 7 , and genetic circuits 8 ), to purify DNA 9 , to clone DNA 10 , and to transform 11 and transfect 12 cells. Multipurpose platforms have been developed that integrate several processes 8,13 .…”

Computational Selection and Experimental Validation of Allosteric Ribozymes That Sense a Specific Sequence of Human Telomerase Reverse Transcriptase mRNAs as Universal Anticancer Therapy Agents

“…4. Replace the oligonucleotide binding site (Kong et al, 2007) from the structure with the same number of artificial nucleotides (n) that are defined to have no binding properties. 5.…”