DropSynth, a one-pot approach to gene synthesis

January 5, 2018 by Bob Yirka, Phys.org report
Credit: CC0 Public Domain

A team of researchers at the University of California has found a way to synthesize multiple genes from a group of microarray-generated oligonucleotides. In their paper published in the journal Science, the group describes their technique, called DropSynth, how well it works, and its drawbacks.

Synthesizing genes has become so popular that there are now companies that do it for a living, but it is still an expensive proposition—current methods require sewing small strands into sequences one at a time after they have been created. In this new effort, the researchers report a one-pot approach to that could lead to lowering costs.

Currently, gene synthesis is done using a microarray, producing DNA oligonucleotides, which then have to be sewn together—the team at UoC also started with a microarray, but they first gave the oligonucleotides an identifying length of , which they describe as a "barcode." Next, they added microbeads with complimentary barcodes that could pull matching oligonucleotides from a pool of different kinds of oligonucleotides. The result was a pool of microbeads, each of which had a small group of attached same-type oligonucleotides. The team then enclosed each of the microbeads (and its group of oligonucleotides) in an emulsion droplet using what they describe as an oligonucleotide and oil mixture vortex—for 30 seconds. After that, enzymes induced all of the oligonucleotides in a single droplet to merge (via polymerase cycling assembly), resulting in a desired sequence. The sequences were then removed from the emulsion ready for use.

The process is better than conventional methods, the team suggests, because it offers access to a gene pool at essentially the same cost as an pool—it does not add much. Unfortunately, there is a pretty big downside. The process, they note, is "messy," because it is only approximately 5 percent efficient—not nearly good enough for use in manufacturing processes. On a more positive note, the method could be used to create large libraries of for various purposes, at very little cost. It could also be used in research efforts, particularly those involved in protein design.

Explore further: Antisense therapy—a promising new way to treat neurological disease

More information: Calin Plesa et al. Multiplexed gene synthesis in emulsions for exploring protein functional landscapes, Science (2018). DOI: 10.1126/science.aao5167

Abstract
Improving our ability to construct and functionally characterize DNA sequences would broadly accelerate progress in biology. Here, we introduce DropSynth, a scalable, low-cost method to build thousands of defined gene-length constructs in a pooled (multiplexed) manner. DropSynth uses a library of barcoded beads that pull down the oligonucleotides necessary for a gene's assembly, which are then processed and assembled in water-in-oil emulsions. We use DropSynth to successfully build >7000 synthetic genes that encode phylogenetically-diverse homologs of two essential genes in E. coli. We tested the ability of phosphopantetheine adenylyltransferase homologs to complement a knockout E. coli strain in multiplex, revealing core functional motifs and reasons underlying homolog incompatibility. DropSynth coupled with multiplexed functional assays allow us to rationally explore sequence-function relationships at unprecedented scale.

Related Stories

Hope for new treatment for Huntington's disease

February 20, 2017

Researchers working at Karolinska Institutet in Sweden and University of Southern Denmark have managed to produce short synthetic DNA analogues – oligonucleotides – that bind directly to the gene that is mutated in Huntington's ...

New method for mass-producing high-quality DNA molecules

June 2, 2013

A new method of manufacturing short, single-stranded DNA molecules can solve many of the problems associated with current production methods. The new method, which is described in the scientific periodical Nature Methods, ...

Unraveling the mechanism of antisense oligonucleotides

February 2, 2017

Stanley T. Crooke, MD, PhD, CEO of Ionis Pharmaceuticals and recipient of the 2016 Lifetime Achievement Award from the Oligonucleotide Therapeutic Society presents a detailed look at the mechanisms that underlie antisense ...

Can toxicity of a DNA drug be predicted and minimized?

August 27, 2013

New classes of therapeutic antisense oligonucleotides can have toxic effects on the liver. A novel machine learning-based approach used to predict the hepatotoxic potential of an antisense drug based on its chemical sequence ...

Recommended for you

Looking for LUCA, the last universal common ancestor

December 18, 2018

Around 4 billion years ago there lived a microbe called LUCA: the Last Universal Common Ancestor. There is evidence that it could have lived a somewhat 'alien' lifestyle, hidden away deep underground in iron-sulfur rich hydrothermal ...

0 comments

Please sign in to add a comment. Registration is free, and takes less than a minute. Read more

Click here to reset your password.
Sign in to get notified via email when new comments are made.