Next-generation DNA synthesis?

[Bryan Bishop]

Hey all,

I was wondering if anyone has leads on next-generation DNA synthesis. I have been trying to find literature on this every once in a while for the past two years, but can't really come up with much other than traditional solid phase oligonucleotide synthesis. Rob or Raymond, maybe one of you have a lead on this?

- Bryan
http://heybryan.org/
1 512 203 0507

[lianchao han]

not sure if these are the next generation, but George Church's lab and  another group came up something interesting in the current issue of Nature Biotechnology:

High-fidelity gene synthesis by retrieval of sequence-verified DNA identified using high-throughput pyrosequencing

The construction of synthetic biological systems involving millions of nucleotides is limited by the lack of high-quality synthetic DNA. Consequently, the field requires advances in the accuracy and scale of chemical DNA synthesis and in the processing of longer DNA assembled from short fragments. Here we describe a highly parallel and miniaturized method, called megacloning, for obtaining high-quality DNA by using next-generation sequencing (NGS) technology as a preparative tool. We demonstrate our method by processing both chemically synthesized and microarray-derived DNA oligonucleotides with a robotic system for imaging and picking beads directly off of a high-throughput pyrosequencing platform. The method can reduce error rates by a factor of 500 compared to the starting oligonucleotide pool generated by microarray. We use DNA obtained by megacloning to assemble synthetic genes. In principle, millions of DNA fragments can be sequenced, characterized and sorted in a single megacloner run, enabling constructive biology up to the megabase scale.

Scalable gene synthesis by selective amplification of DNA pools from high-fidelity microchips

Development of cheap, high-throughput and reliable gene synthesis methods will broadly stimulate progress in biology and biotechnology¹. Currently, the reliance on column-synthesized oligonucleotides as a source of DNA limits further cost reductions in gene synthesis². Oligonucleotides from DNA microchips can reduce costs by at least an order of magnitude^3, 4, 5, yet efforts to scale their use have been largely unsuccessful owing to the high error rates and complexity of the oligonucleotide mixtures. Here we use high-fidelity DNA microchips, selective oligonucleotide pool amplification, optimized gene assembly protocols and enzymatic error correction to develop a method for highly parallel gene synthesis. We tested our approach by assembling 47 genes, including 42 challenging therapeutic antibody sequences, encoding a total of ~35 kilobase pairs of DNA. These assemblies were performed from a complex background containing 13,000 oligonucleotides encoding ~2.5 megabases of DNA, which is at least 50 times larger than in previously published attempts.

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[Bryan Bishop]

On Wed, Dec 15, 2010 at 9:26 AM, lianchao han wrote:
> Scalable gene synthesis by selective amplification of DNA pools from
> high-fidelity microchips

In this method, you have a library of oligonucleotides that you build
up. Then, you selectively ligate these strands together into longer
strands as you need them. There are advantages to do doing this, and
it's probably the only way to reliably build up oligonucleotide DNA
fragments into anything usably large, but I am not sure if this really
qualifies as next-generation DNA synthesis. :-(

[Jordan Miller]

didn't they use a special technique to make the synthetic bacterium?

jordan

[Bryan Bishop]

On Wed, Dec 15, 2010 at 9:58 AM, Jordan Miller wrote:

didn't they use a special technique to make the synthetic bacterium?

Specifically, the chemical oligonucleotide synthesis technique has never been the novelty in Venter's work. In the chemically synthesized genome paper from May [1], he was ordering chemically synthesized oligos from Blue Heron.

Blue Heron overview
http://www.blueheronbio.com/knowledge-center/technology

Their special sauce (if any) is how they assemble multiple oligos into larger fragments. The references Lianchao posted are similar to this in concept (though it sounds like Blue Heron isn't using microchips or microarrays here).

[1] Creation of a bacterial cell controlled by a chemically synthesized genome
http://www.sciencemag.org/content/329/5987/52.abstract
pdf: http://www.sciencemag.org/content/329/5987/52.full.pdf