Next Generation Gene Synthesis by targeted retrieval of bead-immobilized, sequence verified DNA clones from a high throughput pyrosequencing device
Stähler, Peer F.
Leonard, Jack T.
Stähler, Cord F.
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CitationMatzas, Mark, Peer F. Stähler, Nathalie Kefer, Nicole Siebelt, Valesca Boisguérin, Jack T. Leonard, Andreas Keller, et al. 2012. Next generation gene synthesis by targeted retrieval of bead-immobilized, sequence verified dna clones from a high throughput pyrosequencing device. Nature biotechnology 28(12): 1291-1294.
AbstractThe setup of synthetic biological systems involving millions of bases is still limited by the required high quality of synthetic DNA. Important drivers to further open up the field are the accuracy and scale of chemical DNA synthesis and the downstream processing of longer DNA assembled from short fragments. We developed a new, highly parallel and miniaturized method for the preparation of high quality DNA termed “Megacloning” by using Next Generation Sequencing (NGS) technology in a preparative way. We demonstrate our method by processing both conventional and microarray-derived DNA oligonucleotides in combination with a bead-based high throughput pyrosequencing platform, gaining a 500-fold error reduction for microarray oligonucleotides in a first embodiment. We also show the assembly of synthetic genes as part of the Megacloning process. In principle, up to millions of DNA fragments can be sequenced, characterized and sorted in a single Megacloner run, enabling many new applications.
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