High-fidelity gene synthesis by retrieval of sequence-verified DNA identified using high-throughput pyrosequencing.Comments Comment in: Nat Biotechnol. 2010 Dec;28(12):1272-3; PMID: 21139611, Comment in: Nat Methods. 2011 Feb;8(2):114; PMID: 21355126, Comment in: Nat Rev Genet. 2011 Jan;12(1):6; PMID: 21164523

摘要

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.