Accurate gene synthesis with tag-directed retrieval of sequence-verified DNA moleculesView all posters
University of Washington, United States
With increasing scales of oligonucleotide synthesis comes a concomitant need to rapidly screen complex synthetic libraries and selectively retrieve specific error-free sequences. This is currently a significant bottleneck for the field of synthetic biology. To overcome this limitation we have developed dial-out PCR, a highly parallel method for retrieving accurate DNA molecules for gene and genome synthesis. A complex library of DNA molecules is modified with unique flanking tags before massively parallel sequencing. Tag-directed primers then enable the retrieval of molecules with desired sequences by PCR. Dial-out PCR enables multiplex in vitro clone screening, allows for the normalization of target sequence abundance before multiplex assembly steps, and it has the potential to decrease production costs for high-quality, sequence-verified synthetic DNA by over an order of magnitude. The method also supports clone retrieval from other nucleic acid pools, such as in the screening and recovery of specific mutants from a complex mutagenesis library. Dial-out PCR is a compelling alternative to in vivo cloning and Sanger sequencing for large-scale target verification and retrieval.