Accurate gene synthesis with tag-directed retrieval of sequence-verified DNA molecules

Gene synthesis is currently limited by the need to use error-prone oligonucleotide building blocks. Dial-out PCR overcomes the sequence verification bottleneck by using unique sequence tags and massively parallel sequencing to identify and selectively retrieve error-free DNA molecules of interest from complex mixtures. We present dial-out PCR, a highly parallel method for retrieving accurate DNA molecules for gene 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 and is a compelling alternative to in vivo cloning and Sanger sequencing for accurate gene synthesis.