De novo DNA synthesis using single-molecule PCR.

The throughput of DNA reading (i.e., sequencing) has dramatically increased recently owing to the incorporation of in vitro clonal amplification. The throughput of DNA writing (i.e., synthesis) is trailing behind, with cloning and sequencing constituting the main bottleneck. To overcome this bottleneck, an in vitro alternative for in vivo DNA cloning needs to be integrated into DNA synthesis methods. Here, we show how a new single-molecule PCR (smPCR)-based procedure can be employed as a general substitute for in vivo cloning, thereby allowing for the first time in vitro DNA synthesis. We integrated this rapid and high fidelity in vitro procedure into our previously described recursive DNA synthesis and error correction procedure and used it to efficiently construct and error-correct a 1.8-kb DNA molecule from synthetic unpurified oligonucleotides, entirely in vitro. Although we demonstrate incorporating smPCR in a particular method, the approach is general and can be used, in principle, in conjunction with other DNA synthesis methods as well.