Splynthesis sequencing data

In this study, we introduce a new in vitro method for oligonucleotide fragment assembly. Unlike Polymerase Chain Assembly and Ligase Chain Assembly that rely on short, highly purified oligonucleotides, our method, named Splynthesis uses a one-tube, splint-driven assembly reaction. Splynthesis connects standard desalted contig oligos (around 150 nt in length) via shorter splint oligos harboring 5 prime and 3 prime blocking modifications to prevent off-target ligation and amplification events. We demonstrate the Splynthesis method to assemble a 741 bp gene fragment. We verify the assembled PCR product using standard molecular biology techniques and long read Oxford Nanopore sequencing and confirm that the product is cloneable via molecular means and Sanger sequencing. This approach is applicable for synthetic biology, directed evolution, functional protein assays, and potentially even splint-based Ligase Chain Reaction assays.