DOI: 10.6084/m9.figshare.26068327.v1
title: Fully <i>in vitro</i> iterative construction of a 24 kb-long artificial DNA sequence to store digital information

description: In the absence of a DNA template, the <i>ab initio</i> production of long double-stranded DNA molecules of predefined sequences is particularly challenging. The DNA synthesis step remains a bottleneck for many applications such as functional assessment of ancestral genes, analysis of alternative splicing or DNA-based data storage. In this report we propose a fully <i>in vitro</i> protocol to generate very long double-stranded DNA molecules starting from commercially available short DNA blocks in less than 3 days using Golden Gate assembly. This innovative application allowed us to streamline the process to produce a 24 kb-long DNA molecule storing part of <i>the</i> <i>Declaration of the Rights of Man and of the Citizen</i> of 1789 . The DNA molecule produced can be readily cloned into a suitable host/vector system for amplification and selection. Minion-controlled <i>ab initio</i> production of long DNA molecules of predefined sequences from commercially available short DNA blocks in less than 3 days. DNA molecules are easily copied from pre-existing DNA molecules. However, in the absence of a pre-existing DNA template, the <i>ab initio</i> production of long DNA molecules of chosen sequences is particularly challenging and precludes the usage of such molecule for DNA digital data storage. In this report we propose a protocol that is carried out exclusively in a reaction tube to generate very long DNA molecules starting from synthetic short DNA blocks in less than 3 days using Golden Gate assembly. This innovative application allowed us to streamline the process to produce a 24 kb-long DNA molecule storing part of the <i>Declaration of the Rights of Man and of the Citizen</i> of 1789. We developed, using Golden gate assembly, an iterative DNA assembly pipeline to produce long double-stranded DNA molecules of predefined sequences. This pipeline relies on the orderly assembly of multiple 500-nt commercial fragments into medium sized 5-kb BigBlocks that are then assembled in an iterative process. PCR allowed us to select and amplify the full-size molecules. The DNA construction steps were analyzed using single-molecule sequencing, demonstrating that our protocol is highly effective in obtaining correctly assembled molecules. We used this pipeline to store part of the <i>Declaration of the Rights of Man and of the Citizen</i> of 1789 on DNA. To define and evaluate a method of assembly of long DNA molecules of artificial sequence. We devised an iterative method for the <i>in vitro</i> two-step construction of a 24 kb-long artificial sequence DNA molecule. The assembly process was monitored using single-molecule sequencing on a minion sequencer. This study devised an iterative method that allows for the synthesis of long DNA molecules to be used for digital data storage, with our study producing a DNA molecule encoding the <i>Declaration of the Rights of Man and of the Citizen </i>of 1789. Evaluation of the assembly steps by minion sequencing allowed us to rapidly evaluate the faithfulness of the assembly process. Our fully <i>in vitro</i> iterative method can faithfully construct long synthetic DNA molecules by assembling commercially available short dsDNA.