Hierarchical Multiplex Pairwise Golden Gate Assembly: Converting short oligo-pools into longer DNA libraries

Shaozhong Zou, zhien wu, Chunfu Xu

Track: Biology: datasets and/or experimental results

Nature Biotechnology: Yes

Keywords: High-throughput DNA synthesis; Oligo-pools; Golden Gate Assembly

TL;DR: We developed a novel method for the high-throughput assembly of gene-sized DNA sequences, starting from cost-effective chip-synthesized oligo-pools.

Abstract: Large-scale screening and high-throughput experimental data generation are essential for advancing AI-driven biomolecular design. However, these processes are economically unfeasible due to the high costs associated with synthesizing gene-sized DNA sequences at scale. To address this challenge, we developed a novel method for the high-throughput assembly of gene-sized DNA sequences, starting from cost-effective chip-synthesized oligo-pools. In contrast to Polymerase Cycling Assembly (PCA) methods, we employed Golden Gate Assembly (GGA) to facilitate the ligation of short DNA fragments. This approach enabled us to successfully assemble high-quality DNA libraries containing up to 96 gene-sized sequences (600 bp) in a single-pot reaction, with convenient retrieval of individual sequences. If numerous reactions are conducted in parallel---for example, in a 96-well plate---we can readily assemble up to 9,216 (96 x 96)) genes. When combined with advances in automation technologies, this enables the efficient and cost-effective synthesis of gene-sized DNA sequences at scale, thereby accelerating the generation of experimental data for the biomolecular design community.

Anonymization: This submission has been anonymized for double-blind review via the removal of identifying information such as names, affiliations, and identifying URLs.

Presenter: ~Shaozhong_Zou1

Format: Yes, the presenting author will attend in person if this work is accepted to the workshop.

Funding: Yes, the presenting author of this submission falls under ICLR’s funding aims, and funding would significantly impact their ability to attend the workshop in person.

Submission Number: 120