Parallel hierarchical gene assembly from oligonucleotide pools on an integrated digital microfluidic platform.

We report an integrated electrowetting-on-dielectric (EWOD) digital microfluidic (DMF) platform for automated gene assembly directly from oligonucleotide pools (OPs). The system is implemented on a glass chip with through-glass vias (TGVs), enabling dense, individually addressable electrodes for programmable droplet manipulation and integrated thermal control for enzymatic reactions. Barcode probes immobilized on a disposable top plate enable sequence-specific capture, spatial partitioning, and on-chip release of oligonucleotides for polymerase cycling assembly (PCA). The platform supports single-round assembly of ∼1.2 kbp fragments and enables hierarchical two-round assembly of ∼1.8 kbp products from a common OP, whereas direct single-round assembly of longer targets fails. This work establishes a TGV-based DMF platform as a programmable and scalable platform for low-volume, automated, and hierarchical gene assembly from oligonucleotide pools.