title: Integrated two-step gene synthesis in a microfluidic device.
abstract: Herein we present an integrated microfluidic device capable of performing two-step gene synthesis to assemble a pool of oligonucleotides into genes with the desired coding sequence. The device comprised of two polymerase chain reactions (PCRs), temperature-controlled hydrogel valves, electromagnetic micromixer, shuttle micromixer, volume meters, and magnetic beads based solid-phase PCR purification, fabricated using a fast prototyping method without lithography process. The fabricated device is combined with a miniaturized thermal cycler to perform gene synthesis. Oligonucleotides were first assembled into genes by polymerase chain assembly (PCA), and the full-length gene was amplified by a second PCR. The synthesized gene was further separated from the PCR reaction mixture by the solid-phase PCR purification. We have successfully used this device to synthesize a green fluorescent protein fragment (GFPuv) (760 bp), and obtained comparable synthesis yield and error rate with experiments conducted in a PCR tube within a commercial thermal cycler. The resulting error rate determined by DNA sequencing was 1 per 250 bp. To our knowledge, this is the first microfluidic device demonstrating integrated two-step gene synthesis.
authors.affiliation: Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, Singapore 138669.
authors.forename: Mo Chao
authors.initials: MC
authors.lastname: Huang
authors.forename: Hongye
authors.initials: H
authors.lastname: Ye
authors.forename: Yoke Kong
authors.initials: YK
authors.lastname: Kuan
authors.forename: Mo-Huang
authors.initials: MH
authors.lastname: Li
authors.forename: Jackie Y
authors.initials: JY
authors.lastname: Ying
mesh_terms: D004247:DNA / Q000138:chemical synthesis; D004252:DNA Mutational Analysis; D005813:Genes, Synthetic; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation* / Q000379:methods; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction
chemical_list: D004247:DNA