Issue 2, 2009

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.

Graphical abstract: Integrated two-step gene synthesis in a microfluidic device

Supplementary files

Article information

Article type
Paper
Submitted
06 May 2008
Accepted
19 Aug 2008
First published
23 Oct 2008

Lab Chip, 2009,9, 276-285

Integrated two-step gene synthesis in a microfluidic device

M. C. Huang, H. Ye, Y. K. Kuan, M. Li and J. Y. Ying, Lab Chip, 2009, 9, 276 DOI: 10.1039/B807688J

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