Gene Synthesis from Oligonucleotide Mixtures by Solid Phase PCR and Assembly PCR in a Microfluidic Chip System.

Abstract

The conventional gene synthesis methods, chemical or PCR, usually require over 2 weeks because of the separate executions of the different procedures. An integrated microfluidic chip system was designed to reduce this processing time to only 2 days with much less reaction volumes, and experimental reagent and solvent requirements. This fast high throughput gene synthesis method considerably minimizes contamination and simplifies material handling procedures. Our overall aim in this project is using the above-mentioned advantages of this system to synthesize long genes of arbitrary sequence with high purity, and cut the lead times and cost per base from the current values by at least one order of magnitude. In order to do this, four different steps are included in the microfluidic chip system: oligonucleotide synthesis and amplification on solid phase, on-chip purification, long DNA assembly, and gene transformation. The designed oligonucleotides to form the long DNAs were synthesized via light-directed phosphoramidite chemistry, and amplified on solid phase. The amplified products were treated by on-surface hybridization using complementary probes to make single strands and purification. The purified oligonucleotides were assembled into long DNAs on chip, and amplified with polymerase chain reaction in a separate microfluidic chip chamber. Finally, the synthetic target gene was transformed on a chip for gene expression. Our results showed these individual steps in bringing the system capability to a simultaneous production level of tens of double stranded oligonucleotides of lengths ranging from 0.2 to 1.2 kb and the potential of microfluidic gene and protein synthesis system.