Long DNA Synthesis from Oligonucleotide Mixtures by Solid Phase PCR and Assembly PCR In a Microfluidic Chip System
Woonghee Lee1, Jean-Marie Rouillard2, and Erdogan Gulari2. (1) Biomedical Engineering, The University of Michigan Ann Arbor, 2300 Hayward St., 3074 H.H. Dow Building, Ann Arbor, MI 48109, (2) Chemical Engineering, The University of Michigan Ann Arbor, 2300 Hayward St., 3074 H.H. Dow Building, Ann Arbor, MI 48109
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 is 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, single stranded DNA production, followed by on-chip purification, and long DNA assembly. The designed oligonucleotides to form the long DNAs are synthesized via light-directed phosphoramidite chemistry, and amplified on solid phase. The amplified products are treated to make single strands, which are later purified by on-surface hybridization using complementary probes. The purified oligonucleotides are assembled into long DNAs on chip, and amplified with polymerase chain reaction in a separate microfluidic chip chamber. Our current focus in the project is testing these individual steps in bringing the system capability to a simultaneous production level of tens of double stranded oligomers of lengths ranging from 0.2 to 1kb. In this respect, to increase the assembly yield, a single stranded DNA production method from PCR products is also under development along with a purification system which eliminates mismatch containing synthetic oligomers.