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Parallel gene synthesis in a microfluidic device by David Kong et al. Presented by Eric Gomez & Dahlia Alkekhia December 2nd, 2010 Background • Synthesis of custom de novo long DNA strands and genes is very valuable • Issues: accuracy, time, COST – $0.1 per nucleotide for conventionally synthesized oligos – $0.65 – $1.10 per bp for custom gene synthesis services – Example: synthesis of bacterial genomes 106bp in size become prohibitively costly, requiring on the order of $100, 000 in oligos alone Proposed technology: Multi-chambered microfluidic device Why? • Minimizes reaction volumes 500nL instead of 50uL • Reduces sample handling • Enables large number of complex reactions to be preformed in parallel • reduces costs! • Has lower error rate • Can be used in conjunction with other applications The Tiny Reaction : PCA - Starting pool of construction oligos - Thermocycling leads to annealing and extension by DNA polymerase - Multiple thermocycling leads to increasingly extended gene sequences - Complete gene is achieved, amplification can be performed The Tiny Device Fabrication PDMS1 PDMS2 PDMS3 Blue & Green: Gene Synthesis Chamber Yellow: Water Jacket Blue: Fluid Inlet Channel Red: Valve Channel Experimental Procedure - Every microfluidic reaction was also ran in vitro in normal PCR tubes to compare performance Genes selected: – bacterial “alba” gene – bacteriophage “hjc” gene – GFP construct Mixes demonstrating successful synthesis amplified through PCR – Red fluorescent protein (dsRed) - All reaction products analyzed through PAGE - - Amplified products visualized again through PAGE to verify correct amplification - Products sequenced using amplifying primers to confirm correct gene - Errors quantified by vector cloning and transformation Results With 50% higher yield relative to reactions in PCR tubes (In PCR tubes) Error Rates Forty eight clones for both ‘in fluidic’ and in vitro DsRed synthesis yielded: 12.5% of full-length clones were error-free Teaming up with Microarrays synthesis of construction oligos through high density microarrays Incorporated into microfluidic device Cleave and collect Femtomoles = > 200nM or lower per sequence Enough Insufficient for gene forsynthesis conventional in same set ups device (requires 10-25nM) • time amplification • reagents • handling complex pools of oligos • money • introducing more error Gene synthesis/ desired application Peter Carr & David Kong Looking ahead… • Incorporation of existing DNA error correction techniques on-chip • Integration of in vitro protein expression • Assembly of constructs larger than single genes Thank You!