A Microfluidic Oligonucleotide Synthesizer

Overview

Journal Nucleic Acids Res

Publisher Oxford University Press

Specialty Biochemistry

Date 2010 Feb 24

PMID 20176572

Citations 26

Authors

Cheng-Chung Lee

Thomas M Snyder

Stephen R Quake

Affiliations

Soon will be listed here.

Abstract

De novo gene and genome synthesis enables the design of any sequence without the requirement of a pre-existing template as in traditional genetic engineering methods. The ability to mass produce synthetic genes holds great potential for biological research, but widespread availability of de novo DNA constructs is currently hampered by their high cost. In this work, we describe a microfluidic platform for parallel solid phase synthesis of oligonucleotides that can greatly reduce the cost of gene synthesis by reducing reagent consumption (by 100-fold) while maintaining a approximately 100 pmol synthesis scale so there is no need for amplification before assembly. Sixteen oligonucleotides were synthesized in parallel on this platform and then successfully used in a ligation-mediated assembly method to generate DNA constructs approximately 200 bp in length.

Citing Articles

Droplet-based μChopper device with a 3D-printed pneumatic valving layer and a simple photometer for absorbance based fructosamine quantification in human serum.

Kayirangwa Y, Mohibullah M, Easley C Analyst. 2023; 148(19):4810-4819.

PMID: 37605899 PMC: 10530610. DOI: 10.1039/d3an01149f.

Uncertainties in synthetic DNA-based data storage.

Xu C, Zhao C, Ma B, Liu H Nucleic Acids Res. 2021; 49(10):5451-5469.

PMID: 33836076 PMC: 8191772. DOI: 10.1093/nar/gkab230.

Monodisperse drops templated by 3D-structured microparticles.

Wu C, Ouyang M, Wang B, de Rutte J, Joo A, Jacobs M Sci Adv. 2020; 6(45).

PMID: 33148643 PMC: 7673687. DOI: 10.1126/sciadv.abb9023.

Next generation gene synthesis: From microarrays to genomes.

Kuhn P, Wagner K, Heil K, Liss M, Netuschil N Eng Life Sci. 2020; 17(1):6-13.

PMID: 32624724 PMC: 6999524. DOI: 10.1002/elsc.201600121.

3DμF - Interactive Design Environment for Continuous Flow Microfluidic Devices.

Sanka R, Lippai J, Samarasekera D, Nemsick S, Densmore D Sci Rep. 2019; 9(1):9166.

PMID: 31235804 PMC: 6591506. DOI: 10.1038/s41598-019-45623-z.

References

Tian J, Gong H, Sheng N, Zhou X, Gulari E, Gao X . Accurate multiplex gene synthesis from programmable DNA microchips. Nature. 2004; 432(7020):1050-4. DOI: 10.1038/nature03151. View

Au L, Yang F, Yang W, Lo S, Kao C . Gene synthesis by a LCR-based approach: high-level production of leptin-L54 using synthetic gene in Escherichia coli. Biochem Biophys Res Commun. 1998; 248(1):200-3. DOI: 10.1006/bbrc.1998.8929. View

Mueller S, Coleman J, Wimmer E . Putting synthesis into biology: a viral view of genetic engineering through de novo gene and genome synthesis. Chem Biol. 2009; 16(3):337-47. PMC: 2728443. DOI: 10.1016/j.chembiol.2009.03.002. View

Gao X, Gulari E, Zhou X . In situ synthesis of oligonucleotide microarrays. Biopolymers. 2004; 73(5):579-96. DOI: 10.1002/bip.20005. View

Kong D, Carr P, Chen L, Zhang S, Jacobson J . Parallel gene synthesis in a microfluidic device. Nucleic Acids Res. 2007; 35(8):e61. PMC: 1885655. DOI: 10.1093/nar/gkm121. View

Huang M, Ye H, Kuan Y, Li M, Ying J . Integrated two-step gene synthesis in a microfluidic device. Lab Chip. 2008; 9(2):276-85. DOI: 10.1039/b807688j. View

Ye H, Huang M, Li M, Ying J . Experimental analysis of gene assembly with TopDown one-step real-time gene synthesis. Nucleic Acids Res. 2009; 37(7):e51. PMC: 2673447. DOI: 10.1093/nar/gkp118. View

LETSINGER R, Lunsford W . Synthesis of thymidine oligonucleotides by phosphite triester intermediates. J Am Chem Soc. 1976; 98(12):3655-61. DOI: 10.1021/ja00428a045. View

Thorsen T, Maerkl S, Quake S . Microfluidic large-scale integration. Science. 2002; 298(5593):580-4. DOI: 10.1126/science.1076996. View

10.

Stemmer W, Crameri A, Ha K, Brennan T, Heyneker H . Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides. Gene. 1995; 164(1):49-53. DOI: 10.1016/0378-1119(95)00511-4. View

11.

Huang Y, Castrataro P, Lee C, Quake S . Solvent resistant microfluidic DNA synthesizer. Lab Chip. 2006; 7(1):24-6. DOI: 10.1039/b613923j. View

12.

Li G, Ran R, Zhao J, Xu Y . Design, simulation, and optimization of a miniaturized device for size-fractioned DNA extraction. Electrophoresis. 2007; 28(24):4661-7. DOI: 10.1002/elps.200700226. View

13.

Unger M, Chou H, Thorsen T, Scherer A, Quake S . Monolithic microfabricated valves and pumps by multilayer soft lithography. Science. 2001; 288(5463):113-6. DOI: 10.1126/science.288.5463.113. View

14.

Wang Z, Olsen P, Ravikumar V . A novel universal linker for efficient synthesis of phosphorothioate oligonucleotides. Nucleosides Nucleotides Nucleic Acids. 2007; 26(3):259-69. DOI: 10.1080/15257770701257277. View

15.

Paul C, Royappa A . Acid binding and detritylation during oligonucleotide synthesis. Nucleic Acids Res. 1996; 24(15):3048-52. PMC: 146054. DOI: 10.1093/nar/24.15.3048. View

16.

Lee J, Park C, Whitesides G . Solvent compatibility of poly(dimethylsiloxane)-based microfluidic devices. Anal Chem. 2003; 75(23):6544-54. DOI: 10.1021/ac0346712. View

17.

Septak M . Kinetic studies on depurination and detritylation of CPG-bound intermediates during oligonucleotide synthesis. Nucleic Acids Res. 1996; 24(15):3053-8. PMC: 146050. DOI: 10.1093/nar/24.15.3053. View