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Nucleic Acids Research 2004 32(18):5409-5417; doi:10.1093/nar/gkh879
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Published online 11 October 2004

Nucleic Acids Research, Vol. 32 No. 18 © Oxford University Press 2004; all rights reserved

Microfluidic PicoArray synthesis of oligodeoxynucleotides and simultaneous assembling of multiple DNA sequences

Xiaochuan Zhou3,4, Shiying Cai3, Ailing Hong3,4, Qimin You3, Peilin Yu1, Nijing Sheng1, Onnop Srivannavit2, Seema Muranjan3, Jean Marie Rouillard2, Yongmei Xia2, Xiaolin Zhang3,4, Qin Xiang3, Renuka Ganesh1,4, Qi Zhu1, Anna Matejko1, Erdogan Gulari2 and Xiaolian Gao1,*

1 Department of Chemistry, University of Houston, Houston, TX 77004-5003, USA, 2 Department of Chemical Engineering, University of Michigan, Ann Arbor, MI 48109, USA, 3 Xeotron Co. 8275 El Rio, Suite 130, Houston, TX 77054, USA and 4 Atactic Technologies Inc., 2575 W. Bellfort, Suite 270, Houston, TX 77054, USA

* To whom correspondence should be addressed. Tel: +1 713 743 2805; Fax: +1 713 742 2709; Email: gao{at}mail.uh.edu

Received July 21, 2004; Revised September 10, 2004; Accepted September 20, 2004

Large DNA constructs of arbitrary sequences can currently be assembled with relative ease by joining short synthetic oligodeoxynucleotides (oligonucleotides). The ability to mass produce these synthetic genes readily will have a significant impact on research in biology and medicine. Presently, high-throughput gene synthesis is unlikely, due to the limits of oligonucleotide synthesis. We describe a microfluidic PicoArray method for the simultaneous synthesis and purification of oligonucleotides that are designed for multiplex gene synthesis. Given the demand for highly pure oligonucleotides in gene synthesis processes, we used a model to improve key reaction steps in DNA synthesis. The oligonucleotides obtained were successfully used in ligation under thermal cycling conditions to generate DNA constructs of several hundreds of base pairs. Protein expression using the gene thus synthesized was demonstrated. We used a DNA assembly strategy, i.e. ligation followed by fusion PCR, and achieved effective assembling of up to 10 kb DNA constructs. These results illustrate the potential of microfluidics-based ultra-fast oligonucleotide parallel synthesis as an enabling tool for modern synthetic biology applications, such as the construction of genome-scale molecular clones and cell-free large scale protein expression.


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