Microfluidic PicoArray synthesis of oligodeoxynucleotides and simultaneous assembling of multiple DNA sequences
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...
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Published in | Nucleic acids research Vol. 32; no. 18; pp. 5409 - 5417 |
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Main Authors | , , , , , , , , , , , , , , , , |
Format | Journal Article |
Language | English |
Published |
England
Oxford University Press
01.01.2004
Oxford Publishing Limited (England) |
Subjects | |
Online Access | Get full text |
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Summary: | 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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Bibliography: | local:gkh879 To whom correspondence should be addressed. Tel: +1 713 743 2805; Fax: +1 713 742 2709; Email: gao@mail.uh.edu Received July 21, 2004; Revised September 10, 2004; Accepted September 20, 2004 ark:/67375/HXZ-GBX6W4TH-8 istex:4C2801D71FFA0DB6D694868A68237AC3596EB821 ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23 ObjectType-Article-1 ObjectType-Feature-2 ObjectType-Undefined-1 ObjectType-Feature-3 |
ISSN: | 0305-1048 1362-4962 1362-4962 |
DOI: | 10.1093/nar/gkh879 |