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Nucleic Acids Research 2004 32(17):5011-5018; doi:10.1093/nar/gkh793
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Published online 24 September 2004

Nucleic Acids Research, Vol. 32 No. 17 © Oxford University Press 2004; all rights reserved

Amplification and assembly of chip-eluted DNA (AACED): a method for high-throughput gene synthesis

Kathryn E. Richmond1, Mo-Huang Li1, Matthew J. Rodesch2, Madhusudan Patel1,3, Aaron M. Lowe4, Changhan Kim5, Larry L. Chu1, Narasimhar Venkataramaian5, Shane F. Flickinger3, James Kaysen1, Peter J. Belshaw3,6, Michael R. Sussman2,6 and Franco Cerrina1,5,*

1 Center of Nanotechnology, 2 Biotechnology Center, 3 Department of Chemistry, 4 Department of Material Sciences, 5 Department of Electrical and Computer Engineering and 6 Department of Biochemistry, University of Wisconsin, Madison, WI 53706, USA

* To whom correspondence should be addressed. Tel: +1 608 262 7712; Fax: +1 608 265 3811; Email: cerrina{at}nanotech.wisc.edu
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors

Received June 29, 2004; Revised and Accepted August 9, 2004

A basic problem in gene synthesis is the acquisition of many short oligonucleotide sequences needed for the assembly of genes. Photolithographic methods for the massively parallel synthesis of high-density oligonucleotide arrays provides a potential source, once appropriate methods have been devised for their elution in forms suitable for enzyme-catalyzed assembly. Here, we describe a method based on the photolithographic synthesis of long (>60mers) single-stranded oligonucleotides, using a modified maskless array synthesizer. Once the covalent bond between the DNA and the glass surface is cleaved, the full-length oligonucleotides are selected and amplified using PCR. After cleavage of flanking primer sites, a population of unique, internal 40mer dsDNA sequences are released and are ready for use in biological applications. Subsequent gene assembly experiments using this DNA pool were performed and were successful in creating longer DNA fragments. This is the first report demonstrating the use of eluted chip oligonucleotides in biological applications such as PCR and assembly PCR.


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