Published online 6 August 2010 Nucleic Acids Research, 2010, Vol. 38, No. 19 e180 doi:10.1093/nar/gkq677

High-quality gene assembly directly from unpurified mixtures of microarray-synthesized oligonucleotides

Alex Y. Borovkov1,*, Andrey V. Loskutov1, Mark D. Robida1, Kristen M. Day1, Jose A. Cano1, Tien Le Olson1, Hetal Patel1, Kevin Brown1, Preston D. Hunter1 and Kathryn F. Sykes1,2,

1Center for Innovations in Medicine of the Biodesign Institute at the Arizona State University, Tempe, AZ 85287 and 2School of Life Sciences, Arizona State University, Tempe, AZ 85287, USA

Received March 23, 2010; Revised July 19, 2010; Accepted July 21, 2010

ABSTRACT

To meet the growing demand for synthetic genes more robust, scalable and inexpensive gene assembly technologies must be developed. Here, we present a protocol for high-quality gene assembly directly from low-cost marginal-quality microarray-synthesized oligonucleotides. Significantly, we eliminated the time-and money-consuming oligonucleotide purification steps through the use of hybridization-based selection embedded in the assembly process. The protocol was tested on mixtures of up to 2000 oligonucleotides eluted directly from microarrays obtained from three different chip manufacturers. These mixtures containing <5% perfect oligos, and were used directly for assembly of 27 test genes of different sizes. Gene quality was assessed by sequencing, and their activity was tested in coupled in vitro transcription/ translation reactions. Genes assembled from the microarray-eluted material using the new protocol matched the quality of the genes assembled from >95% pure column-synthesized oligonucleotides by the standard protocol. Both averaged only 2.7 errors/kb, and genes assembled from microarray-eluted material without clonal selection produced only 30% less protein than sequence-confirmed clones. This report represents the first demonstration of cost-efficient gene assembly from microarray-synthesized oligonucleotides. The overall cost of assembly by this method approaches 5¢ per base, making gene synthesis more affordable than traditional cloning.

*To whom correspondence should be addressed. Tel: +1 469 774 2263; Email: aborovkov@synbuild.com

Present addresses:

Alex Y. Borovkov, Synbuild LLC, 1833 W. Main Street, Suite 129, Mesa, AZ 85201, USA.

Jose A. Cano and Tien Le Olson, Departmento de Biotecnologia, Psicofarma, Mexico City, Mexico 14050, USA.

Hetal Patel, Kevin Brown and Preston D. Hunter, Covance Laboratories Inc., Chandler, AZ 85249, USA.

� The Author(s) 2010. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.