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

• Published in: Nucleic acids research 2010-10, Vol.38 (19), p.e180-e180
• Main Authors: Borovkov, Alex Y, Loskutov, Andrey V, Robida, Mark D, Day, Kristen M, Cano, Jose A, Le Olson, Tien, Patel, Hetal, Brown, Kevin, Hunter, Preston D, Sykes, Kathryn F
• Format: Article
• Language: English
• Subjects: Genes, Synthetic; Methods Online; Oligonucleotide Array Sequence Analysis - economics; Oligonucleotide Array Sequence Analysis - methods; Oligonucleotides - chemical synthesis; Polymerase Chain Reaction
• ISSN: 0305-1048; 1362-4962; 1362-4962
• DOI: 10.1093/nar/gkq677

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 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[cent sign] per base, making gene synthesis more affordable than traditional cloning.