High-quality gene assembly directly from unpurified mixtures of microarray-synthesized oligonucleotides.
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.
Borovkov
Alex Y
AY
Center for Innovations in Medicine of the Biodesign Institute at the Arizona State University, Tempe, AZ 85287, USA. aborovkov@synbuild.com
Loskutov
Andrey V
AV
Robida
Mark D
MD
Day
Kristen M
KM
Cano
Jose A
JA
Le Olson
Tien
T
Patel
Hetal
H
Brown
Kevin
K
Hunter
Preston D
PD
Sykes
Kathryn F
KF
D005813:Genes, Synthetic; D020411:Oligonucleotide Array Sequence Analysis / Q000191:economics / Q000379:methods*; D009841:Oligonucleotides / Q000138:chemical synthesis*; D016133:Polymerase Chain Reaction
D009841:Oligonucleotides