title: Multiplex pairwise assembly of array-derived DNA oligonucleotides.
abstract: While the cost of DNA sequencing has dropped by five orders of magnitude in the past decade, DNA synthesis remains expensive for many applications. Although DNA microarrays have decreased the cost of oligonucleotide synthesis, the use of array-synthesized oligos in practice is limited by short synthesis lengths, high synthesis error rates, low yield and the challenges of assembling long constructs from complex pools. Toward addressing these issues, we developed a protocol for multiplex pairwise assembly of oligos from array-synthesized oligonucleotide pools. To evaluate the method, we attempted to assemble up to 2271 targets ranging in length from 192-252 bases using pairs of array-synthesized oligos. Within sets of complexity ranging from 131-250 targets, we observed error-free assemblies for 90.5% of all targets. When all 2271 targets were assembled in one reaction, we observed error-free constructs for 70.6%. While the assembly method intrinsically increased accuracy to a small degree, we further increased accuracy by using a high throughput 'Dial-Out PCR' protocol, which combines Illumina sequencing with an in-house set of unique PCR tags to selectively amplify perfect assemblies from complex synthetic pools. This approach has broad applicability to DNA assembly and high-throughput functional screens.
authors.affiliation: Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA.
authors.forename: Jason C
authors.initials: JC
authors.lastname: Klein
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.
authors.forename: Marc J
authors.initials: MJ
authors.lastname: Lajoie
authors.affiliation: Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA.
authors.forename: Jerrod J
authors.initials: JJ
authors.lastname: Schwartz
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.
authors.forename: Eva-Maria
authors.initials: EM
authors.lastname: Strauch
authors.affiliation: Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.
authors.forename: Jorgen
authors.initials: J
authors.lastname: Nelson
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, WA 98195, USA Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195, USA.
authors.forename: David
authors.initials: D
authors.lastname: Baker
authors.affiliation: Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195, USA shendure@u.washington.edu.
authors.forename: Jay
authors.initials: J
authors.lastname: Shendure
mesh_terms: D000465:Algorithms; D004247:DNA / Q000737:chemistry; D017931:DNA Primers / Q000138:chemical synthesis; D020224:Expressed Sequence Tags / Q000737:chemistry; D059014:High-Throughput Nucleotide Sequencing / Q000295:instrumentation / Q000379:methods; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D009841:Oligonucleotides / Q000138:chemical synthesis* / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D017931:DNA Primers; D009841:Oligonucleotides; D004247:DNA