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Nucleic Acids Res. 2016 Mar 18;44(5):e43. doi: 10.1093/nar/gkv1177. Epub 2015 Nov 8.

Multiplex pairwise assembly of array-derived DNA oligonucleotides.

Author information

1
Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA.
2
Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.
3
Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.
4
Department of Biochemistry, University of Washington, Seattle, WA 98195, USA Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195, USA.
5
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.

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.

PMID:
26553805
PMCID:
PMC4797260
DOI:
10.1093/nar/gkv1177
[Indexed for MEDLINE]
Free PMC Article

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