July 19, 2019  |  

SMRT Gate: A method for validation of synthetic constructs on Pacific Biosciences sequencing platforms.

Authors: D'Amore, Rosalinda and Johnson, James and Haldenby, Sam and Hall, Neil and Hughes, Margaret and Joynson, Ryan and Kenny, John G and Patron, Nicola and Hertz-Fowler, Christiane and Hall, Anthony

Current DNA assembly methods are prone to sequence errors, requiring rigorous quality control (QC) to identify incorrect assemblies or synthesized constructs. Such errors can lead to misinterpretation of phenotypes. Because of this intrinsic problem, routine QC analysis is generally performed on three or more clones using a combination of restriction endonuclease assays, colony PCR, and Sanger sequencing. However, as new automation methods emerge that enable high-throughput assembly, QC using these techniques has become a major bottleneck. Here, we describe a quick and affordable methodology for the QC of synthetic constructs. Our method involves a one-pot digestion-ligation DNA assembly reaction, based on the Golden Gate assembly methodology, that is coupled with Pacific Biosciences' Single Molecule, Real-Time (PacBio SMRT) sequencing technology.

Journal: BioTechniques
DOI: 10.2144/000114565
Year: 2017

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