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



D'Amore, Rosalinda ORCID: 0000-0002-2638-4466, Johnson, James ORCID: 0000-0002-8849-0993, Haldenby, Sam, Hall, Neil, Hughes, Margaret ORCID: 0000-0001-7251-6821, Joynson, Ryan, Kenny, John G, Patron, Nicola, Hertz-Fowler, Christiane ORCID: 0000-0002-0729-6479 and Hall, Anthony
(2017) SMRT Gate: A method for validation of synthetic constructs on Pacific Biosciences sequencing platforms. BIOTECHNIQUES, 63 (1). pp. 13-20.

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Abstract

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.

Item Type: Article
Uncontrolled Keywords: Golden Gate assembly, synthetic biology, next-generation sequencing (NGS), high-throughput, plasmid QC
Depositing User: Symplectic Admin
Date Deposited: 26 Feb 2018 09:39
Last Modified: 19 Jan 2023 06:52
DOI: 10.2144/000114565
Related URLs:
URI: https://livrepository.liverpool.ac.uk/id/eprint/3010522