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PLoS One. 2009 Jul 30;4(7):e6441. doi: 10.1371/journal.pone.0006441.

Circular polymerase extension cloning of complex gene libraries and pathways.

Author information

1
Department of Biomedical Engineering & Institute for Genome Sciences and Policy, Duke University, Durham, North Carolina, United States of America.

Abstract

High-throughput genomics and the emerging field of synthetic biology demand ever more convenient, economical, and efficient technologies to assemble and clone genes, gene libraries and synthetic pathways. Here, we describe the development of a novel and extremely simple cloning method, circular polymerase extension cloning (CPEC). This method uses a single polymerase to assemble and clone multiple inserts with any vector in a one-step reaction in vitro. No restriction digestion, ligation, or single-stranded homologous recombination is required. In this study, we elucidate the CPEC reaction mechanism and demonstrate its usage in demanding synthetic biology applications such as one-step assembly and cloning of complex combinatorial libraries and multi-component pathways.

PMID:
19649325
PMCID:
PMC2713398
DOI:
10.1371/journal.pone.0006441
[Indexed for MEDLINE]
Free PMC Article

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