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PLoS One. 2015 Sep 11;10(9):e0137652. doi: 10.1371/journal.pone.0137652. eCollection 2015.

AQUA Cloning: A Versatile and Simple Enzyme-Free Cloning Approach.

Author information

1
Faculty of Biology, University of Freiburg, Freiburg, Germany; Spemann Graduate School of Biology and Medicine (SGBM), University of Freiburg, Freiburg, Germany.
2
Faculty of Biology, University of Freiburg, Freiburg, Germany.
3
BIOSS Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany; IMTEK, Department of Microsystems Engineering, University of Freiburg, Freiburg, Germany.
4
Faculty of Biology, University of Freiburg, Freiburg, Germany; Spemann Graduate School of Biology and Medicine (SGBM), University of Freiburg, Freiburg, Germany; BIOSS Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany.
5
Faculty of Biology, University of Freiburg, Freiburg, Germany; BIOSS Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany.

Abstract

Assembly cloning is increasingly replacing conventional restriction enzyme and DNA-ligase-dependent cloning methods for reasons of efficiency and performance. Here, we describe AQUA (advanced quick assembly), a simple and versatile seamless assembly cloning approach. We demonstrate the applicability and versatility of AQUA Cloning in selected proof-of-principle applications including targeted insertion-, deletion- and site-directed point-mutagenesis, and combinatorial cloning. Furthermore, we show the one pot de novo assembly of multiple DNA fragments into a single circular plasmid encoding a complex light- and chemically-regulated Boolean A NIMPLY B logic operation. AQUA Cloning harnesses intrinsic in vivo processing of linear DNA fragments with short regions of homology of 16 to 32 bp mediated by Escherichia coli. It does not require any kits, enzymes or preparations of reagents and is the simplest assembly cloning protocol to date.

PMID:
26360249
PMCID:
PMC4567319
DOI:
10.1371/journal.pone.0137652
[Indexed for MEDLINE]
Free PMC Article

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