ExoCET: exonuclease in vitro assembly combined with RecET recombination for highly efficient direct DNA cloning from complex genomes. (Erratum)
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Authors
Wang, HailongLi, Zhen
Jia, Ruonan
Yin, Jia
Li, Aiying
Xia, Liqiu
Yin, Yulong
Müller, Rolf
Fu, Jun
Stewart, A Francis
Zhang, Youming
Issue Date
2018-03-16
Metadata
Show full item recordAbstract
The exponentially increasing volumes of DNA sequence data highlight the need for new DNA cloning methods to explore the new information. Here, we describe ‘ExoCET’ (Exonuclease Combined with RecET recombination) to directly clone any chosen region from bacterial and mammalian genomes with nucleotide precision into operational plasmids. ExoCET combines in vitro exonuclease and annealing with the remarkable capacity of full length RecET homologous recombination (HR) to retrieve specified regions from genomic DNA preparations. Using T4 polymerase (T4pol) as the in vitro exonuclease for ExoCET, we directly cloned large regions (>50 kb) from bacterial and mammalian genomes, including DNA isolated from blood. Employing RecET HR or Cas9 cleavage in vitro, the directly cloned region can be chosen with nucleotide precision to position, for example, a gene into an expression vector without the need for further subcloning. In addition to its utility for bioprospecting in bacterial genomes, ExoCET presents straightforward access to mammalian genomes for various applications such as region-specific DNA sequencing that retains haplotype phasing, the rapid construction of optimal, haplotypic, isogenic targeting constructs or a new way to genotype that presents advantages over Southern blotting or polymerase chain reaction. The direct cloning capacities of ExoCET present new freedoms in recombinant DNA technology.Citation
Nucleic Acids Res. 2018 Mar 16;46(5):2697. doi: 10.1093/nar/gkx1296.Affiliation
HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany.Publisher
Oxford University PressJournal
Nucleic Acids ResearchPubMed ID
29272442Type
Erratum to https://dx.doi.org/10.1093/nar/gkx1249 / http://hdl.handle.net/10033/621234Language
enISSN
1362-4962ae974a485f413a2113503eed53cd6c53
10.1093/nar/gkx1296
Scopus Count
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