ExoCET: exonuclease in vitro assembly combined with RecET recombination for highly efficient direct DNA cloning from complex genomes.

2.50
Hdl Handle:
http://hdl.handle.net/10033/621234
Title:
ExoCET: exonuclease in vitro assembly combined with RecET recombination for highly efficient direct DNA cloning from complex genomes.
Authors:
Wang, Hailong; Li, Zhen; Jia, Ruonan; Yin, Jia; Li, Aiying; Xia, Liqiu; Yin, Yulong; Müller, Rolf ( 0000-0002-1042-5665 ) ; Fu, Jun; Stewart, A Francis; Zhang, Youming
Abstract:
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.
Affiliation:
HIPS, Helmholtz-Institut für pharmazeutische Forschung Saarland, Universitätscampus E8.1, 66123 Saarbrücken, Germany.
Citation:
ExoCET: exonuclease in vitro assembly combined with RecET recombination for highly efficient direct DNA cloning from complex genomes. 2017 Nucleic Acids Res.
Journal:
Nucleic acids research
Issue Date:
12-Dec-2017
URI:
http://hdl.handle.net/10033/621234
DOI:
10.1093/nar/gkx1249
PubMed ID:
29240926
Type:
Article
Language:
en
ISSN:
1362-4962
Appears in Collections:
publications of the department of microbial natural substances ([HIPS]MINS)

Full metadata record

DC FieldValue Language
dc.contributor.authorWang, Hailongen
dc.contributor.authorLi, Zhenen
dc.contributor.authorJia, Ruonanen
dc.contributor.authorYin, Jiaen
dc.contributor.authorLi, Aiyingen
dc.contributor.authorXia, Liqiuen
dc.contributor.authorYin, Yulongen
dc.contributor.authorMüller, Rolfen
dc.contributor.authorFu, Junen
dc.contributor.authorStewart, A Francisen
dc.contributor.authorZhang, Youmingen
dc.date.accessioned2018-01-16T10:22:50Z-
dc.date.available2018-01-16T10:22:50Z-
dc.date.issued2017-12-12-
dc.identifier.citationExoCET: exonuclease in vitro assembly combined with RecET recombination for highly efficient direct DNA cloning from complex genomes. 2017 Nucleic Acids Res.en
dc.identifier.issn1362-4962-
dc.identifier.pmid29240926-
dc.identifier.doi10.1093/nar/gkx1249-
dc.identifier.urihttp://hdl.handle.net/10033/621234-
dc.description.abstractThe 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.en
dc.language.isoenen
dc.rights.urihttp://creativecommons.org/licenses/by-nc-sa/4.0/*
dc.titleExoCET: exonuclease in vitro assembly combined with RecET recombination for highly efficient direct DNA cloning from complex genomes.en
dc.typeArticleen
dc.contributor.departmentHIPS, Helmholtz-Institut für pharmazeutische Forschung Saarland, Universitätscampus E8.1, 66123 Saarbrücken, Germany.en
dc.identifier.journalNucleic acids researchen

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