ExoCET: exonuclease in vitro assembly combined with RecET recombination for highly efficient direct DNA cloning from complex genomes.
Cast your vote
You can rate an item by clicking the amount of stars they wish to award to this item.
When enough users have cast their vote on this item, the average rating will also be shown.
Your vote was cast
Thank you for your feedback
Thank you for your feedback
Stewart, A Francis
MetadataShow full item record
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.
CitationExoCET: exonuclease in vitro assembly combined with RecET recombination for highly efficient direct DNA cloning from complex genomes. 2017 Nucleic Acids Res.
AffiliationHIPS, Helmholtz-Institut für pharmazeutische Forschung Saarland, Universitätscampus E8.1, 66123 Saarbrücken, Germany.
JournalNucleic acids research
The following license files are associated with this item:
- Creative Commons
Except where otherwise noted, this item's license is described as http://creativecommons.org/licenses/by-nc-sa/4.0/
- Full-length RecE enhances linear-linear homologous recombination and facilitates direct cloning for bioprospecting.
- Authors: Fu J, Bian X, Hu S, Wang H, Huang F, Seibert PM, Plaza A, Xia L, Müller R, Stewart AF, Zhang Y
- Issue date: 2012 May
- RecET driven chromosomal gene targeting to generate a RecA deficient Escherichia coli strain for Cre mediated production of minicircle DNA.
- Authors: Tolmachov O, Palaszewski I, Bigger B, Coutelle C
- Issue date: 2006 Mar 10
- ExoCET: exonuclease in vitro assembly combined with RecET recombination for highly efficient direct DNA cloning from complex genomes.
- Authors: Wang H, Li Z, Jia R, Yin J, Li A, Xia L, Yin Y, Müller R, Fu J, Stewart AF, Zhang Y
- Issue date: 2018 Mar 16
- [The application of Red/ET recombination to high efficient gene-targeting vector construction].
- Authors: Wang JP, Zhang YM
- Issue date: 2005 Nov
- DNA cloning by homologous recombination in Escherichia coli.
- Authors: Zhang Y, Muyrers JP, Testa G, Stewart AF
- Issue date: 2000 Dec