Full-length RecE enhances linear-linear homologous recombination and facilitates direct cloning for bioprospecting

@article{Fu2012FulllengthRE,
  title={Full-length RecE enhances linear-linear homologous recombination and facilitates direct cloning for bioprospecting},
  author={Jun Fu and X. Bian and Shengbaio Hu and Hailong Wang and F. Huang and P. M. Seibert and A. Plaza and L. Xia and R. M{\"u}ller and A. Stewart and Youming Zhang},
  journal={Nature Biotechnology},
  year={2012},
  volume={30},
  pages={440-446}
}
Functional analysis of genome sequences requires methods for cloning DNA of interest. However, existing methods, such as library cloning and screening, are too demanding or inefficient for high-throughput application to the wealth of genomic data being delivered by massively parallel sequencing. Here we describe direct DNA cloning based on the discovery that the full-length Rac prophage protein RecE and its partner RecT mediate highly efficient linear-linear homologous recombination… Expand
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References

SHOWING 1-10 OF 66 REFERENCES
RecA-mediated affinity capture: a method for full-length cDNA cloning.
TLDR
An improved method for rapid cloning of full-length cDNA from cDNA libraries based on the ability of Escherichia coli RecA protein to form a stable nucleoprotein complex with a linear single-Stranded DNA probe and homologous sequences in circular double-stranded DNA is described. Expand
Selective isolation of genomic loci from complex genomes by transformation-associated recombination cloning in the yeast Saccharomyces cerevisiae
TLDR
A protocol for the selective isolation of any genomic fragment or gene of interest up to 250 kb in size from complex genomes as a circular yeast artificial chromosome (YAC) based on transformation-associated recombination in the yeast Saccharomyces cerevisiae. Expand
An improved recombineering approach by adding RecA to λ Red recombination
TLDR
It is reported that transient RecA co-expression enhances the total numer of successful recombinations in bacterial artificial chromosomes (BACs), mostly because the E. coli host is more able to survive the stresses of DNA transformation procedures. Expand
An improved recombineering approach by adding RecA to lambda Red recombination.
TLDR
It is reported that transient RecA co-expression enhances the total number of successful recombinations in bacterial artificial chromosomes (BACs), mostly because the E. coli host is more able to survive the stresses of DNA transformation procedures. Expand
DNA cloning by homologous recombination in Escherichia coli
TLDR
The cloning of foreign DNA in Escherichia coli episomes is a cornerstone of molecular biology and a different principle, using ET recombination, for directed cloning and subcloning is described, which offers a variety of advantages. Expand
GENETIC ENGINEERING USING HOMOLOGOUS RECOMBINATION 1
■ Abstract In the past few years, in vivo technologies have emerged that, due to their efficiency and simplicity, may one day replace standard genetic engineering techniques. Constructs can be madeExpand
High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides
TLDR
It is demonstrated in this paper that Beta protein of phage λ generates recombinants in chromosomal DNA by using synthetic single-stranded DNAs as short as 30 bases long, which provides new avenues for studying and modifying genomes ranging from bacterial pathogens to eukaryotes. Expand
Lambda Red Recombineering in Escherichia coli Occurs Through a Fully Single-Stranded Intermediate
TLDR
This work proposes an alternative in which lambda exonuclease entirely degrades one strand, while leaving the other strand intact as single-stranded DNA, which then recombines via beta recombinase-catalyzed annealing at the replication fork. Expand
Heterologous expression of a myxobacterial natural products assembly line in pseudomonads via red/ET recombineering.
TLDR
A straightforward strategy that combines the power of advanced DNA engineering in Escherichia coli with the utility of pseudomonads as the heterologous host for the analysis and mutagenesis of known and unknown secondary metabolite pathways is described. Expand
A recombineering pipeline for functional genomics applied to Caenorhabditis elegans
TLDR
The strategy that is described adapts the power of recombineering in Escherichia coli for fluent DNA engineering to a format that can be directly scaled up for genomic projects. Expand
...
1
2
3
4
5
...