Consequently, the implications for a new DNA engineering technology were not obvious. However, Murphy's experiments required expression of RecA and also employed long homology arms. The lambda Red recombination system is now most commonly used and the first demonstrations of Red in vivo genetic engineering were independently made by Kenan Murphy and Francis Stewart. Recombineering is based on homologous recombination in Escherichia coli mediated by bacteriophage proteins, either RecE/RecT from Rac prophage or Redαβδ from bacteriophage lambda. Recombination was observed to take place with oligonucleotides as short as 20 bases. In addition, recombination with single-strand oligonucleotides (oligos) was first shown in Saccharomyces cerevisiae. Recombineering is widely used for bacterial genetics, in the generation of target vectors for making a conditional mouse knockout, and for modifying DNA of any source often contained on a bacterial artificial chromosome (BAC), among other applications.Īlthough developed in bacteria, much of the inspiration for recombineering techniques came from methods first developed in Saccharomyces cerevisiae where a linear plasmid was used to target genes or clone genes off the chromosome. Recombineering ( recombination-mediated genetic engi neering) is a genetic and molecular biology technique based on homologous recombination systems, as opposed to the older/more common method of using restriction enzymes and ligases to combine DNA sequences in a specified order.
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