Summary

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Recombineering is a highly efficient and precise method for genetically engineering DNA in vivo. With recombineering one can make gene replacements, deletions, insertions, inversions, and single and multiple point mutations. Gene cloning and gene/protein tagging is also possible. Recombineering is catalyzed by the bacteriophage Red or similar phage homologous recombination systems and can utilize both double- and single-stranded linear DNAs as substrates. All genetic alterations are precise to the base pair as designed by the user and are mediated efficiently by DNA containing target homologies of ~50 bases. These homologies are short enough to be incorporated into commercially available single-stranded DNA oligonucleotides. Since recombineering is directed by sequence homology, not convenient restriction sites, it can be used to create genetic alterations on large DNA molecules such as BACs or chromosomes more precisely than was possible with classical in vitro genetic engineering techniques. Because of this, recombineering has expanded the analytical genetic capability of many organisms. Recombineering with ssDNA can be >100-fold more efficient than with dsDNA as a substrate. Using ssDNA it is possible to achieve recombination frequencies of over 50% of the cells that survive electroporation, thus a selection is not necessary for finding the desired mutations. Key to these high frequencies is avoiding the methyl-directed mismatch repair system, which normally removes greater than 99% of the incorporated changes. Avoiding mismatch repair can be easily achieved, even in wild type cells, with careful oligonucleotide design.


 © RECOMBINEERING 2016