The Stewart lab showed that these homologous recombination systems mediate efficient recombination of linear DNA molecules flanked by homology sequences as short as 30 base pairs (40-50 base pairs are more efficient) into target DNA sequences in the absence of RecA.
Now the homology could be provided by oligonucleotides made to order, and standard recA cloning hosts could be used, greatly expanding the utility of recombineering.
In the second stage, a second counterselection marker (e.g. sacB) on the cassette is selected against following introduction of a target fragment containing the desired modification.
Alternatively, the target fragment could be flanked by loxP or FRT sites, which could be removed later simply by the expression of the Cre or FLP recombinases, respectively.
[13] Recombineering with ssDNA provided a breakthrough both in the efficiency of the reaction and the ease of making point mutations.
[1] This technique was further enhanced by the discovery that by avoiding the methyl-directed mismatch repair system, the frequency of obtaining recombinants can be increased to over 107/108 viable cells.
It also forgoes multiple cloning stages for generating intermediate vectors and therefore is used to modify DNA constructs in a relatively short time-frame.
Recently, recombineering has been developed for high throughput DNA engineering applications termed 'recombineering pipelines'.
[23] Recombineering pipelines support the large scale production of BAC transgenes and gene targeting constructs for functional genomics programs such as EUCOMM (European Conditional Mouse Mutagenesis Consortium) and KOMP (Knock-Out Mouse Program).