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Requirements for recombineering.

Recombineering requires that the linear DNA contain homologies to the target DNA of ~50 bases, which are short enough to be built into readily available commercial DNA oligonucleotides (oligos). The linear DNA can be either double-stranded (dsDNA) or single-stranded (ssDNA), and is introduced, generally via electroporation, into the recombineering-competent bacterial cell. For simplicity, we concentrate on the λRed system which consists of three proteins, Gam, Exo and Beta. The Gam protein inhibits the E. coli RecBCD and SbcCD exonucleases that normally degrade all linear dsDNA. Gam is not absolutely required for recombineering but increases the frequency of dsDNA recombination about 10-fold. Exo is a 5’λ3’ double-strand DNA specific exonuclease and is only required for dsDNA recombination. The Beta protein, a ssDNA annealing protein, is the central player in recombineering and is required for both dsDNA and ssDNA-mediated recombination. Importantly, host recombination functions, including the key recombination protein RecA, are not required for recombineering.

In most cases, the target DNA must be replicating: active DNA replication of the target molecule is required for most recombineering reactions. The target DNA can be the bacterial chromosome, plasmids, bacterial artificial chromosomes (BAC) or phage. Thus any DNA, even DNA from human cells cloned into a BAC, can be placed into a recombineering-competent cell and efficiently modified.