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Recombineering with dsDNA.

Routinely, dsDNA cassettes encoding a drug-resistance are used to make gene knockouts. Typically, the drug-resistant cassette is made by PCR using bi-partite primers. These primers consist of (from 5’λ3’) 50 bases of homology to the target region, where the cassette is to be inserted, followed by ~20 bases to prime the drug-resistant cassette (Figure 1).

Figure 1. Using dsDNA to generate gene replacement knockouts and deletions. I.) A drug-resistant cassette is made by PCR using two, 70-base hybrid primers. The 5’ end of the primer has homology to the desired target (50-base) and the 3’ end of the primer (~20-base) has sequence to prime the drug-resistance template gene (DrugR). Primer design determines the junctions of the final construct. II.) The linear drug-resistant cassette made by PCR is transformed into recombination-competent cells and Red-mediated recombination occurs. Depending on what PCR product is used, (a) or (b), the final recombinant will be a gene swap, IIIa or a more substantial deletion, IIIb, respectively. The drug cassette could be inserted between two adjacent bases as well.

The junction sequence created by the primer design determines precisely how the recombination occurs. For example, the cassette can be inserted between two adjacent bases, the coding sequence of a gene can be replaced to create an in-frame, non-polar knockout, or a whole operon can be deleted. The deletions can range in size from a base pair to >10kb. The PCR fragment is then transformed into electro-competent cells that have been induced for the λ Red system. Having the recombination system under tight regulation yet highly expressed when desired is key for achieving optimal recombination frequencies and preventing unwanted rearrangements. The Red system under control of its own regulated promoter, pL, is much better for this than other systems where the recombination functions are expressed by less tightly controlled promoters.

dsDNA recombineering is very useful for making replacement knockouts and deletions, which are important for many genetic analyses. These recombinants can be found at a frequency of greater than 104 per 108 viable cells ( >104/108 viable). With modifications to the protocol, deletions and point mutations can be made such that there is no drug marker or other alteration left behind. This is accomplished by completing two rounds of dsDNA recombineering using a selection/counter-selection dual cassette. In the first step, the dual cassette is inserted and the (drug) marker is selected. In the second recombination event, the entire cassette is removed by counter-selection. The end result can be a clean deletion, a point mutation created in a gene of interest with no scar left behind, or a tag such as GFP or His. If mutagenic PCR is used to create the linear substrate for the second step, random mutations can be made in the region of interest. The second step can also be done with ssDNA (see below).