Figure 9.
Gene replacement and verification of recombinants using recombineering. (A) Outline of the basic steps involved in recombineering. (B) Primer design for gene replacement and verification. The 3′ ends of primers 1 and 2 contain 20 bp for amplification of the drugR marker (including regulatory regions), while the 5′ ends of the primers contain 40 to 50 bp of sequence that flank the target gene (red lines). Primers 5 and 6 are used to verify the 5′ junction of the recombinant, and primers 7 and 8 are used to verify the 3′ junction of the recombinant. Primers 5 and 8 can be used to verify loss of wild-type sequence (either by agarose gel or restriction enzyme analysis). Alternatively, primers 3 and 4 are designed to generate an internal fragment of the target gene. This product should be absent in the recombinant.