FIG. 2.

PCR identification for specific gene construction in mutants. (A) PCR for ΔpknG; (B) PCR for ΔrelAL; (C) PCR for ΔrelAS; (D) PCR for Δlsr2. Lane 1, DNA size marker; lane 2, wild type (M. avium subsp. paratuberculosis K10); lane 3, mutant in M. avium subsp. paratuberculosis K10; lane 4, mutant in M. avium subsp. paratuberculosis K10-GFP. The primer sites for ΔpknG (A) and ΔrelAL (B) PCRs were located in the Hyg gene (inserted gene) for the forward primer and outside the downstream homologous region of each disrupted gene for the reverse primer. Note that the wild-type gene was not amplified in panels A and B because of the primer design. The primer sites for ΔrelAS (C) and Δlsr2 (D) PCRs were located outside up- and downstream homologous regions of each disrupted gene, which allowed identification of mutants based on the sizes of the amplified fragments.