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. 2005 Jan;79(1):299–313. doi: 10.1128/JVI.79.1.299-313.2005

FIG. 2.

FIG. 2.

Genomic analysis of pYEbac102 mutants. (A) Diagnostic PCR to confirm the insertion of the relevant gene cassettes in place of the deleted genomic regions in UL20 (A, panel i) UL27 (gB) (A, panel ii), and UL53 (gK) (A, panel iii). (B) KpnI restriction fragment analysis of pYEbac102 mutant BACs in comparison to wild-type pYEbac102. The black arrow demarcates the position of the KpnI DNA fragment altered by the ΔgB-Kan mutation, while the gray arrow points to the missing 9,432-bp fragment resulting from the ΔUL20-GFPzeo mutation. (C and D) Southern blot analysis of pYEbac102 mutant BACs. The KpnI-restricted BACs from panel B were hybridized with either a kanamycin resistance gene (C) or GFP-zeocin resistance gene (D) biotinylated probe. The biotinylated kanamycin resistance gene probe hybridized to an estimated 4,835-bp DNA fragment in pYEbacΔgB, pYEbacΔgBΔgUL20, and pYEbacΔgBΔgK DNA, which was absent in the wild-type pYEbac102 DNA (indicated by a black arrow in panel C). The biotinylated GFP-zeocin probe identified a predicted 10,992-bp DNA fragment (gray arrow) in pYEbacΔUL20 and pYEbacΔgBΔgUL20 DNA corresponding to the ΔUL20-GFPzeo mutation, and a predicted 8,956-bp fragment (white arrow) in pYEbacΔgK and pYEbacΔgBΔgK DNA corresponding to the ΔgK-GFPzeo mutation (D).